Home

MetaMorph NX User Guide

1. Measurement Definition The distance around the edge of the object measuring from the mid points of each pixel that defines its border Perimeter Centroid X and Y in calibrated units The X and Y coordinates of the centroid of the object The centroid is the center of mass of the object ignoring pixel intensities Depending on the shape of the object the centroid may not be inside the object for example if the object is concave Centroid X and Y in pixel units The X and Y coordinates of the centroid of the object expressed in pixels If the calibrated unit is 1 this measurement is the same as the Centroid X and Y in calibrated units measurement The angle between the longest chord through the object and the horizontal axis Orientation Angles are measured from the three o clock East position and range from 90 to 90 degrees Downward angles are expressed 5011685 B 135 Chapter 8 Measuring and Analyzing Images Measurement Definition as positive numbers and upward angles are expressed as negative numbers 90 Note In the MetaMorph Software version 7 7 and earlier in the Integrated Morphometry Analysis application module downward angles were expressed as negative numbers and upward angles were expressed as positive numbers A value from 0 zero to 1 representing how closely the object represents a circle A value near 0 indicat
2. Open the Graph Settings dialog box Selectthe measurements Selecta layout for Selecta marker style to select the measurements forthe X and Y ais and the current graph forthe current graph included on the graph set the range configure the range of data forthe axes type a title and select to display on each ais other style and layout options 5011685 B 13 Chapter 1 Navigating the Workspace Script Tab Cancel the Select and run script that is a saipt running Acquisition Mode os i Create Runthe Resume Cancel Script scnptfile Scri Script Open the saipt Resume a editor to greate paused saipt a new saipt Hardware Setup Tab Setthe Open the dialog box for Assign software acquisition area creating and managing commands to a Callate ofthe camerato hardware illumination or Current stage and Z Install Load installed keypad keyboard or exposure time the full chip area magnification settings motor positions devices devices mouse Acquisition Mode Mod Full Chip E Current View Snapasinge Displayalive Set the acquisition area of Move devices to positions Openthe Stage Motor Configurethe Configure the mouse image using the view of the the camera to the area defined in the selected Designations dialog boxto active camera wheel to move a spedfied binning image usingthe displayed in the Latest hardware illumination designate motors for specific device spedfied binning Image tab set the area to or magnification settin
3. 1 Configure the measurement results so that the data table contains only the data that you want to export 2 Onthe Measure tab click Export to File 3 Inthe File Path field verify or type the location of the file into which the data will be exported By default the MetaMorph NX Software saves the file with the open dataset 4 Inthe File Type field select Excel In the Worksheet Title field verify or type the name of the new worksheet 6 Click OK To export data to a text file You can export data directly to a text file comma separated values format The text file does not automatically open when you export data to it A CAUTION If you export data to an existing file the exported data overwrites all existing data in the file the data is not appended to the file 1 Configure the measurement results so that the data table contains only the data that you want to export 2 Onthe Measure tab click Export to File 3 Inthe File Path field verify or type the location of the file into which the data will be exported By default the MetaMorph NX Software saves the file with the open dataset 4 Inthe File Type field select Csv 5 Click OK Graphing Measurement Results The MetaMorph NX Software provides two types of graphs e Scatter plot shows the relationship between any two measurements including o the settings or input used to make the measurement for example the low and high threshold values o
4. If the data table for the measurement set is open when you click on a point in a graph the data table highlights the row that corresponds to the point and if the image is in view the image highlights the corresponding object Likewise when you select a row in the data table the graph highlights the point that corresponds to the row and the image highlights the corresponding object When you click on an object in an image the graph and table highlight the corresponding point and row To select more than one object row or point use lt CTRL gt click to select each item When you double click a point on a graph or a row in the data table the image display updates so that the corresponding object is in view While graphs are open in a Dataset View you can select a measurement set on the Measure tab to view the data in the measurement set as configured for that graph and then select another measurement set to view the data as configured for that graph and so on When you close an experiment the MetaMorph NX Software remembers the settings used for the last graph that you created The next time you open the same experiment or a different one the first graph that you create will display the same settings as the last graph that you created If you want to compare similar data between datasets and experiments you can do so without needing to reconfigure the settings for each graph This section describes how to e Create a graph of meas
5. l just acquired images using the Multidimensional acquisition mode so why is there no Plane Information for the new dataset on the Measure tab When you acquire images the Latest Image tab appears Because the Latest Image tab does not contain a full dataset plane information is not available when the Latest Image tab is displayed To view the plane information for the new dataset you must switch from the Latest Image tab to the new Dataset tab click on the new dataset in the filmstrip and then click Plane Information in the Measure tab How do I threshold an image Use the Measure Using Threshold measurement function to threshold an image using set values Although the thresholded image is not saved as a separate image it is stored with the measurement set that is created when you use the Measure Using Threshold measurement function See Measuring Morphometry of Objects l installed focus stabilization hardware but I do not see the Focus Stabilizer options on the tab or in the Acquisition dialog box Why Open the Stage Motor Designations dialog box and make sure that the Z motor that will be used for focus stabilization is designated as the same motor that will be used for autofocusing For example if the Z motor that will be used for focus stabilization is designated as the Z2 motor then you must also designate the Z2 motor as the motor to use for autofocusing See Designating Z Motors and Stages for Acquisition Why am I not
6. 24 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Suppose for example that you want to compare images taken at the first and last time points of a lengthy time lapse image acquisition Rather than scroll back and forth through the dataset you can display the same dataset in a second window scroll to the last time point and then place the two windows side by side For example Note Only one experiment can be open at a time This section describes how to e Move or group tabbed windows e Move windows e Hide a window e Auto Hide a dialog box e Close a tabbed window or close all tabbed windows except the selected one To move or group tabbed windows 1 Right click on a tab name and select New Horizontal Tab Group or New Vertical Tab Group 2 To move a tabbed window from one group of windows to another group of windows right click a tab name and select Move to Next Tab Group or Move to Previous Tab Group To move windows The data table and graphs can be docked at the left side right side top or bottom of a Dataset View Dialog boxes can be docked anywhere in the MetaMorph NX Software workspace 5011685 B 25 Chapter 2 Viewing Images 1 Click anywhere in the tab or title bar of the window and then drag it until dim arrows appear on the left right top and bottom of the Dataset View or workspace For example The arrows may also appear as a target in the midd
7. Click Start Continuous Focus While continuous focus is on the Olympus ZDC or ZDC2 continuously adjusts the lens position to maintain focus Note You cannot adjust the Z motor position using the microscope controls or change objectives while continuous focus is on Click Acquire When the image acquisition is complete click Stop Continuous Focus When continuous focusing is off you can control the Z motor using the knobs on the microscope To use the Olympus ZDC or ZDC2 for a multidimensional image acquisition without a stage series 114 1 Configure the settings for the multidimensional acquisition such as channels and if applicable image autofocus and a Z series If your acquisition includes more than one channel on the Mode Multidimensional tab select the first channel in the channel series Note The focus stabilization system uses the first channel to calculate the focus adjustment value Subsequent channels are offset from the first channel In Multidimensional Acquisition click Configure to open the Acquisition dialog box In the Acquisition dialog box select the Focus Stabilizer check box to indicate that the acquisition should use the focus stabilizer hardware Olympus ZDC or ZDC2 and then click Focus Stabilizer to display the related fields If your acquisition includes a time series in the Autofocus Frequency field Select To re establish focus Every Point at every time point may slow
8. Inthe Calibration field type the size of one pixel Typically a pixel is measured in microns If you do not know how many microns equal a pixel use a stage micrometer to measure a pixel or ask your lens or camera dealer for this information The calibration should be defined for a non binned image When acquiring images that have been binned the MetaMorph NX Software will automatically adjust the spatial calibration to reflect the binning Spatial Calibration Calibration x Binning The spatial calibration of an image is recorded in the Plane Information measurement set In the Magnification field type the magnification of the objective Inthe Z Escape Distance field type the distance to move the Z motor before changing the objective The Z motor returns to its original position after the objective is changed Using an escape distance helps prevent the objective from contacting the slide while the objective is changing Optionally select the Serialize Moves check box to indicate that no other devices should move when the selected device moves If additional components are available you can click Other Peripherals to expand the list of components and review or change their properties See Device and Component Settings 5011685 B 59 Chapter 6 Installing and Configuring Hardware topics for specific settings information 14 Click Save Current Setting to save your changes The new magnification setting is added to the list of mag
9. MetaMorph NX Microscopy Automation amp Image Analysis Software Version 1 1 User Guide 5011685 B December 2010 Mol I OQoevices This document is provided to customers who have purchased Molecular Devices Inc Molecular Devices equipment software reagents and consumables to use in the operation of such Molecular Devices equipment software reagents and consumables This documentis copyright protected and any reproduction of this document in whole or any part is strictly prohibited except as Molecular Devices may authorize in writing Software that may be described in this document is furnished under a license agreement It is against the law to copy modify or distribute the software on any medium except as specifically allowed in the license agreement Furthermore the license agreement may prohibit the software from being disassembled reverse engineered or decompiled for any purpose Portions of this document may make reference to other manufacturers and or their products which may contain parts whose names are registered as trademarks and or function as trademarks of their respective owners Any such usage is intended only to designate those manufacturers products as supplied by Molecular Devices for REGISTERED incorporation into its equipment and does not imply any right and or license to use or COMPANY permit others to use such manufacturers and or their product names as trademarks Molecular
10. Note The focus stabilization system uses the first channel to calculate the focus adjustment value Subsequent channels are offset from the first channel In Acquisition click Configure to open the Acquisition dialog box In the Acquisition dialog box select the Focus Stabilizer check box to indicate that the acquisition should use the focus stabilizer hardware and then click Focus Stabilizer to 5011685 B 117 Chapter 7 Acquiring Images o oN 10 11 display the related fields Ge You can also use the options in the Focus group on the Mode Interactive tab to configure focus stabilization Changes that you make on the tab are reflected in the Acquisition dialog box and changes that you make in the dialog box are reflected on the tab Inthe Continuous Frequency field enter the intervals in seconds at which the Zeiss Definite Focus should re establish focus Click Live to display a live view of the image Use the focusing knobs on the microscope to bring the image into focus Click Memorize Position Click Start Continuous Focus Note You cannot adjust the Z motor position using the microscope controls or change objectives while continuous focus is on Click Acquire The Zeiss Definite Focus maintains focus of the image as the image is acquired Click Stop Continuous Focus to turn off continuous focusing When continuous focusing is off you can control the Z motor using the knobs on the microscope To us
11. 1 Use the display options on the Home tab to display the image or images exactly the way you want them to appear in a PowerPoint slide See Arranging and Displaying Images for more information 2 Onthe Home tab in Dataset View click Export to PowerPoint Microsoft PowerPoint opens and displays the images that you exported in a new slide The dataset name and the date it was created as well as any dimension information that is available for example stage position or time point appears in a text box above the image 3 To export another image to a new slide in the same presentation keep Microsoft PowerPoint open and repeat Steps 1 2 33 Chapter 4 Configuring a Keypad Keyboard or Mouse This chapter includes the following topics e Using the Mouse Wheel to Control Device Movement e Configuring an Input Accessory Such as a Keypad or Mouse Using the Mouse Wheel to Control Device Movement If your mouse has a wheel you can use the Mouse Wheel Control option to specify that a device should move when you scroll the mouse wheel For example you could configure the wheel control so that a Z motor moves up or down when you scroll the mouse wheel up or down The devices that the mouse wheel can move depend on the type of hardware that is installed If you have two mice one can be used as the primary system mouse and the other can be used as an input accessory Keep in mind that when the Mouse Wheel Control option i
12. CurrentZ position Confi gure the settings for Z steps at which images will be acquired Set the interval time and duration for image acquisition Open the Acquisition dialog bax to configure additional settings fora multidimensional acquisition for sample add a time segment or set spedfic camera parameters per channel Acquisition Mode Mode Multidimensional Measure Hardware Srt Green2 Shutter en Focus F Interval Duration 2 f s ms G gt Configure Acquire Stop Multidimensional Acquisition Select the Configure the channels Setthe acquisition area of Configure the magnificatontouse touse to acquire images the camera to the full chip settings for to acquire images and adjust exposure area ofthe camera orto stage positions time if needed the area displayed in the at which images Latest Image tab set the area to full chip and then zoomin on the image to displ ay just the area that you wantto acquire Mode Interactive Tab Display a live view of will be acquired the image or acquire a Confi gure the Select the singe image using the settings for image magnification to use spedfied binning and Acquire images autofocus and to acquire images selected channel usingall channels focus stabilization ZN w Home Acquisition Mode Mode Interactive Meare Script Hardware Setup Channel Series Camera Configure the channels Setthe acquisition area o
13. MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide 20 If you are using different values for the channel in the list of settings for the channel type or select the new value for the camera setting 21 If the acquisition includes a Z series select the Center Z Only check box to acquire an image at just the center Z position using the channel or clear the check box to acquire the entire Z series using the channel See Configuring a Z Series Acquisition 22 Totest the current device positions and settings at the top of the Acquisition dialog box click the Snap button that corresponds to the channel that you want to use for testing or double click the icon of the channel on the Mode Multidimensional tab The MetaMorph NX Software opens the Latest Image tab and displays a single new image using the selected channel You can use the snapped image as a guide for adjusting your settings See Snapping an Image 23 Optionally configure other parts of the acquisition before acquiring images See Configuring a Time Series Acquisition Configuring a Stage Series Acquisition Configuring Image Autofocus Using Focus Stabilization or Configuring a Z Series for Acquisition 24 Select the Do Not Update Display check box to indicate that images should not be displayed as they are acquired in this case the new dataset is not added to the experiment until the acquisition is complete or clear the check box to indicate th
14. To save images into a dataset In Interactive acquisition mode you can save acquired images into an existing dataset or into a new dataset Each time you save an image into a dataset the image is saved as a separate stage position Images whether multi channel or single channel can be saved together as a separate stage 72 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide position in one dataset or in separate datasets Saving as stage positions in a single dataset may allow for easier comparisons of measurements made after acquisition Saving as individual datasets may make organization and reviewing simpler if you have acquired only a few images Only images that are consistent such as having the same number of channels and same number of pixels can be saved together The filmstrip provides guidance on which images can be saved together When you select double click an image in the filmstrip an orange border appears around the image If you select other images acquired with different channels the orange border remains around the images that can be saved together To save more than one image into a dataset the images must have the same e number of channels e binning value e camera area e width and height If you attempt to save an image into an existing dataset that contains images with a different number of channels binning camera area or size the MetaMorph NX Software automatically cre
15. To use auto expose before acquiring an image 1 On the Mode Interactive tab or Mode Multidimensional tab click Configure to open the Acquisition dialog box Inthe Acquisition dialog box in the Camera Settings section type the binning value to use for the auto expose test image Inthe Acquisition dialog box in the Channels section select the channel that you want to auto expose In the Target Intensity field type the maximum intensity value that auto exposure should attempt to attain for the brightest pixel in the image The target intensity determines the exposure time during auto expose Typically the target intensity value should be approximately 75 of the maximum intensity value that the camera driver is able to obtain Be careful to not set the target intensity too close to the saturation point of the camera Lower intensity values will help prevent bleaching of the sample In the Initial Exposure field type the length of the first exposure attempt Set an exposure time that is well below the typical exposure time required for this type of experiment generally the initial exposure time should be approximately one tenth of the typical exposure time If you do not know the required exposure time enter 1 or 5 or use the default value Inthe Maximum Exposure field type the longest exposure time that you want to allow On the Mode Interactive tab Mode Multidimensional tab or Hardware Setup tab click Auto Expose The
16. accommodate brightfield fluorescence and other microscopy techniques such as differential interference contrast DIC and phase contrast For each channel you can set a variety of specific camera settings You can also indicate whether a Z series of images should be acquired using the channel and you can specify a Z motor adjustment for the channel Images are acquired with each channel using auto expose or the exposure time that you specify 78 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Example You can use multiple channels to acquire a transmitted light image and two fluorescent images You can set different exposure times for each channel You can specify that auto expose be used for some or all channels A channel series can be used alone to acquire multiple channels at one location at one time point Or a channel series may be part of an acquisition that acquires images with multiple channels at multiple locations over time You can configure as many channels as you need After images are acquired you can view them as separate images using the individual channels or as composite images using some or all of the channels See Viewing Images in a Dataset Use the options in the Channel Series group on the Mode Multidimensional tab to define basic settings for one or more channels Use the Acquisition dialog box to define basic and advanced settings for one or more channels Mode Multidim
17. di Cached Data Each dataset inthe d Datasetl experiment has its lt lt own folder di Dataset2 _ Experiment mexp This is the file that you select when you open the experiment in the MetaMorph NX software You can rename the experiment mexp file as long as you keep the mexp file extension and as long as the file name is valid does not contain special characters such as exclamation points You can also move or copy the experiment folder to another computer running the MetaMorph NX Software and then work with the experiment on that computer If you move or copy the experiment folder you must move or copy all of its contents You can specify the name of a dataset only before it is created When you acquire images using the Interactive acquisition mode you use the options on the Mode Interactive tab to specify the prefix of the dataset folder name before saving images into the dataset When you acquire images using the Multidimensional acquisition mode the MetaMorph NX Software saves the images into a dataset using the dataset name prefix specified in the Administration dialog box See Creating a New Experiment for information about how to change the prefix for the dataset folder name for multidimensional acquisitions A CAUTION You can rename or move experiment folders Do not rename or delete dataset folders The experiment file contains references to all of the data in all datasets in the experiment If you rename or delete
18. down to zoom out To use the zoom options on the Home tab e click Zoom To Fit Space to resize the display of the images so that the entire image fits into each grid in the image grid Latest Image tab or Live Display tab e select or type a percentage of the original image display size and then press lt Enter gt e click or to increase or decrease the current zoom level by 20 percent When you zoom in more than 100 percent of the original image display size a Zoom control window appears in the upper right corner of the image grid Latest Image tab or Live Display tab The zoom control window shows you exactly which part of the image is in view For example Zoomcontrol window Zoomed area You can use your mouse to click and drag the zoomed area within the zoom control window to view other parts of the image 5011685 B 31 Chapter 2 Viewing Images Displaying Series Information on Images in the Image Grid You can display an overlay of information on each image in the image grid and in the filmstrip The overlay lists the dataset to which the image belongs the channel used to acquire the image and the Z step number time point number and stage position number at which the image was acquired For example Dataset Dataset2 Z 4 Time 1 Stage 2 Channel 2 In this example the overlay indicates that the image is part of Dataset2 and that it was acquired at the fifth Z step at the second time point at the third stag
19. 2222 e cece eeeee 163 Chapter 11 Checking for Software Updates 2 2 0 0 eee cece cece eee eee 165 Chapter 12 Microscopy and Image Processing Resources 2 167 Web sites l 0 000000000000 0000000000000000 ce cee eee Aaaa AAAA E aa aa eanan arenen 167 VOUWINAIS ose op vccauntedorieuccsedennns E a a aE EONAR 167 BOOKS sacs el a ee chee es eee es ete ee E E E 167 MNO ete eee ae ete ts ee i es cea ee ee ee 169 5011685 B 7 Chapter 1 Navigating the Workspace This chapter includes the following topics e Tourof the Workspace e Creating Shortcuts for Ribbon Options on the Quick Access Toolbar e Minimizing the Ribbon Tour of the Workspace With its integrated Dataset View the MetaMorph NX Software makes it easy to navigate through and compare sets of images explore relationships between images dimensions and data and prepare images and data for presentations The workspace is flexible and can be arranged in a variety of layouts You can move group resize and hide windows Two main areas comprise the MetaMorph NX Software workspace e The ribbon across the top which contains tabs and options for performing tasks e The Dataset View which contains images and optionally a filmstrip the data table and one or more graphs that belong to a dataset Ribbon Dataset View Chapter 1 Navigating the Workspace More than one Dataset View can be open at a time
20. Acquiring Images 80 Multidimensional tab are reflected in the Acquisition dialog box and changes that you make in the dialog box are reflected on the tab In the Acquisition dialog box select the Channels check box to indicate that the acquisition will include a channel series and then click Channels to expand the list of settings Click lt None gt to configure the first channel or click Add Channel to configure additional channels An icon for the channel appears in the Channel Series group on the Mode Multidimensional tab The illumination setting that you select in Step 7 will be used as the channel name In the Exposure field If you Then do not plan to use auto expose for type the exposure time to be used for the channel this channel plan to use auto expose for this type an exposure time that will be used if auto expose channel fails to achieve the target intensity Note The Shutter button on the ribbon which opens and closes the shutter is available only if the shutter is set to Active in the illumination setting used with the selected channel The Shutter button will open and close all shutters that are set to Active in the illumination setting See Creating Illumination Settings for more information In the Illumination Setting field select a setting from the list Illumination settings contain pre defined information such as the wavelength and positions of various components For information about
21. Acquisition 0 000000002 c occ cece cece cece cece cece cece eee eeeees 121 To pause an acquisition 2 2 222222 e eee cceeccceeeteeeees 121 To stop an acquisition 0 00000000000000 000000000000000 e cece cece eeceeccceeeeeeeees 121 Saving and Loading Acquisition Settings Protocols 2 00 000 eee eee eee eee 122 To save acquisition settings 0 00 e eee e ce ccecececceeeeeeees 122 To load saved acquisition settings 000 00 eee cece ee eeeeeeeee 122 Chapter 8 Measuring and Analyzing Images 0 cece cece cece eeeeeeeeeees 125 OVEIVIEW 22 eee cee ee cee cece cece cece ccc eee ee eecsceeececeeeeeeeeececeeesceersseseetes 125 General Workflow 2 2 0 2 2 c eee c cece cece cece ce ceccceecceceeeceeeeeceeeeececeeteeseeseees 127 Viewing Plane Information 2000 e cece cee aara aaora onanan 127 To view plane information for a dataset 2 222 222 cece cece eee cece eee ccceeeeeees 128 Plane information data definitions 2 00 0 2 c eee c cece cece anaoa anana onanan 128 Measuring Morphometry of Objects Measure Using Threshold 2 2 2 130 To measure morphometry using thresholding 0 0 eee eee eee cece eee 130 Measurement definitions 002 222 2222 c eee cece eee cece LaLa LaLa aLaaa a annaa 132 Area measurements 22 aoaaa aaan cence cece cece cece ceeececeeececeesee
22. B 2 MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide To switch between ascending and descending order click on the column heading again To include or exclude a column 1 2 On the Measure tab click Configure Results In the Configure Results dialog box expand the list of columns that contains the column that you want to include or exclude To exclude a column from the data table clear the check box next to the column name to include a column in the measurement data table select the check box next to the column name Click OK The MetaMorph NX Software updates the measurement data table to show only the columns you selected To filter data 1 6 On the Measure tab select the measurement set that you want to filter and then click Configure Results In the Configure Results dialog box click Filters In the Filters section click Add Filter Inthe Column Name field select the column measurement in the data table that contains data that you want to filter In the Filter Type field Select To MinFilter include all values in the column that are more than or above the minimum value that you specify in the Min Value field that appears MaxFilter include all values in the column that are less than or below the maximum value that you specify in the Max Value field that appears include all values in the column between the minimum and maximum values that you spe
23. Configure and at the top of the Acquisition dialog box click the Snap button that is next to the channel that you want to use for the snapped image The snapped image appears in the Latest Image tab and a thumbnail of the image appears in the filmstrip Each subsequently snapped image is also placed in the filmstrip so that you can compare images snapped with different settings Snapped images are saved in the Scratch folder on the hard drive of your computer Images that are snapped in Multidimensional acquisition mode cannot be saved in the same dataset as images that are snapped in Interactive acquisition mode The filmstrip displays a separator between snapped images that cannot be saved to the same dataset for example if the images were 96 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide acquired using different acquisition modes or different binning values or if the size of the images is different Using Auto Expose Auto expose calculates the appropriate exposure time for image acquisition using a channel that you You can use auto expose e to quickly verify or calculate exposure time before acquiring an image or while configuring hardware settings see below e for some or all channels during a interactive acquisition see Acquiring Images in Interactive Mode e for some or all channels during a multidimensional acquisition see Configuring a Channel Series Acquisition
24. Devices makes no warranties or representations as to the fitness of this equipment for any particular purpose and assumes no responsibility or contingent liability including indirect or consequential damages for any use to which the purchaser may put the equipment described herein or for any adverse circumstances arising therefrom For research use only Not for use in diagnostic procedures The trademarks mentioned herein are the property of Molecular Devices Inc or their respective owners These trademarks may not be used in any type of promotion or advertising without the prior written permission of Molecular Devices Inc Product manufactured by Molecular Devices Inc 1311 Orleans Drive Sunnyvale California United States of America 94089 Molecular Devices Inc is ISO 9001 registered 2010 Molecular Devices Inc Allrights reserved Printed in the USA Contents Chapter 1 Navigating the Workspace 0 0 0000000 e eee 9 Tour of the Workspace 2 2 2 e cece adodo odana aranana nannan 9 Creating Shortcuts to Options Using the Quick Access Toolbar 22 15 To add an item to the toolbar 2 2 2 e cece cece ccc ce cee eeeees 15 To remove an item from the toolbar 00 0000 e cece eee ee 15 To move the toolbar above or below the ribbon 222000 022222222 e eee ee eee eee 16 Minimizing the Ribbon 2 0 2 2 eee cece cece eee ce ccc cccecceccccecce
25. How can compare images acquired at various stage positions Using the filmstrip or the drop down list on the Home tab select a stage position Then open another tabbed window click the sign on the extra tab and select another stage position You can arrange the tabbed windows in the workspace so that you can compare images side by side You can open and rearrange as many Dataset Views and tabbed windows as needed See Rearranging Dataset Views acquired 10 images so why are just 5 images displayed in the Dataset View The current view mode may not show one of the dimensions in the images that you just acquired For example if you acquired a Z series of 5 steps with 2 channels and the view mode is Time vs Z the Data View will display just the images taken at the 5 Z positions with the first channel but not the second channel In this case you must change the view mode to Channel vs Z to display the images taken at the 5 Z positions with both channels See Viewing Images in a Dataset My dataset contains numerous images but even when am not using Single View mode only one image is displayed How can I display multiple images If the value in the row and column fields next to the view mode on the Home tab is set to 1 try changing the value to a higher number and then adjust the value until you see the correct number of images Why does the data in the measurement results table not reflect what see in the images in th
26. If the data table is already open the summary appears at the bottom of the table If the data table is not open the data table opens and the summary appears at the bottom of the table To hide the statistical summary click Show Summary again To display the original Source image on the Measure tab select Plane Information You can draw additional regions on the original image or on the segmentation of the image and re measure the regions as many times as needed Each time you measure regions the measurement results are stored ina new measurement set To delete the measurement set in the list of measurement sets click the X that is next to the measurement set Region measurement definitions The MetaMorph NX Software calculates the following types of region measurements Area Measurements Dimension Measurements Shape Position and Orientation Measurements Intensity Measurements Statistical Summary Measurements Area measurements Area Total Area Definition Measurement The area of the entire region Hole Area Relative Hole Not applicable for regions Area Standard Area Count The number of times larger that the region is than the value defined as the standard area If an object has a standard area count less than 1 0 it will count as a single object 5011685 B 141 Chapter 8 Measuring and Analyzing Images Measurement Definition standard area value o 8 1 0 units 3 75
27. Inthe Camera Configuration dialog box select the camera driver and click Activate Camera A Configure Camera button appears next to the camera name 3 Click Configure Camera to verify and optionally change settings for the camera See Camera Settings for information about the settings that are available for most of the cameras that the MetaMorph NX Software supports 60 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Camera Settings This section provides information about the settings that are common to most of the cameras that MetaMorph NX supports as well as settings for the demonstration camera driver e Basic Camera Settings e Defect Correction e Demonstration Camera Driver For information about settings that are specific to a certain camera and to view a list of currently supported cameras visit the MetaMorph NX Support web site at http support meta moleculardevices com hardware hardware php Basic Camera Settings The settings that are available will vary depending on the camera that you are using Setting Description Defines the operational mode of the camera Digitizer Sets the range of speed within which the digitizer will acquire images Gain Sets the range of sensitivity and signal strength for the camera Intensifier Gain Controls the output gain or the gain of image intensifiers on certain cameras Bit Depth Sets the image bit depth You can expand the
28. MetaMorph NX Software calculates the exposure time updates the exposure setting for the channel and displays an image using the calculated exposure time The image appears in the Latest Image tab and a thumbnail of the image appears in the filmstrip 5011685 B 97 Chapter 7 Acquiring Images Note You may not be allowed to access the Hardware Setup tab or you may need a password to access it See your system administrator for more information If auto expose does not achieve the expected results one of the messages below may appear Error message Description No exposure was attained or the exposure exceeded the target intensity For example the camera or controller is off the light source is off the shutter is off or the wrong shutter is specified or the maximum exposure duration is too small for the sample Low Signal An exposure exceeded the target intensity but no exposure was within range of the target Possible causes include changes in illumination or the sample while the exposure was being calculated Unable to achieve target intensity Configuring Image Autofocus Image autofocus finds the best focus image and if configured for an acquisition corrects the Z motor position to keep the image in focus while images are acquired For example during an image acquisition autofocusing helps compensate for a sample that has drifted or experienced thermal changes over time or small changes in the stage l
29. NX Microscopy Automation amp Image Analysis Software System Administrator Guide for more information Note If you specify a dataset name prefix for a multidimensional image acquisition and then you specify a new dataset name prefix for an interactive image acquisition the new prefix overwrites the existing prefix Before you acquire a multidimensional image make sure the dataset name prefix is correct You cannot rename or delete datasets once they are created In the dataset folder the image file name reflects the time point stage position channel and Z step of the image Example The filename t2s1c1 tif is an image that was acquired at the third time point in the time series at the second stage position in the stage series using the second channel in the channel series Keep in mind that the MetaMorph NX Software begins counting time points stage positions Z steps and channels at zero Plane information for the new dataset is listed on the Measure tab See Viewing Plane Information 5011685 B 77 Chapter 7 Acquiring Images You can combine up to four types of acquisition series to create a multidimensional image e Channel series a multidimensional acquisition must include at least one channel e Stage series e Zseries e Time series Setting the Camera Area for Acquisition The MetaMorph NX Software acquires images using either the full chip area of the camera or the area that is displayed in the image grid When y
30. On the Hardware Setup tab in Manage Settings click Illumination Settings 2 Inthe Illumination Settings dialog box do one of the following e Tocreate anew setting click Add New Setting and type a name for the setting in the blank field that appears in the list of settings e Tomodify asetting click the name of the setting e Torename a setting double click the name of the setting and type a new name 3 Inthe Wavelength field type the collection emission wavelength for the setting If the image is full spectrum or if wavelength does not apply for other reasons set the value to 0 zero The wavelength will be recorded in images collected with this illumination setting The wavelength can be important for some image processing techniques The color box shows the color that corresponds to the wavelength This color will be used in the brightest pixels when the image display color is specified as Set by Wavelength in the 5011685 B 55 Chapter 6 Installing and Configuring Hardware 56 Channel Display group on the Home tab If you set the wavelength outside the visible range 380 780 the color box tums white The position of a component appears next to the component s name To set the position of the component whenever the illumination setting is used select the check box to the left of the component Then optionally select or type the position to be applied when the setting becomes active when it is used as part of a
31. Software upgrades and other resources If you do not find the answers you are seeking follow the links to the Technical Support Request Form Fill out the form to send an e mail toa MetaMorph NX Technical Support Representative The MetaMorph NX Software saves configuration and error related information for each session You can quickly create a WinZip file that contains information about the current session which may help a support representative diagnose issues The WinZip file contains A configuration file with information about the state of the software when it was started A configuration file with information about the state of the software when the WinZip file was created Hardware profiles Camera configuration files Status output log files You can create the WinZip file in one of two ways e From the Application menu select MetaMorph NX Administration and then click Capture MetaMorph Configuration e f an error is displayed in the notification area at the bottom of the MetaMorph NX workspace click the error message and then click Capture MetaMorph Configuration When the file is created the following message appears Settings captured and saved to disk C Users username Documents MetaMorph NX MetaMorphNXReport YYYY MM DD Time zip 163 Chapter 11 Checking for Software Updates You can quickly check for and download updates to the MetaMorph NX Software in one of two ways e On the ribbo
32. Table 000000000 cece cece cece ee eee 149 To open the data table fora measurement Set 2 2 0 2 2 22222 c eee e eee ee cece eee 150 To sort data in a column by ascending or descending values 2 220222 150 To include or exclude a COIUMN 2 22 oe eee ee ee cece naaa LaLa LaLa a aoaaa oana 151 To filter data 20 2 c cece LALALALA DALALDALA DALAL aLa naana 151 Exporting Measurement Data 0 cece ccc c cece ccc cceccecceceeeeeeeeeeeeeeeeennes 152 To export data to an open Excel spreadsheet 22 2 cece ccccceeeceecccececceeeees 152 To export data to an Excel file while Microsoft Excel is closed or not installed 152 To export data toa text file 2 2 oll cece eee cece cccceeeeeccceeceeeececereeeeees 153 Graphing Measurement Results 2 2 00 e cece cece eeececeeceeeeeees 153 To create a graph of measurement results 2 22 22 2 eee eee 154 To filter data for a graph 0000000 e cece ee eeeeeeeeeeeeees 155 To configure the graph style and range of data 20 00 eee 156 To change the graph type 00 eee ee eee cece eeeeeeeeeeeeeees 157 Adaptive Background Correction 22 2 2 002000 e cece eeeeeeeeeeeees 157 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Chapter 9 Frequently Asked Questions 02 02 cece cece cece cece ccceeceeeeees 159 Chapter 10 Obtaining Support 2 22 0
33. You must be able to access the Hardware Setup tab to perform the tasks described in this section See your system administrator for more information Rather than define settings for the multiple hardware devices that affect illumination each time you carry out an experiment you can define specific collections of device and component settings and save them as illumination settings Illumination settings allow the specification of multiple device positions so that each channel is illuminated appropriately Then when you are configuring channels for an image acquisition you can simply select a saved illumination setting You can create as many illumination settings as you need The components that are available depend on what hardware devices are connected and configured for use with the MetaMorph NX Software In the Illumination Settings dialog box the components and settings listed first are those that the MetaMorph NX Software considers most relevant to illumination At the bottom of the list are other components the settings for which can also be defined and saved as part of the illumination setting Components commonly used to control illumination include e shutters e wheels and filters e lamps Prerequisites Before you can create an illumination setting the appropriate hardware devices and components must be installed and loaded See Installing and Loading Hardware Device Drivers To create or modify an illumination setting 1
34. a dataset folder you will not be able to open the experiment If you delete any files including image files you will not be able to access measurements and images will not display If an image was linked to an experiment the experiment folder contains the path name to the location from where the image was linked The folder does not contain the actual image If the experiment folder is moved to a location that cannot access the image data you will not be able to open your experiment You can move the linked image data along with the experiment folder but it must use the correct path for the experiment and dataset folders 40 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Each dataset folder contains the items described in the table below Item File name Description Images t s z c tif The image file name includes the time point stage position channel and Z step Individual TIFF files can be opened of the image For example the filename e directly from the hard drive on t2s1c1 tif is an image that was acquired your computer at the third time point in the time series at the second stage position in the stage series using the second channel in the channel series Keep in mind that the e using version 7 7 of the MetaMorph NX Software begins counting MetaMorph Software time points stage positions Z steps and channels at zero e using the MetaMorph NX Software Plane Information I
35. able to measure an image that I just acquired using the Interactive acquisition mode An image must be saved into a dataset before it can be measured In Interactive acquisition mode images are not saved automatically you select the images to save into an existing or new dataset in the open experiment See Acquiring Images in Interactive Mode How do open images that were acquired with Version 7 7 of the MetaMorph Software To open an image that is not part of an experiment created with the MetaMorph NX Software you can link the image to an existing or new MetaMorph NX Software experiment From the Application menu select Link Image and then select the current experiment or a new experiment 5011685 B 161 Chapter 9 Frequently Asked Questions See Linking an Image to an Experiment How do I change the background color of a graph On the Graph tab click the dialog box launcher Ii in the Graph Types group or right click anywhere on the graph to open the Graph Settings dialog box In the Graph Settings dialog box click Data Display and then select a background color See Graphing Measurement Results How do I remove the regions from my source image On the Measure tab scroll to the bottom of the list of region tools and select Clear all regions See Graphing Measurement Results 162 5011685 B Chapter 10 Obtaining Support The web site http support meta moleculardevices com provides links to technical notes
36. below contains two time segments 5011685 B 89 Chapter 7 Acquiring Images T Interval Duration 1 10 sec 40 sec If an acquisition contains more than one time segment the interval between the last time point in the current segment and the first time point in the next segment is the interval defined in the current time segment For example if the first time segment contains three time points with a five second interval and the second time segment contains three time points with a one second interval five seconds will pass between the end of the first segment and the start of the second segment An acquisition can run for as long as necessary The duration of a time series acquisition is limited only by the viability and light sensitivity of the sample or by the amount of disk space available for acquired images Example You can use atime series acquisition to acquire an image every 10 seconds using an exposure of 100 milliseconds and then every 5 seconds using the same exposure Or the time series that you configure might be one part of a lengthy experiment that acquires a Z series of images at 5 stage positions every 15 minutes for 48 hours using DAPI and FITC channels Use the options in the Time Series group on the Mode Multidimensional tab to define one time segment Use the Acquisition dialog box to define more than one time segment and to add or remove time segments To configure a time series 1 On
37. calculate the background intensity estimates in the locality of each nucleus The Count Nuclei measurement function uses the Adaptive Background Correction system to help correct uneven image backgrounds This section describes e How to count nuclei e Count Nuclei measurement definitions To count nuclei 1 Open the dataset that contains the images Se 2 On the Measure tab in the list of measurement functions select Count Nuclei Ael The options for configuring the measurement appear in a popup area You can configure the measurement in the popup area or if you prefer you can click Configure and open a dialog box that contains the same options 3 Inthe Source field select the channel to use to create the segmentation for counting nuclei Measurements are made using a single channel 4 Inthe Intensity Above Local Background field type the difference in intensity between the dimmest part of a nucleus and the background adjacent to it This value controls the sensitivity of the detection of nuclei The lower the value the more sensitive the detection 5 Inthe Approximate Minimum Width field type the approximate minimum cell nuclei width in calibrated units to include in the count Nuclei patterns in the image much less than this width will be considered to be noise patterns 5011685 B 147 Chapter 8 Measuring and Analyzing Images 10 11 12 13 14 Inthe Approximate Maximum Width field type the app
38. change the experiment default settings 000 0000 eee cece e cece eens 42 Opening an Experiment 0 0 cece cece cece cece cece cece cece cece ccc cceceeeeeeeeeees 43 To open an experiment 22 22 222 eee c eee e cece cece eeeeeeeeeeeeeeeeeeees 43 Linking an Image to an Experiment 00 2 e cece cee renren ennenen 43 To link AN IMAGE 2 e cece cece cee cc cece cece ececceceeceeeeeeeeeeeeeeeeeeeeteeeeneenees 44 Chapter 6 Installing and Configuring Hardware 000 0 0e cece ee eee 45 General Workflow 000 eee cece ee eee cece cece cece eee eececeeceseceeeeeees 45 Preparing to Install Hardware Device Drivers 0 cece cece e cece ee eeeeeeeee 46 Installing Device Drivers 2 2 2 2 0 222 c cece ccc ee cece ecccccecceeeeeees 47 Prer quisitesS ook ots Sait ah alee ed Nee ete at et chr tect a 47 To install a device driver 0 002022 c cece cece cece ccc ccc ccc cece eee e cece ee aonaran 47 Loading Device Drivers 0 2 2 2 2 222 cece cece cece cece eee c ccc cece cece oraora naaar rainn 49 PrereguisiteS ese hee es et te ate SN ete See at Rt es eae et eect 49 To load a device driver 2 2 2 eee 49 Device and Component Settings 00 00000 e cece eeeeeeeeeeees 50 Device Calibration Settings 2 0 0 0 cece cece cc cecccccceceecccccceececcccceceteetcccceeeeeeees 50 Discrete Component Position Settings 0 00 0000 eeeeeee
39. criteria If you create a filter for a graph that excludes certain data that data will also be excluded from the data table Likewise when you create a filter for a data table that excludes certain data that data cannot be included on a graph When configuring filter criteria for a line graph do not exclude time measurements line graphs always plot time measurements on the X axis To open the data table for a measurement set 1 On the Measure tab in the list of measurement sets select the measurement set 2 Onthe Measure tab in Measurement Display click Show Table 3 To display the measurement summary data in the data table on the Measure tab click Show Summary to hide the summary data click Show Summary again After the data table opens you can configure the data as described in this section You can also resize or move the table to another place in the Dataset View so that it takes up less space or so that you can compare it to the data table of another measurement set See Rearranging Dataset Views If you select another measurement set while the data table is open the table updates its display with the data of the selected measurement set To sort data in a column by ascending or descending values 1 Click on a column heading The data in the column is arranged in ascending order the lowest value in the column appears at the top and the highest value appears at the bottom A triangle appears in the column 150 5011685
40. described in this section See your system administrator for more information Before installing and configuring a hardware device driver and related components for use with the MetaMorph NX Software follow the steps below 1 Create a list of the devices connected to your computer and the corresponding serial parallel USB or 1394 port assignments a list of the hardware for which you will install hardware device drivers and a list of any port assignment requirements noted in the documentation supplied with the devices 2 Connect the hardware device to your computer as instructed in the documentation supplied with the device You may need to install related Software 3 Turn on the device as instructed in the documentation supplied with the device 46 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Installing Device Drivers You must be able to access the Hardware Setup tab to perform the tasks described in this section See your system administrator for more information To use the MetaMorph NX Software with a connected hardware device and its related components you must first install the hardware device driver After installing the device driver you can rename and change the settings of the device and its related components These names and settings are then used as the default values when loading the device drivers Component names and settings may be changed again when you load the devic
41. display a live view of the image 4 Inthe Multidimensional Acquisition click Configure In the Acquisition dialog box select the Focus Stabilizer check box to indicate that the acquisition should use the focus stabilizer hardware The focus stabilizer options appear in the Stage Series section 6 Inthe Stage Series section click Go To to move the stage to a stage position 7 Use the focusing knobs on the microscope to bring the image into focus 8 Inthe Stage Series section in the row that corresponds to the current stage position click Set Focus The Focus Adjustment field displays the surface to focal plane distance sometimes called the offset 9 Optionally slightly adjust the focus by using the knob on the Nikon PFS controller 10 Repeat steps 6 9 for each stage position 11 Inthe Focus Stabilizer section in the Autofocus Frequency field Select To have the Nikon PFS continuously focus Every Point at every time point focusing at every time point may slow down acquisition First at only the first time point Every Nth Point at specific time intervals specified in the next step for example you could focus at every third time point 12 If you selected EveryNthPoint in the previous step in the Autofocus Rate field type the time point interval at which continuous focusing should occur for example type 3 to focus at every third time point Keep in mind that the first time point in a time series is a
42. down acquisition First at only the first time point Every Nth Point at specific time intervals specified in the next step for example you could focus at every third time point 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide 6 If you selected EveryNthPoint in the previous step in the Autofocus Rate field type the time point interval at which focusing should occur for example type 3 to focus at every third time point Keep in mind that the first time point in a time series is always zero 0 7 Click Live to display a live view of the image 8 Use the focusing knobs on the microscope to bring the image into focus 9 Inthe One Shot Range field type the total distance above and below the current Z motor position that the Z motor is allowed to move while the ZDC or ZDC2 is locating the surface of the dish or slide For example if you type 100 the Z motor will be allowed to move 100 microns above and 100 microns below its current position This distance should not let the Z motor go above the near limit position specified in the objective component settings Best Practice Use the default one shot range If you set a very large range the ZDC or ZDC2 may locate something other than the surface and focusing will not be possible 10 Click Set Focus to set the focus at the current focal plane The Olympus ZDC or ZDC2 displays the surface to focal plane distance in the Focus Adjustment
43. field and beeps once to indicate that it is configured correctly Note The Olympus ZDC or ZDC2 emits three beeps if it did not successfully calculate the lens focus adjustment value Try to set the focus again If you do not hear any beeps the ZDC or ZDC2 is not configured correctly Make sure that the observation method for the objective is set to something other than None check that you are using the correct lens and check that the IX2 Software is configured correctly 11 Optionally slightly adjust the focus by clicking the buttons or by typing a value in the Focus Adjustment field 12 Just before starting the image acquisition if the image is out of focus click Go To Focus The Olympus ZDC or ZDC2 re establishes focus at the focal plane that you set in Step 10 Ga You can also use the options in the Focus group on the Mode Multidimensional tab to set the focus and to re establish focus 13 Once you have configured all of the settings for the multidimensional acquisition click Acquire During the image acquisition the Olympus ZDC or ZDC2 re establishes focus at the specified time intervals To use the Olympus ZDC or ZDC2 for a multidimensional image acquisition with a stage series 1 Configure the settings for the multidimensional acquisition at least the stage positions and the settings that affect Z motor position and illumination such as the channels and if applicable image autofocus and a Z series 50
44. for the brightest pixel in the image The default target intensity value is 75 of the maximum intensity value that the camera driver is able to obtain The target intensity will determine the exposure time during auto expose 10 Inthe Initial Exposure field type the length of the first exposure attempt Type an exposure time that is well below the typical exposure time required for this type of experiment generally the initial exposure time should be approximately one tenth of the typical exposure time If you do not know the required exposure time type 1 or 5 or use the default value 11 Inthe Maximum Exposure field type the longest exposure time that you want to allow Be careful not to over expose your sample which may result in bleaching photo damage or photo toxicity 12 Inthe Z Offset field specify the difference between the focus position of the Z motor in this channel and the focus position of the Z motor in the first channel If the acquisition includes a Z series the entire Z series will be shifted by the Z offset distance Note The Z Offset field is not available for the first channel that you define in the Channels section In the example below the position of the Z motor when the sample is in focus using Channel 1 is 500 The position of the Z motor when the sample is in focus using Channel 2 is 700 The difference between the two positions is 200 so the Z offset distance is 200 700 7 postion for Samp
45. for the setting in the blank field that appears in the list of settings e Tomodify asetting click the name of the setting e Torename a setting double click the name of the setting and type a new name 3 The position of a component appears next to the component s name To set the position of the component whenever the hardware setting is used select the check box to the left of the component Then optionally select or type the position that you want to be applied when the setting becomes active for example when it is selected to test a microscope setup 4 Optionally select the Serialize Moves check box to indicate that no other devices should move when the selected device moves 5 If additional properties for a component are available you can click Other Peripherals to expand the list of properties See Device and Component Settings topics for specific settings information 6 Click Save Current Setting to save your changes The new hardware setting is saved in the list of settings on the Hardware Setup tab and the Acquisition Mode tab 7 To move the devices to the positions defined in the hardware setting on the Hardware Setup tab in Select Setting select the hardware setting and click Apply Or on the Acquisition Mode tab in Hardware Settings select the setting and click Apply 54 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Creating and Modifying Illumination Settings
46. illumination settings and magnification settings hardware settings are not part of the image acquisition process Hardware settings are mostly useful for initializing the position of a microscope for a pre set condition For example you may want to set all the device positions so they are the same at the start of every session or you may want to set up the illumination path for an experiment 5011685 B 53 Chapter 6 Installing and Configuring Hardware that uses DIC differential interference contrast Because these settings including the objective position are not used as part of an acquisition configuration it is important to make sure that magnification and illumination settings are correct after you use a hardware setting and before acquiring images You can define and save the settings of any available hardware component and you can create as many hardware settings as you need The components that are available depend on what hardware devices are connected and configured for use with the MetaMorph NX Software Prerequisites Before you can create hardware settings the appropriate hardware devices and components must be installed and loaded See Installing and Loading Hardware Device Drivers To create or modify a hardware setting 1 On the Hardware Setup tab in Manage Settings click Hardware Settings 2 Inthe Hardware Settings dialog box do one of the following e Tocreate anew setting click Add New Setting and type a name
47. leave the lamp voltage component unchecked The lamp voltage will be adjusted only if the check box for the component is selected Optionally select the Serialize Moves check box to indicate that no other devices should move when the selected device moves If additional properties fora component are available you can click Other Peripherals to expand the list of properties See Device and Component Settings topics for specific settings information Click Save Current Setting to save your changes The new illumination setting is added to the list of illumination settings on the Hardware Setup tab in the Select Setting group The setting is also included in the list of illumination settings that are available to use with channels for image acquisition in the Channel Settings group on the Mode Multidimensional tab and Mode Interactive tab Tomove the devices to the positions defined in the illumination setting on the Hardware Setup tab in Select Setting select the illumination setting 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Creating and Modifying Magnification Settings You must be able to access the Hardware Setup tab to perform the tasks described in this section See your system administrator for more information A magnification setting is a group of device settings that will affect the area covered by each pixel in an image Rather than define individual settings for the har
48. list of channel settings a specific value in each channel Note The binning value applies to all channels and cannot be set per channel 15 If you are using different camera setting values for the channel in the list of settings for that channel type or select the new value for the camera setting 16 Optionally configure focus stabilization 17 Optionally configure image autofocus 18 After you have configured the acquisition do one of the following To acquire an image On the Mode Interactive tab double click the channel or select the channel and click Snap using all channels click Acquire All Channels If you configured more than one channel the Latest Image tab displays slots for each channel in the image grid If you acquired all channels an image acquired with each channel appears in each slot and a composite image appears in the filmstrip if you acquired one channel its slot updates to display the image that was acquired using that channel As you acquire subsequent images using various channels each image updates in the corresponding slot in the image grid using one channel If you acquire additional images with different attributes for example fewer channels or a larger binning value the filmstrip displays a green vertical line to separate images that have different attributes As explained in the next section only images acquired with the same attributes can be saved into the same dataset
49. on an image the region is drawn simultaneously on all images in the dataset Note If you draw a region within a region or if you overlap regions each region is measured separately That is the overlapping area of the regions is counted as part of each region amp Use the wheel on your mouse to zoom in on an image to draw a more precise region Then in the zoom control window drag the zoom area to another location and draw more regions To delete all regions in Regions scroll to the end of the list of tools and select Clear all regions After you have drawn the regions that you want to measure in the list of measurement er functions on the Measure tab select Measure Regions a f The options for configuring region measurements appear in a popup area You can configure the measurement in the popup area or if you prefer you can click Configure and open a dialog box that contains the same options Inthe Region Source field select the channel to use to create the segmentation for measuring the regions Measurements are made using a single channel unless you select the All channels check box as described in Step 10 In the Standard Area Value field enter the size in microns of a typical cell Measuring objects is often complicated by the fact that objects overlap or clump together when thresholded resulting in counts that are lower than the actual number of objects The Standard Area Value helps compensate for overlapping
50. or clustered objects The Standard Area Value determines the standard area count reported in the measurement results To measure the intensity of all channels in the region select the All channels check box or if you want to measure the intensity of just the channel selected in the Region Source field clear this check box On the Measure tab click Measure The MetaMorph NX Software converts the regions to objects creates a segmentation of the image measures the objects and stores the measurement results in a new measurement set The progress bar at the bottom of the workspace shows the status of the measurement Once the measurement is finished the new measurement set appears at the bottom of the list of measurement sets on the Measure tab Note To cancel a measurement click anywhere in the progress bar at the bottom of the workspace and then click the Cancel button that appears The data that was calculated before you canceled the measurement is stored ina new measurement set 5011685 B 11 12 13 14 15 16 MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide To display the segmentation of the image that shows the regions as objects on the Measure tab select the new measurement set To display the measurement results click Show Table See Displaying Measurement Results in a Table To display the statistical summary of all measurements in the measurement set click Show Summary
51. plan to use two Z motors for image acquisition you must designate which Z motor should be used for a Z series autofocus and travel offset as described in Designating Z Motors and Stages for Acquisition 5011685 B 87 Chapter 7 Acquiring Images To configure a Z series 88 1 On the Acquisition Mode tab select Multidimensional select the camera channel and optionally select a hardware setting if one is needed to set the microscope devices to pre defined positions On the Mode Multidimensional tab in Magnification select the magnification you want to use for the acquisition On the Mode Multidimensional tab click Configure to display the Acquisition dialog box In the Acquisition dialog box select the Z Series check box to indicate that the acquisition will include a Z series and then click Z Series to expand the list of settings Ge You can also specify the Z series step size and the Z range in the Z Series group on the Mode Multidimensional tab Move the Z motor so that it is focused on approximately the center of the sample The Current Z Position field updates as you move the Z motor Typically just manually positioning the Z motor so that the center of the sample is in focus is sufficient However to calculate the exact center of the sample move the Z motor so that the bottom of the sample is in focus and note the Z position Then move the Z motor so that the top of the sample is in focus and note the Z pos
52. positions are shown in the X and Y fields on the Hardware Setup tab and click Set Origin for the X and Y motors The positions in the X and Y fields on the Hardware Setup tab change to 0 zero to indicate that the current position of the stage is now the point of origin Displaying a Live View As you adjust devices and illumination conditions you can test the current settings by displaying a live view of the image Although the live view continuously displays new images the MetaMorph NX Software does not automatically save these images E Note The Shutter button 2 on the ribbon which opens and closes the shutter is available only if the shutter is set to Active in the illumination setting used with the selected channel The Shutter button will open and close all shutters that are set to Active in the illumination setting See Creating Illumination Settings for more information This section describes how to e Display a live view while configuring hardware settings e Display a live view while configuring an image acquisition To display a live view while setting up the microscope You must be able to access the Hardware Setup tab to perform the tasks described in this section See your system administrator for more information 94 On the Mode Multidimensional tab or Mode Interactive tab select the channel to use for the live view In the Live Binning field Ek type the binning value to use for the live view Note The c
53. presentations documents and notes The experiment folder also contains the experiment file which contains references to all of the experiment data that you work with when you open the experiment in the MetaMorph NX Software Experiment Images Measurement sets Plane information Acquisition settings TIE zal Presentations E mail Notes ee Experiment file Every time you create a new experiment the MetaMorph NX Software creates a folder for the experiment on the hard drive of your computer When you create a new experiment the experiment folder name is incremented by one number For example E My Documents d My Experiments d Experimentl d Experiment2 dd Experiment3 39 Chapter 5 Creating and Managing Experiments By default experiments are saved in C Users username My Documents My Experiments You can change the name and location of an experiment folder either before or after it is created To change the prefix of the experiment folder name or the location where experiments are saved before an experiment is created follow the instructions in Creating a New Experiment To change the experiment folder name or the location of an experiment after the experiment is created rename the folder on the hard drive or move the folder to a new location An experiment folder contains a subfolder for each dataset in the experiment For example the Experiment folder shown above might contain the following
54. the Ribbon A check appears next to the menu option To re display the ribbon 1 On the Quick Access Toolbar located just above the Home tab click the down arrow to display its menu options 2 Select Minimize the Ribbon The check is removed from the menu option and the ribbon appears again 16 5011685 B Chapter 2 Viewing Images This chapter includes the following topics e Displaying Images in a Dataset e Rearranging Dataset Views e Scaling Images e Zooming in on Images e Displaying Series Information on Images in the Image Grid Arranging and Displaying Images The Home tab provides many ways to control how images are displayed in the image grid This section describes how to e Display a specific dataset in the open experiment e Display images acquired at a specific stage position Z position or time point e Navigate between images in the image grid e Use the view modes to display and arrange multiple images in the image grid or display a single composite image e Change the display color of a channel e Turnthe display of a channel on or off For information about scaling channels in an image or zooming in on and out of images see Scaling Images or Zooming in on Images To display a specific dataset If the open experiment contains more than one dataset you can select the dataset that you want to view in one of two ways e Onthe Home tab in Dataset View select the dataset e Inthe filmstrip in
55. the View Mode field select Dataset and then double click the thumbnail image that represents the dataset 17 Chapter 2 Viewing Images To display images acquired at a stage position Z position or time point You can display the images in a dataset that were acquired at a specific stage position in one of two ways e Onthe Home tab in Dataset View select the stage position e Inthe filmstrip in the View Mode field select Stage and then double click the thumbnail image that represents the stage position To compare images acquired at various stage positions open another tabbed window click the sign on the extra tab and select another stage position You can arrange the tabbed windows in the workspace so that you can compare images side by side See Rearranging Dataset Views for more information To display images that were acquired at a specific Z position or time point use the filmstrip view modes When you select a filmstrip view mode the thumbnail images update to represent images in the dataset according to the option that you selected time Z or stage For example if you select Z in the View Mode field in the filmstrip the thumbnail images that appear in the filmstrip represent the images acquired at each Z position Filmstrip View Mode zz After you select a view mode in the filmstrip double click a thumbnail image to move the images in the image grid so that the image is in view Place the
56. the last full time point in a new dataset 5011685 B 121 Chapter 7 Acquiring Images Saving and Loading Acquisition Settings Protocols Every time you acquire multidimensional images the MetaMorph NX Software automatically saves the acquisition settings or protocol as a separate file in the dataset folder on the hard drive of your computer When you acquire individual images in Interactive acquisition mode you can save the acquisition settings manually Later you can save time by using previously saved acquisition settings to configure an acquisition The acquisition protocol includes all settings as they are configured in the Acquisition dialog box including camera parameters The protocol does not save device configuration information such as the magnification setting that was used for the acquisition the configuration of illumination and magnification settings or the stage and Z motor configurations If you save a protocol and then make changes to an illumination setting that was saved with the protocol the next time you load the protocol the illumination setting will include the changes that you made This section describes how to e Save acquisition settings e Load acquisition settings To save acquisition settings 1 On the Mode Interactive tab or the Mode Multidimensional tab click Configure to open the Acquisition dialog box 2 Inthe Acquisition dialog box click Export Acquisition Protocol Ge You can also cli
57. the near limits See Installing Device Drivers and Settings For Specific Devices and Components 110 5011685 B 3 MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Objective Labels Refraction Medium Index Num Aperture Objective 1 70 25 r Jf 0 25 Objective 2 10K70 75 r yh 075 Objective 3 20K77 25 ain f 25 Objective 4 40K 71 75 Air f E Objective 5 63x 2 25 oi zih Objective 6 007275 o tw Objective Parameters Open Control Dialog OK Cancel After you have configured the driver and components open the illumination setting you plan to use for your acquisition Make sure that the observation method component setting is set to something other than None see the Olympus documentation for recommendations See Creating and Modifying Illumination Settings Designate the focus stabilizer hardware as the Z motor for autofocusing See Designating Z Motors and Stages for Acquisition To use the Olympus ZDC or ZDC2 for an interactive acquisition using one shot focus 1 oa fF Q Configure all of the interactive acquisition settings for the channels and if applicable image autofocus If your acquisition includes more than one channel on the Mode Interactive tab select the first channel in the channel series Note The focus stabilization system uses the first channel to calculate the focus adjustment value Subsequent channels are offse
58. the next column All of the images displayed in the image grid were acquired at successive time points at one Z position Scroll up or down to see images acquired at successive time points at other Z positions Of all the view modes this view shows the largest range of time 21 Chapter 2 Viewing Images View Mode Multi Z Description Displays a different Z step in each grid position The image acquired at the first Z position is displayed in the upper left grid position The image that was acquired next is displayed in the grid position to the right of the first image The order of images starts over in the next row All of the images acquired at the Z positions displayed in the image grid were acquired at one time point Scroll left or right to see the images acquired at Z positions at other time points Multi Channel Displays a different channel in each grid position All of the images acquired with the channels displayed in the image grid were acquired at one time point Scroll left or right to see images acquired with these channels at other time points Scroll up or down to see images acquired with these channels at other Z steps Note In a sparse acquisition images are not necessarily acquired at every possible Z step stage position or time point and they may not be acquired with every channel For example to save time or data storage space your experiment may use part
59. the range of the threshold values you specified pixels with intensity values between the threshold values will not be included in the segmentation 7 Select the Exclude objects touching edge check box to exclude objects that touch the edge of the image from the segmentation Clear the check box to include objects that touch the edge of the image 8 Select the Fill Holes check box to fill objects that contain holes in the segmentation Clear the check box to leave holes in objects empty in the segmentation Note The centers of some objects have intensity values similar to the background of the image and do not threshold well Filling holes in objects ensures that they can be accounted for in the measurement results 9 Doone of the following e To measure just one image or the images that can fit in the image grid on the Home tab use the view modes to display just the image or images that you want to measure e To measure the images that were acquired at all time points Z positions or stage positions on the Measure tab select the All Time All Z or All Stage check box you can select more than one check box Note Measurements are made on either the images displayed in the image grid or the images acquired at all time points Z positions or stage positions If you want to measure a range of images that cannot fit into the image grid measure all the images and then filter the measurement results 10 Onthe Measure tab c
60. to the color currently assigned to the channel and select a new color For example SS ee DIC Green2 Channel Display Display Settings for Channel Red1 The color changes for display purposes only the MetaMorph NX Software does not save the new color with the image or change any information about the wavelength that was used to acquire the image Note When you change channel display colors only the channels in the current selected dataset change colors The changes do not affect the display of channels in other open datasets 5011685 B 23 Chapter 2 Viewing Images To turn the display of a channel on or off You can control which channels are displayed in the images in the image grid of the open and selected dataset On the Home tab in the Channel Display group double click the name of a channel to hide the channel or to display it If the channel is not displayed the word Hidden appears in the channel icon For example Eg E y DIC Redi Green2 Channel Hidden Display a Channel Display Rearranging Dataset Views You can arrange Dataset Views side by side either vertically or horizontally and you can group Dataset Views together into a tab group Each Dataset View can have one data table and one or more graphs for displaying measurements You may want to put dialog boxes on one side of the Dataset View images in the middle and the table and graphs on the other side For example
61. to use for the snapped image On the Hardware Setup tab in Select Settings select the hardware and magnification setting that you want to use for the snapped image and if needed make adjustments to the stage and Z motor positions On the Hardware Setup tab if needed click Auto Expose to automatically calculate and set the exposure time for the live view See Using Auto Expose On the Hardware Setup tab in Camera type the binning value to use for the snapped image inthe Snap Binning field E Note The camera driver may only support certain binning values Click Snap The snapped image appears in the Latest Image tab and a thumbnail of the image appears in the filmstrip Each subsequently snapped image is also placed in the filmstrip so that you can compare images snapped with different settings Snapped images are saved in the Scratch folder on the hard drive of your computer To snap an image while in Multidimensional acquisition mode In Multidimensional acquisition mode you can snap an image using one channel 1 On the Acquire tab in Camera type the binning value to use for the snapped image in the Snap Binning field E Note The camera driver may only support certain binning values Do one of the following e In Channel Series select the channel to use for the snapped image if needed adjust the exposure time and then double click the icon of the channel or click Snap e In Multidimensional Acquisition click
62. units 1 0 units rounded to 4 0 objects Dimension measurements Measurement Definition Width The horizontal dimension of the region The vertical dimension of the region The span of the longest chord through the region QSs _ Note This measurement is calculated slightly differently in the MetaMorph Software version 7 7 and earlier The MetaMorph NXSoftware uses a more comprehensive system for determining maximum possible length The distance of a line that was drawn with the line multiple point line or freehand line tool If the region is a closed region for example a closed Line Length 142 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Measurement Definition rectangle the Line Length is zero Breadth The caliper width of the region perpendicular to the longest chord B Note Due to changes in the way that Length is measured this measurement is calculated slightly differently in the MetaMorph Software version 7 7 and earlier See the description of Length above Fiber Length The length of a region assuming that it is a fiber Fiber Length i P VP 16A Where P Perimeter and A Area C oO The width of a region assuming that it is a fiber Fiber Breadth P VP 16A Fiber Breadth Where P Perimeter and A Area Cf Shape position and orientation measurements Measurement Defin
63. using each objective to find the focus adjustment value This section describes how to e Configure the Nikon PFS for an interactive acquisition e Configure the Nikon PFS fora time lapse multidimensional acquisition without a stage series e Configure the Nikon PFS fora time lapse multidimensional acquisition with a stage series Prerequisites The appropriate Nikon device driver and the Z motor component must be installed and configured See Installing Device Drivers and Settings For Specific Devices and Components The focus stabilizer hardware must be designated as the Z motor for autofocusing See Designating Z Motors and Stages for Acquisition To use the Nikon PFS for an interactive acquisition 1 oa F Q 106 Configure all of the interactive acquisition settings for the channels and if applicable image autofocus If your acquisition includes more than one channel on the Mode Interactive tab select the first channel in the channel series Note The focus stabilization system uses the first channel to calculate the focus adjustment value Subsequent channels are offset from the first channel Click Live to display a live view of the image Use the focusing knobs on the microscope to bring the image into focus Inthe Acquisition click Configure Inthe Acquisition dialog box select the Focus Stabilizer check box to indicate that the acquisition should use the focus stabilizer hardware and then click Focus Stabiliz
64. you re measure the same images the MetaMorph NX Software uses the source images for measurements not the most recently created segmentation of the images Measurement definitions The MetaMorph NX Software can calculate the following types of morphometric measurements Area Measurements Dimension Measurements Shape Position and Orientation Measurements Intensity Measurements Statistical Summary Measurements Area measurements Measurement Definition Total Area The area of the entire object including any holes present whether or not hole filling is selected for the measurement Area The area of the object in calibrated units The area inside holes is not included unless hole filling is selected for the measurement 132 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Measurement Definition OR Filled holes Hole Area The area of the holes in the object If hole filling has been selected for the measurement this area is zero 0 Relative Hole Area The ratio of the hole area to the total area of the object A relative hole area of 0 indicates that the object has no holes whereas a relative hole area near 1 indicates that the object consists mostly of holes Hole Area Relative Hole Area Total Area If hole filling is selected the Relative Hole Area is zero Standard Area Count The number of times larger that the object i
65. you start an acquisition the MetaMorph NX Software will create one to contain the new dataset A separate window within the Latest Image tab displays the status of the acquisition as well as a histogram for each channel The MetaMorph NX Software reports the number of images acquired as well as the current time point in a time series if applicable stage position if applicable channel and Z position if applicable Other statistics may also be reported to help you monitor progress At any time you can pause the acquisition adjust certain settings and then continue the acquisition If you stop the acquisition all of the images that were acquired through the last full time point are automatically saved See Pausing or Stopping an Acquisition Once the multidimensional image acquisition is complete the MetaMorph NX Software automatically saves the acquisition settings or protocol as a separate file in the dataset folder Later when you want to configure the same or a similar experiment you can save time by loading the saved acquisition protocol By using a saved acquisition protocol you can ensure that an experiment is repeated with the same acquisition settings See Saving and Loading Acquisition Settings The MetaMorph NX Software saves acquired images from a multidimensional acquisition in a new dataset in a dataset folder To specify the prefix for new dataset names use the options in the Administration dialog box See the MetaMorph
66. 00 lt Original range of intensity values _ _ _ _______ White Black Highest alue Lowest Value Subset of original range of intensity values that will be redistributed from lowest to highest The histogram below shows that the Normalize scaling method will redistribute intensity values using only the pixels with intensity values between 7 5 and 96 of the original range of intensity values 75 E To scale channels in an image This section describes how to scale images using the options in the Channel Display dialog box which opens when you click Channel Display on the Home tab Most of the same scaling options are available in the Scaling toolbar which is located below the image grid as shown below 28 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide 1 Onthe Home tab click Channel Display The Channel Display dialog box shows a histogram for each channel 2 Inthe Channel Display dialog box select a scaling mode The type of scaling Camera Normalize or Fixed that you select will be used for all channels in the image That is you cannot select Fixed scaling for one channel and Normalize scaling for another channel However if you select Fixed or Normalize you can specify different intensity ranges for each channel Method Camera Normalize Fixed 5011685 B Description Automatically scale
67. 11685 B 115 Chapter 7 Acquiring Images 116 10 11 12 13 14 If your acquisition includes more than one channel on the Mode Multidimensional tab select the first channel in the channel series Note The focus stabilization system uses the first channel to calculate the focus adjustment value Subsequent channels are offset from the first channel Click Live to display a live view of the image In Multidimensional Acquisition click Configure to open the Acquisition dialog box In the Acquisition dialog box select the Focus Stabilizer check box to indicate that the acquisition should use the focus stabilizer hardware The focus stabilizer options appear in the Stage Series section Inthe Focus Stabilizer section in the One Shot Range field type the total distance above and below the current Z motor position that the Z motor is allowed to move while the ZDC or ZDC2 is locating the surface of the dish or slide For example if you type 100 the Z motor will be allowed to move 100 microns above and 100 microns below its current position This distance should not let the Z motor go above the near limit position specified in the objective component settings In an acquisition with multiple stage positions make sure that the range is large enough to accommodate all stage positions Inthe Stage Series section click Go To to move the stage to a stage position Use the focusing knobs on the microscope to bring the image into fo
68. If the step size in microns is Magnification of objective 10X Narrower 6 1 5 40X Narrowest 1 5 0 25 The image autofocus system first moves the objective along the Z axis in larger steps to determine where along the Z axis the sample is emitting a strong signal Then to refine its search and find the best focus image the autofocus system moves the objective in smaller and smaller steps until it detects the strongest signal from the sample The following illustration shows an example of an autofocus configuration with a range of 4 microns initial steps of 1 micron each and an accuracy distance of 0 15 microns Step 5 Step 4 Accuracy Step 3 Distance ee ae a ia center Step 2 Step 1 Low Sample Strength High This section describes how to e Use autofocus before acquiring an image e Use autofocus in a multidimensional image acquisition e Use autofocus in an interactive image acquisition Prerequisites To use image autofocus the appropriate hardware device driver for your Z motor must be installed and configured See Installing and Loading Hardware Device Drivers If more than one Z motor is installed or you plan to use two Z motors for image acquisition you must designate which Z motor should be used for autofocus as described in Designating Z Motors and Stages for Acquisition 5011685 B 99 Chapter 7 Acquiring Images To configure image autofocus before acquiring an image 1
69. Marzhauser Stage x Marzhauser Stage Y i Marzhauser Z Motor Adan Ad lt lt Remove etting Status 1 devices available 1 devices claimed Remove All Apply OK Cancel 5011685 B 49 Chapter 6 Installing and Configuring Hardware 3 To rename a device or component in the list of claimed devices select the device or component and type a new name 4 To change the settings of a component in the list of claimed devices select the component and click Settings In the Settings dialog box make the changes and click OK See Device and Component Settings for specific settings information for the component Note Device settings can be changed and components can be removed or added only in the Install System Devices dialog box as described in Installing Device Drivers Component settings can be changed in both the User Settings for Default configuration hardware dialog box and the Install System Devices dialog box Changes that you make to component settings in the User Settings for Default configuration hardware dialog box do not affect the settings defined in the Install System Devices dialog box 5 Inthe User Settings for Default configuration hardware dialog box click Apply to save your changes without closing the dialog box if you want to load and configure other devices or click OK to save your changes and close the dialog box Ge To uninstall a device driver you do not have to first unload it Yo
70. Olympus ZDC and ZDC2 0 c cece cece cece eeeeeeeeeees 110 Preregu isites e ss ea a Sta a eae eee ee 110 To use the Olympus ZDC or ZDC2 for an interactive acquisition using one shot focus 111 To use the Olympus ZDC or ZDC2 for an interactive acquisition using continuous WOCUS ERE ters es eS ee ek pe ge tee ase sen nee beeen ceceas sues AE See 112 To use the ZDC or ZDC2 for a simple time lapse acquisition without a Z series or stage series continuous focus 00 0000 o cece cee eee ee cece cece eeeeeeeeeeeeees 113 To use the Olympus ZDC or ZDC2 for a multidimensional image acquisition without a Stage SOMOS oils alee eet ale Spee See et Oe he a a ee 114 To use the Olympus ZDC or ZDC2 for a multidimensional image acquisition with a stage Series l 0 00000000000000 000000000 e cece cece cece AA L araar aroraa ana 115 Zeiss Definite Focus 2 22 2 cece eee cece aa eadera raare daaraan nnna 117 Prerequisites e essee a he e tee eet een ee et 117 To use the Zeiss Definite Focus for an interactive acquisition 222 117 5011685 B 5 To use the Zeiss Definite Focus for a multidimensional image acquisition without a Stage series 2 2 eee eee cece eee eee ee eee cece cece eeeeeeeeeseeeees 118 To use the Zeiss Definite Focus for a multidimensional image acquisition with a stage SONOS 2222 22325225 is iat ees soto tte et eet ice tote eee eet ahead a aa 119 Pausing or Stopping an
71. On the Mode Interactive tab or Mode Multidimensional tab in Focus click Configure 2 Inthe Focus Configuration dialog box select the Display Only Best Focus Image check box to display just the best focus image in the Latest Image tab after autofocusing is complete or clear the Display Only Best Focus Image check box to display each focus test image in the filmstrip as it is acquired during autofocusing 3 Inthe Autofocus Step Size field type the step size a larger distance than the accuracy distance between each focus test image that will be acquired The step size is affected by the depth of field of the objective and the signal strength of the sample If the step size is too small extra images will be collected during autofocus If the step size is too large the focus position may be inaccurate Generally you should set a step size that may be too small rather than too large if the step size is too large the autofocus algorithm may not be able to locate the sample 4 Inthe Autofocus Range field type the total distance along the Z axis that the Z motor will move while acquiring focus test images A CAUTION Be aware of the distance between the microscope objective and your slide Specifying a large autofocus range may allow the objective to collide with the slide which could damage your sample and microscope The units of measurement for the Z axis positions are based on the Z motor configuration For information about device
72. This section describes e How to view plane information e Plane information data definitions To view plane information for a dataset 1 Open the dataset that contains the image or images 2 Onthe Measure tab in Measurement Set select Plane Information and then click Show Table The MetaMorph NX Software displays the plane information for the dataset Gs If you want to view plane information for images in another dataset in the experiment keep the plane information data table open and in the filmstrip double click the other dataset The data in the plane information data table will update to reflect the other dataset Plane information data definitions Note Some of the data described below may not always appear in the plane information data table you may need to configure the plane information to display additional columns See Displaying Measurement Results in a Table Data Description ZStep The Z step in the order defined by the Z series at which the image was acquired TStep The time point in the order defined by the time series at which the image was acquired ChannelStep The channel used in the order defined by the channel series to acquire the image StagePositionStep The stage position in the order defined by the stage series to acquire the image Width The width of the image in pixels Height The height of the image in pixels Depth The range of grayscale values or color values in the imag
73. ab select the first channel in the channel series Note The focus stabilization system uses the first channel to calculate the focus adjustment value Subsequent channels are offset from the first channel Inthe Multidimensional Acquisition click Configure Inthe Acquisition dialog box select the Focus Stabilizer check box to indicate that the acquisition should use the focus stabilizer hardware and then click Focus Stabilizer to display the related fields 5011685 B 107 Chapter 7 Acquiring Images 5 10 11 12 Inthe Autofocus Frequency field Select To have the Nikon PFS continuously focus at every time point focusing at every time point may slow down acquisition First at only the first time point Every Nth Point at specific time intervals Specified in the next step for example you could focus at every third time point Every Point If you selected EveryNthPoint in the previous step in the Autofocus Rate field type the time point interval at which continuous focusing should occur for example type 3 to focus at every third time point Keep in mind that the first time point in a time series is always 0 zero Click Live to display a live view of the image Use the focusing knobs on the microscope to bring the image into focus In the Acquisition dialog box in the Focus Stabilizer section click Start Continuous Focus Optionally slightly adjust the focus by using the knob on the Niko
74. adjust camera and channel settings such as gain and exposure before resuming the acquisition Some options are not available while the acquisition is paused For example you cannot change the number of stage positions but you can change the X Y and Z positions of the existing stage positions Example Ina time series acquisition the acquisition ends as soon as the duration ends even if the acquisition has not acquired images at all time points To extend the duration of an acquisition while images are being acquired you can pause the acquisition extend the duration and then resume the acquisition When you click Pause on the Mode Multidimensional tab all images channels Z series are acquired for the current stage position before the acquisition pauses Stopping an acquisition halts the acquisition immediately and saves all images acquired up through the last time point You cannot resume an acquisition once you have stopped it This section describes how to e Pause an acquisition e Stop an acquisition To pause an acquisition 1 On the Mode Multidimensional tab click Pause The Pause button changes to a Resume button 2 Make any adjustments that are needed and then click Resume To stop an acquisition 1 On the Mode Multidimensional tab click Stop 2 Inthe dialog box that appears click Yes to confirm that you want to stop the acquisition The MetaMorph NX Software saves all images that were acquired up through
75. age position Repeat Steps 6 10 for each stage position that you want to add to the series Optionally configure other parts of the acquisition before acquiring images See Configuring a Time Series Acquisition Configuring Channels for Acquisition Configuring Image Autofocus Using Focus Stabilization or Configuring a Z Series for Acquisition Select the Do Not Update Display check box to indicate that images should not be displayed as they are acquired in this case the new dataset is not added to the experiment until the acquisition is complete or clear the check box to indicate that the Latest Image tab should display the most recently acquired image and that the images should appear in a new Dataset tab as they are acquired 5011685 B 85 Chapter 7 Acquiring Images 14 When you have configured the acquisition click Acquire If the Do Not Update Display check box is cleared the MetaMorph NX Software opens the Latest Image tab which displays the most recently acquired image This image helps you gauge the quality of the images as they are acquired which can be especially useful for lengthy time series acquisitions During acquisition the MetaMorph NX Software saves the acquired images into a new dataset You can review images as they are acquired by displaying the Dataset tab See Viewing Images in a Dataset Note If an experiment is not open when you start an acquisition MetaMorph NX will create one to contain the new da
76. ages using some or all of the channels See Viewing Images in a Dataset This section describes how to e Configure an interactive acquisition e Save images into a dataset 5011685 B 69 Chapter 7 Acquiring Images Prerequisites Before you configure a channel for an acquisition determine if you plan to use an illumination setting See Creating Illumination Settings To configure an interactive acquisition 1 70 On the Acquisition Mode tab select Interactive Acquisition select the camera channel and optionally select a hardware setting if one is needed to set the microscope devices to pre defined positions On the Mode Interactive tab in Magnification select the magnification that you want to use for the image acquisition On the Mode Interactive tab click Configure to open the Acquisition dialog box Ge You can also add remove and configure channels using the options that appear when you select a channel in the Channel Series group on the Mode Interactive tab All settings that you select to define per channel in Step 14 also appear in the options in the Channel Series group Changes that you make to channel settings on the Mode Interactive tab are reflected in the Acquisition dialog box and changes that you make in the dialog box are reflected on the tab Inthe Acquisition dialog box click Channels to expand the list of configured channels Click lt None gt to configure the first channel or click Add Channel
77. allowing you to compare the images data table and graphs of multiple datasets and different stage positions Z positions or time points The example below shows two Dataset Views each displaying a different stage position of the same dataset You can open as many Dataset Views as you need See Rearranging Dataset Views As you move the mouse cursor over an image information about the image appears below the image grid This information includes the intensity values of the pixels for each channel the X Y coordinates and if applicable the stage position number time point number and Z step number of the image For example Stage position 2 position X Y coordinates Intensity values for each displayed in displayed in in image where channel in image where image grid image gid mouse is located mouse is located Stage 0 of 0 2 Z30f0 4 339 864 M173 E 189 E 205 Total number of Total number of Z stage positions in positions in the the dataset in dataset in this this example 0 2 example 0 4 indicates the indicates the dataset contains dataset contains 5 3 stage positions zZ positions If the image is a single image it was acquired at one time point at one stage position at one Z position then Single Image appears below the image grid rather than stage Z and time point 10 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Home Tab Organize the display Display a s
78. amera driver may only support certain binning values On the Hardware Setup tab if needed click Auto Expose to automatically calculate and set the exposure time for the live view See Using Auto Expose Click Live The live view appears in the Live Display tab 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Ge You can adjust the binning value while the Live Display tab is open you can zoom in on the live view and you can scale the channel that is displayed 5 To stop the live display click Live again or close the Live Display tab To display a live view while configuring an image acquisition 1 On the Mode Multidimensional tab or Mode Interactive tab select the channel to use for the live view 2 Inthe Live Binning field E type the binning value to use for the live view Note The camera driver may only support certain binning values 3 If needed adjust the exposure time for the channel or click Auto Expose to automatically calculate and set the exposure time for the live view See Using Auto Expose 4 Click Live The live view appears in the Live Display tab Ge You can adjust the binning value while the Live Display tab is open you can zoom in on the live view and you can scale the channel that is displayed 5 Tostop the live display click Live again or close the Live Display tab Snapping an Image As you adjust devices and illumination conditions
79. and as you configure a multidimensional acquisition you can quickly test the current settings by snapping an image If you are using the Interactive acquisition mode snapping an image acquires a single image You can snap an image in one of three ways e while configuring hardware settings described below e using a specific channel while in Multidimensional acquisition mode described below e using one or all channels while in Interactive acquisition mode see Acquiring Images in Interactive Mode The MetaMorph NX Software also snaps an image when you test auto exposure or image autofocus before acquiring an image or when you set the camera area for acquisition These snapped images are not saved as part of a dataset but are saved in the Scratch folder on the hard drive of your computer See Using Auto Expose Configuring Image Autofocus or Setting the Camera Area for Acquisition Note If you manually move a filter wheel or other illumination device and then snap an image the snapped image reflects the actual position of the device rather than the illumination setting that is selected 5011685 B 95 Chapter 7 Acquiring Images To snap an image while configuring hardware settings You must be able to access the Hardware Setup tab to perform the tasks described in this section See your system administrator for more information 1 On the Mode Interactive tab or Mode Multidimensional tab select the channel that you want
80. at the Latest Image tab should display the most recently acquired image and that the images should appear in a new Dataset tab as they are acquired 25 Click Acquire If the Do Not Update Display check box is cleared the MetaMorph NX Software opens the Latest Image tab which displays the most recently acquired image This image helps you gauge the quality of the images as they are acquired which can be especially useful for lengthy time series acquisitions During acquisition the MetaMorph NX Software saves the acquired images into a new dataset You can review images as they are acquired by displaying the Dataset tab See Viewing Images in a Dataset for more information Note If an experiment is not open when you start an acquisition MetaMorph NX will create one to contain the new dataset A separate window within the Latest Image tab displays the status of the acquisition as well as a histogram for each channel The MetaMorph NX Software reports the number of images acquired as well as the current time point in a time series if applicable stage position if applicable channel and Z position if applicable Other statistics may also be reported to help you monitor progress Configuring Stage Positions for a Multidimensional Acquisition If you have a stage motor you can designate the stage positions specific sites in your sample where images will be acquired Images are acquired at each stage position in the order that the position
81. at you specify The Zeiss Definite Focus works best with air objectives or samples with water around the sample Consult the Zeiss documentation for more information Note The search range along the Z axis and surface location are different with each objective As aresult the focus adjustment value which is the surface to focal plane distance some manufacturers call this the offset will not be the same for all objectives The sample must be brought into focus using each objective to find the focus adjustment value This section describes how to e Configure the Zeiss Definite Focus for an interactive acquisition e Configure the Zeiss Definite Focus for a time lapse multidimensional acquisition without a stage series e Configure the Zeiss Definite Focus for a time lapse multidimensional acquisition with a stage series Prerequisites The Zeiss device driver and focus component must be installed and configured See Installing Device Drivers and Settings For Specific Devices and Components The focus stabilizer hardware must be designated as the Z motor for autofocusing See Designating Z Motors and Stages for Acquisition To use the Zeiss Definite Focus for an interactive acquisition 1 Configure all of the interactive acquisition settings for the channels and if applicable image autofocus If your acquisition includes more than one channel on the Mode Interactive tab select the first channel in the channel series
82. ates a new dataset and saves the image into it Initially saving images is a two step process you set up the saving options and then you save the images However you do not need to configure the saving options each time you save an image you can change the options only when you want to save an image in a different manner Save the 1 Configure the displayed image Saving options Pe Save as Individuals Saving to DatasetFLUOR1 x asu Save Save into Groups Name Prefix DatasetFLUOR New Clear Dataset Display 1 Set up the saving options If you want to Then click Save as Individuals and in the Name Prefix field type the prefix to use for the name of the dataset that will be created for the image automatically create a new dataset each time you save an image The MetaMorph NX Software appends a number to the prefix and increments the number by one each time you save an image using this option For example if the name prefix is DatasetFLUOR the dataset that is created when you first save an image will be named DatasetFLUOR1 and then when you save a second image the dataset that is created will be named DatasetFLUOR2 and so on 5011685 B 73 Chapter 7 Acquiring Images 74 2 If you want to Then Once you create a dataset you can add images to it using the Save into Groups option described below save an image into click Save into Groups and do one of t
83. ays the settings stored in the acquisition protocol file that you loaded 5011685 B 123 Chapter 8 Measuring and Analyzing Images This chapter includes the following topics e Overview of Measuring and Analyzing Images e General Workflow for Measuring and Analyzing Images e Viewing Plane Information e Measuring Morphometry of Objects e Drawing and Measuring Regions e Counting Nuclei e Displaying Measurement Results in a Table e Exporting Measurement Data e Graphing Measurement Results e Adaptive Background Correction Overview The MetaMorph NX Software provides several ways to obtain information about images and to measure and evaluate objects in an image or a set of images Depending on the measurement function that you choose and the settings that you select measurements can include the area dimensions shape position orientation and intensity of objects in an image Each measurement function also includes a statistical summary of the measurements made Once measurements are made on an image you can display sort or graph the results You can then export the measurement results to Microsoft Office Excel or to a text file You can measure just the images that are displayed in the image grid or all of the images ina dataset that are of a specific series type Z time or stage If an image contains multiple channels region measurements can be made on either a specific channel or all channels One dataset
84. but that have not yet been saved into a dataset Initially the Scratch 5011685 B 41 Chapter 5 Creating and Managing Experiments 42 folder is in C Users usermame AppData Local Temp You can move or rename the Scratch folder by designating a new location or name in the Administration dialog box See the MetaMorph NX Microscopy Automation amp Image Analysis Software System Administrator Guide for more information The file name format of images in the Scratch folder is different than the file name format of images that are saved in a dataset In the Scratch folder the file name includes the date on which the image was snapped or acquired and a number that increments by one with each image that is snapped or acquired For example Image_20101122 0000000015 tif is the fifteenth image in a series of images that were snapped on November 22 2010 Creating a New Experiment You can create an experiment that will contain images that you plan to acquire or to which you plan to link images The MetaMorph NX Software creates an experiment for you if one is not already open when you acquire or link images The MetaMorph NX Software saves and names the new experiment folder as specified by the experiment default settings This section describes how to e Create anew experiment e Change the experiment default settings Note All images acquired using the multidimensional acquisition mode and all measurement data in an experiment are automat
85. calculation Maximum The maximum pixel intensity value in the region Minimum The minimum pixel intensity value in the region Statistical summary measurements Measurement Definition The total number of regions measured Minimum The minimum value of the measurement that was made Maximum The maximum value of the measurement that was made Total The sum of values for a specific measurement For example for width the total value is the sum of width measurements for all regions measured Average The sum of all measures divided by the number of measures The 146 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Measurement Definition average is calculated by the formula Fee n where n number of measures Standard deviation A measure of the variability of a set of measurements around their average The standard deviation is defined as the positive square root of the variance Counting Nuclei The Count Nuclei measurement function identifies and isolates cell nuclei through image segmentation The segmented image displays each nucleus in yellow Any nuclei like pattern in the image less than the specified minimum width will be considered to be background noise and will be excluded from the segmentation The value you specify for the maximum width can be used to exclude any blobs larger than the specified size to help divide touching cells and is used in to
86. camera image bit depth range to fit within the bit depth range of the acquired image Set this value to 24 bit to enable color image acquisition from qualified color cameras Sharpness Controls camera hardware to increase or decrease camera sharpness Camera Shutter Sets the state of the camera shutter to one of the following states Open for Exposure Always Closed or Always Open Clear Mode Defines when to clear the camera chip Clear Count Specifies the number of frames to clear when clearing the camera chip Frames to Average Field Specifies the number of frames to combine for frame averaging cameras Offset Adjusts the black level reference above the zero level to 5011685 B 61 Chapter 6 Installing and Configuring Hardware 4 reduce or eliminate background noise Sensitivity Turns camera sensitivity capability on or off and specifies a sensitivity value to control the camera s internal image intensifier Light Mode Specifies the relative brightness of the light source and raises or lowers the camera s sensitivity Cooler On Activates internal camera cooling Use Contrast Knobs Enables the camera mounted contrast controls External Trigger Trigger Enables the external trigger capability of the camera Mode Flat Field Correction Activates the internal shading correction capability of the camera Get Flatfield Acquires an image for flatfield shading correction Noise Filter Activates the internal noise c
87. ccececceuceceeueeeeeee 27 To scale channels in an image 2222 e cece cece cece cc cece ccccccecceeceeeeeeeeeeeees 28 Zooming in on IMAQES 2 eee eee eee cece cece cece cece cece cece ce ceceeeeeeeeeees 31 Displaying Series Information on Images in the Image Grid 222 2222200 32 To display series information on images in the image grid 32 Chapter 3 Exporting Images to Microsoft Office PowerPoint 33 To export images to PowerPoint 00000 0c cece e cece eeeeeeeee 33 Chapter 4 Configuring a Keypad Keyboard or Mouse 35 Using the Mouse Wheel to Control Device Movement 2 2e cece cece eee 35 To configure the wheel Control 2 2 0 0 2 eee eee cece cccccccecccccccceeeccccceeeeeeeees 35 To use the wheel Control 00 cece cece cece cece eee eeeceeeeeteeteeeneeeeneees 36 Configuring an Input Accessory Such as a Keypad Keyboard or Mouse 36 To configure an input ACCeESSOTY 22 2222 e cece ee eeees 37 Chapter 5 Creating and Managing Experiments 220 22200 22 39 About Experiment and Dataset Folders and Files 222000 2 cc ceeeee cece eee eee 39 Creating a New Experiment 002 0 00 ee eee cece ee eeeeeeeeeeeees 42 To create anew experiment 2 222222 e cece cece cece cc ceeeeececceceeeeees 42 To
88. ce to indicate that it is configured correctly Note The Olympus ZDC or ZDC2 emits three beeps if it did not successfully calculate the lens focus adjustment value Try to set the focus again If you do not hear any beeps the ZDC or ZDC2 is not configured correctly Make sure that the observation method for the objective is set to something other than None check that you are using the correct lens and check that the IX2 Software is configured correctly Optionally slightly adjust the focus by clicking the buttons or by typing a value in the Focus Adjustment field Just before acquiring your image if your image is out of focus click Go To Focus The Olympus ZDC or ZDC2 re establishes focus using the focus adjustment value that you set in Step 8 Click Acquire To use the Olympus ZDC or ZDC2 for an interactive acquisition using continuous focus 112 1 Configure all of the interactive acquisition settings for the channels and if applicable image autofocus If your acquisition includes more than one channel on the Mode Interactive tab select the first channel in the channel series Note The focus stabilization system uses the first channel to calculate the focus adjustment value Subsequent channels are offset from the first channel Click Live to display a live view of the image Use the focusing knobs on the microscope to bring the image into focus If you are using the Olympus ZDC2 on the Mode Interactiv
89. ceceeeeeeeeeeeeees 61 Defect Correction 2 20 0000 e cece cece oa aa eecccccseeeeeeeees 62 Demonstration Camera Driver 20 02 2222 e cece cece ccc eeeccecccccceeceees 63 Chapter 7 Acquiring Images 2 22 2 occ cece cece cece cece ee cceecceccceceeeeees 65 OVENVICW 2 srece wise kas 32088 2 Gube ee cis gciggn 3 Lda beste snk ae oa F Beaes Mote esa aeS 66 General Workflow 2 22 2 0c ccc cece cece eee eeeeeeecceeecceececccecsccsccecccceucecceseeeeeaaees 67 Selecting an Acquisition Mode 22 2 2 2 eee cece cece cece cece cece cecccccceeeeees 68 Acquiring Images in Interactive Mode 0 022222 e cece eee e cece cece ccc cece cece cece eeeeeeees 69 PrereGuisites erce oot ga es ee ot ee Bk ee tesa ie eet na 70 To configure an interactive acquisition 00 000000022 cece cece cece cc ccc cece cece eee eeee 70 To save images into a dataset 2 222 72 Acquiring Images in Multidimensional Mode 0 00 0000 c cece cece ccc e eee eeeees 75 Setting the Camera Area for Acquisition 0000 0000 00 eee e cece eeeeeeees 78 To set the camera area ee 78 Configuring Channels for a Multidimensional Acquisition 2 2 22222222222 222 78 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Prerequisites sc ruze edie ek noe ele oer E wad alse ouea 79 To configure a channel 000000 e cece eee ee cee cece cece eee
90. ceceseseeseseees 132 Dimension measurements 2 2 2 2 22 oaaao aooaa aaao aLa LaLa LALALA aLa annaa 134 Shape position and orientation measurements 222222 2c002 eee cece eceeeeee 135 Intensity measurements 0 000202 e eee cece cece cece cee cece ee eeeeeeeteeeeeeees 136 Statistical summary measurements 000 eee eee eee cece e eee eeeeeeeee 137 Drawing and Measuring Regions 2 e cece ee eecceecceceeseeeeees 138 To draw and measure regions 22 22 2022 eee ec e cee ec ece idara ranana nann 139 Region measurement definitions 0 00000000 e ee eeeeeeeeees 141 Area measurements 2 2 2 2 2 eee eee cee eee c cece ccc eececeeececeeeseceessceeesseseessesees 141 Dimension measurements 2 2 2 2 0 22 2 eee cece cece cee cecccecececeecseceesetseeteesees 142 Shape position and orientation measurements 2222222 20202 e cece cece eeeeee 143 Intensity measurements 000222 e cece ccc cece cee cece cece ee eeeeeeeeeeeeeeeeees 146 Statistical summary measurements 0 0 0c eee eee cece cece ee annann 146 Counting Nuclei 0 000000 e cece cece eeeeeeeeceecececeeeseeeees 147 To count nuclei a oa aoaaa aana eee eee cee cece cece cece cceeeeecceeseceeeeseseeeseseeees 147 Count Nuclei measurement definitions 022 02 cece eee ec cece ee ceeeeeceeeeeeee 149 Displaying Measurement Results in a
91. ces 58 5011685 B 10 11 12 13 MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide 40X Magnification 20X Magnification Point of Origin Oo 25 210 10 200 x x Y Y x offset distance for the 20X Magnification setting 15 25 10 Y offset distance forthe 20X Magnification setting 10 210 200 Best Practice Select one objective as a reference objective ideally this is the objective with the highest magnification because its focal position is very specific Using the reference objective note the X and Y positions of the point of origin in your sample and set the offset values in the magnification setting for this objective to 0 zero Then switch to the objective with a lower magnification and note the stage X and Y values of the point of origin The offset distance values for the magnification setting of the objective with the lower magnification are the difference between the X and Y values of the point of origin when using the reference objective and the X and Y values of the point of origin when using the objective with a lower magnification Repeat this process for each objective Inthe Z Offset field type the distance to move the Z motor to keep the point of origin in its original focal position when using this objective The Z offset distance helps compensate for non parfocal systems When a magnification setting is selected the Z motor position is adjusted by the offset distance
92. cify in the Min Value and Max Value fields that appear RangeFilter MatchFilter include all values in the column that are the same as the value that you specify in the Max Value field that appears Select the Use Inclusive check box to include the values that you entered in the filter criteria in Step 5 For example if you select this check box and enter 30 in the Min Value field and 50 in the Max Value field the filter includes all values between 30 and 50 as well as the values 30 5011685 B 151 Chapter 8 Measuring and Analyzing Images and 50 if you do not select this check box the filter includes all values between 30 and 50 but excludes the values 30 and 50 7 To create additional filters repeat Steps 3 through 6 8 Click OK The MetaMorph NX Software updates the measurement data table to show only the data that meets the criteria that you specified in the filters Exporting Measurement Data You can export measurement data as it is configured in the data table to either a Microsoft Office Excel 2003 or 2007 spreadsheet or a text file The MetaMorph NX Software exports just the data that is in the table when you export it that is if you have configured the measurement results so that certain data is excluded the data that was excluded will not be exported The order in which the data is sorted in the data table is not preserved when you export the data This section describes how to e Export data to an open Excel s
93. ck Export Acquisition Protocol on the Acquisition Mode tab to save the current acquisition settings 3 Inthe Save As dialog box navigate to the location where you want to save the acquisition protocol file name the file and click Save You can give the file any name as long as you keep the xml extension and the name is valid that is does not contain any special characters such as exclamation points The MetaMorph NX Software saves the acquisition protocol as an XML file in the dataset folder on the hard drive of your computer See Experiment and Dataset Folders and Files An acquisition protocol file can be used on any computer running the MetaMorph NX Software To load saved acquisition settings 1 Onthe Mode Interactive tab or the Mode Multidimensional tab click Configure to open the Acquisition dialog box 2 Inthe Acquisition dialog box click Import Acquisition Protocol Ge You can also click Import Acquisition Protocol on the Acquisition Mode tab 3 Inthe Open dialog box select the acquisition protocol file and click Open 122 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide If you import an interactive acquisition protocol using the option on the Acquisition Mode tab the Mode Interactive tab appears if you import a multidimensional image acquisition protocol the Mode Multidimensional tab appears If the Acquisition dialog box is open when you import a protocol it displ
94. cks the position of the surface of the slide or dish and maintains focus at the specified distance focus adjustment value above the surface Sample Focal plane Focus adjustment value offset Slide dish surface Objective Although the configuration of each system varies slightly in general you simply bring the sample into focus and set the focus position The focus stabilization system uses the focus position to calculate the focus adjustment value which it maintains throughout your experiment If you are configuring an acquisition with a stage series at each stage position you must bring the sample into focus so that the system can calculate each focus adjustment value The MetaMorph NX Software supports the following focus stabilization systems e Nikon Perfect Focus e Olympus ZDC and ZDC2 e Zeiss Definite Focus 5011685 B 105 Chapter 7 Acquiring Images Nikon Perfect Focus The Nikon Perfect Focus System PFS is a focus stabilization system for the Nikon Eclipse TE2000 and Eclipse Ti microscopes The Nikon PFS can only be used with certain objective models Consult the Nikon documentation for more information Note The search range along the Z axis and surface location are different with each objective As aresult the focus adjustment value which is the surface to focal plane distance some manufacturers call this the offset will not be the same for all objectives The sample must be brought into focus
95. configuration see Installing and Loading Hardware Device Drivers Images are acquired from the bottom of the range to the top 5 Inthe Autofocus Accuracy field type the smallest distance that the Z motor should move to refine its search for the best focus image This value is based on the depth of field and the numerical aperture of the objective 6 Inthe Autofocus Algorithm field select an algorithm Algorithm Description Brenner Standard Recommended for most imaging conditions and works well for a number of different microscopy techniques Measures contrast in the image Directional Average Recommended if the Standard algorithm produces slightly different results than expected Most accurate when using objectives with magnifications of 20X or greater 7 Click Autofocus The best focus image is displayed in the Latest Image tab The Z motor moves to the Z position at which the best focus image was acquired this position becomes the current Z position and is displayed on the ribbon and in the Z series section of the Acquisition dialog box 100 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide If autofocusing does not produce in focus images An exposure time may be too short especially if the sample is dim in which case the autofocus system may not be able to find the sample You may need to increase the exposure time or change camera settings so that the autofocus syste
96. croscopy Randy O Wayne Academic Press 2008 Methods in Cellular Imaging Ammasi Periasamy American Physiological Society 2001 Video Microscopy The Fundamentals Shinya Inou and Kenneth R Spring Springer Publishing 1997 Video Microscopy Greenfield Sluder and David E Wolf Academic Press 1998 168 5011685 B Index sss aS A Acquisition Designating motors for 92 Interactive 69 Mode 66 68 Multidimensional 75 Overview 66 Pausing or stopping 121 Protocol settings 122 Setting camera area for 78 Activating a camera 60 Area measurement 132 141 149 Auto expose 78 97 Autofocus 98 Average Intensity measurement 130 138 147 B Background color of graph changing 153 Binning 72 82 94 96 Breadth measurement 134 143 Brenner algorithm 102 104 C Calibration 57 Camera specific settings 61 Camera area for acquisition 78 Camera channel 60 Camera scaling 27 Camera settings 69 78 Cancelling a measurement 130 138 147 Centroid X and Y measurement 130 138 Channel vs Z View Mode 21 Channels Configuring for acquisition 78 Scaling for display 27 Showing and hiding changing color 9 17 Clearing regions 138 Color of graph changing 153 Commands assigning to input accessory 36 Contrast adjusting 27 Count Nuclei measurement function 147 Count summary measurement 130 138 147 Current view camera area 78 D Data Table 149 Dataset View 9 17 24 Datasets Naming in Interactive mode 69 Overview of 39 Storage
97. cus Inthe Stage Series section in the row that corresponds to the current stage position click Set Focus The Focus Adjustment field displays the surface to focal plane distance sometimes called the offset Optionally slightly adjust the focus by clicking the buttons or by typing a value in the Focus Adjustment field Repeat steps 7 10 for each stage position Inthe Focus Stabilizer section in the Autofocus Frequency field Select To re establish focus Every Point at every time point focusing at every time point may slow down acquisition First at only the first time point Every Nth Point at specific time intervals specified in the next step for example you could focus at every third time point If you selected EveryNthPoint in the previous step in the Autofocus Rate field type the time point interval at which focusing should occur for example type 3 to focus at every third time point Keep in mind that the first time point in a time series is always zero 0 Once you have configured the multidimensional acquisition click Acquire 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide The Olympus ZDC or ZDC2 maintains focus of the image throughout the image acquisition by re establishing focus at the stage positions Zeiss Definite Focus The Zeiss Definite Focus is a continuous focus system that re establishes focus at intervals in seconds th
98. descend before the stage moves After the stage moves to the next position the objective will return to its initial position Move the stage to the first position at which images will be acquired and then click Add The stage position coordinates appear in a new row in the stage series You can also type the coordinates of a stage position in the X and Y fields and Z or Z2 fields if applicable Note If the acquisition includes a Z series the Z position defined in the stage position will be the center position of the Z series The center Z position may vary between stage positions See Configuring a Z Series for Acquisition Ge Go To moves the stage to the defined stage position To edit an existing stage position so that it uses the current X Y and Z positions click Update If you are using focus stabilization hardware bring the image into focus and click Set Focus Depending on the type of hardware that you are using either the Focus Adjustment field displays the surface to focal plane distance or the Memorized field displays Yes to indicate that the focus is set See Using Focus Stabilization In the Label field leave the stage position label as is or type your own label name This label will be included in the plane information that is saved with the images when they are acquired at this stage position Optionally select the Use Travel Offset check box to instruct the motor to descend before moving to the specified st
99. dware devices and components that control magnification each time you acquire images you can define specific collections of settings and save them as magnification settings Then when you configure an image acquisition you can select the appropriate magnification setting The MetaMorph NX Software uses magnification settings to apply the correct spatial calibration to images as they are acquired Even if the objectives are changed manually you should define magnification settings to ensure correct spatial calibration of images Specific data included in a magnification setting such as refractive index and numerical aperture are recorded in the Plane Information measurement set these values are often important in deconvolution operations and other calculations The calibration should be defined for a non binned image When acquiring images that have been binned the MetaMorph NX Software will automatically adjust the spatial calibration to reflect this binning Best Practice Magnification settings are not camera specific If you use multiple cameras include the name of the camera in the name of the magnification setting so that it is clear that a particular magnification setting should be used with a particular camera Different cameras may require different calibrations so if you create the magnification setting based on just the magnification then the calibration could be incorrect when you switch cameras You can create as many magnif
100. e S key If you type in a Microsoft Office Word document while the MetaMorph NX Software is open every time you type the letter S the MetaMorph NX Software will snap an image To configure an input accessory 1 Connect the input accessory to your computer 2 If you have access to the Hardware Setup tab click Input Accessory Setup Or if the Quick Access Toolbar contains a shortcut to the Input Accessory Setup option click the shortcut Ei Inthe Input Accessory dialog box click Select Input Accessories 4 Press any key wheel or button on the input accessory that you want to configure The name of the accessory appears in the Last Device Input statement at the bottom of the Select Input Accessories dialog box Note the name of the accessory For example Selected Keyboard Keyboard VID_062A amp PID_0201 amp MI_00 Keyboard Input Accessory lt None gt Selected Mouse lt None gt Mouse Input Accessory lt None gt Refresh Last Device Input Keyboard VID_062A amp PID_0201 amp MI_00 Key S Cancel 5 Inthe Select Input Accessories dialog box select the input accessory from the list The Last Device Input statement at the bottom of the dialog box updates to display the name of the device you used to select the accessory from the list typically a mouse the Last Device Input statement is for informational purposes only and does not indicate that you have selected the device for confi
101. e although in most situations you probably need to change names or settings only once when installing the device Prerequisites Make sure you have completed the tasks described in Preparing to Install Hardware Device Drivers To install a device driver 1 On the Hardware Setup tab click Install Devices 2 Inthe Install System Devices dialog box in the list of available hardware select the device that you want to install and click Install seen one lk Available Hardware Installed Devices AE Acton SpectraPro Marzhauser MC2000 Stage Z FS Al Signatech aia Marzhauser Stage X A Arcturus XT ait Marzhauser Stage Y AE Asahi Spectra MAX301 att Marzhauser Z Motor AE AS Pw 1000 E a Ludi MAC5000 E Asi MS 2000 aia Ludi Stage x FB Auto Focus Sensor Aia Ludl Stage Y FE Cain Optoscan atin LudlZ A Cain Rotor aia Ludi Slide Loader AE canvi at Ludl Wheel 1 A Cell Robotics Stage atin Ludi Wheel 2 E Chamlide ogy Ludl Shutter 1 E Chuo Seiki Focus atta Ludl Shutter 2 __Instal gt gt lt lt Remove Settings Status Marzhauser MC2000 Stage Z on COM1 Remove Al IN EMULATION MODE Apply OK Cancel 5011685 B 47 Chapter 6 Installing and Configuring Hardware 3 7 48 Verify that the port assignment shown in the Status area if applicable is not already assigned to another device and that the port assignment is consistent with any requirements listed in the documentation supplied with the device To re
102. e time or change camera settings so that the autofocus system can find the dim site e The step size may be too small in which case the autofocus system may not be able to accurately distinguish noise from intensity signals from the sample as a result the autofocus system may focus on something other than the sample e The range may be too small You may need to increase the range so that the autofocus system can find the sample e Anexposure time may be too long causing the autofocus camera to become saturated and unable to differentiate intensity signals from the sample You may need to reduce the exposure time or change camera settings e You may need to use the other autofocus algorithm To use image autofocus in an interactive acquisition 1 Onthe Mode Interactive tab click Configure 2 Inthe Acquisition dialog box select the Autofocus check box to indicate that the acquisition should use autofocus and then click Autofocus to expand the list of autofocus settings 3 Inthe Autofocus Range field type the total distance range along the Z axis that the Z motor will move while acquiring focus test images The units of measurement for the Z axis positions are based on the Z motor configuration See Installing and Loading Hardware Device Drivers A CAUTION Be aware of the distance between the microscope objective and your slide Specifying a large Z axis range may result in damage to your specimen and microscope 4 Inthe Au
103. e command assignments for the keyboard or keypad To configure a mouse wheel in the Keypad Mouse Commands section select the name of the device and then in the Spin Unit field type the increments by which the device will move when you scroll the wheel The spin unit is not a specific unit of measurement but is a value that reflects the amount that the device will move each time you scroll the mouse wheel For example if the spin unit for a Z motor is 2 the Z motor position will move 2 units at a time when you scroll the mouse wheel for example from 10 to 12 from 12 to 14 from 14 to 16 and so on Inthe Command column select commands for the left and right mouse buttons The mouse button commands are enabled as soon as you assign them The MetaMorph NX Software remembers the command assignments when you close the software The next time you start the MetaMorph NX Software you can immediately use the command assignments that you configured 38 5011685 B Chapter 5 Creating and Managing Experiments This chapter includes the following topics e About Experiment and Dataset Folders and Files e Creating a New Experiment e Opening an Experiment e Linking an Image to an Experiment About Experiment and Dataset Folders and Files The MetaMorph NX Software organizes your images and related data into a simple hierarchy An experiment is a folder that contains one or more datasets as well as supporting files such as emails
104. e exact Z range You can just set the range so that the Z series encompasses the area that you want to acquire 5011685 B 10 11 12 13 MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Inthe Z Steps field type the total number of images that will be acquired an image is acquired at each Z step To test the current device positions and settings at the top of the Acquisition dialog box click the Snap button that corresponds to the channel that you want to use for testing or double click the icon of the channel on the Mode Multidimensional tab The MetaMorph NX Software opens the Latest Image tab and displays a single new image using the selected channel You can use the snapped image as a guide for adjusting your settings See Snapping an Image Optionally configure other parts of the acquisition before acquiring images See Configuring a Time Series Acquisition Configuring a Stage Series Acquisition Configuring Image Autofocus Using Focus Stabilization or Configuring Channels for Acquisition Select the Do Not Update Display check box to indicate that images should not be displayed as they are acquired in this case the new dataset is not added to the experiment until the acquisition is complete or clear the check box to indicate that the Latest Image tab should display the most recently acquired image and that the images should appear in a new Dataset tab as they are acquired When yo
105. e image grid You may have zoomed in on the images so that regions on the images are out of the field of view The MetaMorph NX Software may be measuring regions that you do not see 159 Chapter 9 Frequently Asked Questions Why does my dataset contain duplicate images Ina sparse acquisition images are not necessarily acquired at every possible Z step stage position or time point and they may not be acquired with every channel For example to save time or data storage space your experiment may use particular channels only occasionally In this case the display fills in missing images by duplicating an existing image from the same channel The duplicate images appear in the image grid and are included in all measurements How do I remove a dataset from an experiment You should not remove a dataset or delete any files from an experiment folder The experiment file mexp file contains references to all of the data in all datasets If you remove a dataset or delete any files including image files measurements and image displays may not be accurate or you may not be able to open the experiment Why has an X replaced some of the images in the image grid Why am not able to make any measurements on the dataset that is missing images If you delete images from a dataset on your hard drive those images are replaced with an X in the image grid You cannot make measurements on an incomplete dataset Why is the Save but
106. e in bits for example 8 or 16 SpatialCalibrationState Indicates if the image was calibrated The check box in this column is selected if the image was calibrated 128 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Data Description SpatialCalibration X The X and Y calibrations of the image Spatial calibration is SpatialCalibrationY calculated by multiplying the calibration for the magnification by the binning used for the image when it was acquired If the image was not calibrated this value is 1 SpatialCalibrationUnitName The unit of measurement used to calibrate the image If the image was not calibrated no value is displayed ExposureTimeMilliseconds The exposure time used to acquire the image IIluminationSettingName The name of the illumination setting used to acquire the image MagnificationSettingName_ The name of the magnification setting used to acquire the image CameraBitDepth The bit depth of the camera used to acquire the image if known MagnificationNA The numerical aperture used to acquire the image MagnificationRI The refractive index used to acquire the image Annotation Comments about the image CreationMonth The month the image was acquired CreationDay The day of the month the image was acquired CreationY ear The year the image was acquired CreationTime The time the image was acquired StagePositionX The X and Y coordinates of the stage at wh
107. e in the progress bar at the bottom of the workspace and then click the Cancel button that appears The data that was calculated before you canceled the measurement is stored ina new measurement set To display the segmentation of the image on the Measure tab select the new measurement set To display the measurement results click Show Table See Displaying Measurement Results in a Table To display the statistical summary of all measurements in the measurement set click Show Summary If the data table is already open the summary appears at the bottom of the table If the data table is not open the data table opens and the summary appears at the bottom of the table To hide the statistical summary click Show Summary again To display the original Source image on the Measure tab select Plane Information To delete the measurement set in the list of measurement sets click the X that is next to the measurement set If you re measure the same images the MetaMorph NX Software uses the source images for measurements not the most recent segmentation of the images 148 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Count Nuclei measurement definitions Measurement Definition Area Total area for a specific nucleus Integrated Intensity The integrated total intensity for a specific nucleus Average Intensity The average intensity for a specific nucleus Statistical Summary M
108. e long such as 500 ms to account for the time it takes for the bulb to achieve a stable brightness Close Delay Delay in milliseconds between the time the MetaMorph NX Software sends the shutter the command to close and the time that the MetaMorph NX Software will start another command This delay is needed to ensure that the shutter has closed completely See illustration below Shutter i i Time ms i Open Delay i Exposure Close Delay ee nc _R _V_OMNMNwyys gt I I I Shutter Exposure Exposure ends Next begins begins and shutter command to open begins to close begins Shutter Parameters Open Shutter Control Dialog Opens the Control Shutter dialog box which provides the following options for testing the shutter Open Opens the shutter Close Closes the shutter Toggle Opens and then closes the shutter or closes and then opens the shutter Creating and Modifying Hardware Settings You must be able to access the Hardware Setup tab to perform the tasks described in this section See your system administrator for more information Rather than define settings for hardware devices and components each time you set up your microscope for a particular type of experiment you can define and save specific collections of settings as hardware settings and re use them as needed You may also want to use a saved hardware setting to quickly test and compare the effects of adjusting certain components Unlike
109. e position using the third channel defined in the channel series Keep in mind the MetaMorph NX Software starts counting time points stage positions Z steps and channels at zero 0 To display series information on images in the image grid 1 Onthe Application menu click MetaMorph NX Administration 2 Click Image Grid Overlay Configuration and then select the Show Image Grid Overlay check box 32 5011685 B Chapter 3 Exporting Images to Microsoft Office PowerPoint If Microsoft Office PowerPoint 2003 or 2007 is installed on your computer you can export images to a PowerPoint presentation The MetaMorph NX Software duplicates the displayed resolution and size of the images so that images are exported exactly as they are displayed in the image grid Exported images are embedded in the PowerPoint slide they are not linked as files in PowerPoint Once the images are in PowerPoint you can manipulate the images as you would any other picture Note You can also use version 7 7 of the MetaMorph Software to open images that were acquired with the MetaMorph NX Software To export images to PowerPoint If more than one Dataset View is open make sure you select the Dataset View that contains the images that you want to export Click once anywhere in the Dataset View to select it An orange border appears around the selected Dataset View Only the images that are displayed in the image grid of the selected Dataset View are exported
110. e tab in Focus click Set Continuous Focus If you are using the Olympus ZDC skip this step and proceed to Step 7 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide The Focus Adjustment field displays the lens focus adjustment value and the Olympus ZDC2 beeps once to indicate that it is configured correctly Note The Olympus ZDC or ZDC2 emits three beeps if it did not successfully calculate the lens focus adjustment value Try to set the focus again If you do not hear any beeps the ZDC or ZDC2 is not configured correctly Make sure that the observation method for the objective is set to something other than None check that you are using the correct lens and check that the IX2 Software is configured correctly 6 Optionally slightly adjust the focus by clicking the buttons or by typing a value in the Focus Adjustment field 7 Click Start Continuous Focus While continuous focus is on the Olympus ZDC or ZDC2 continuously adjusts the lens position to maintain focus Note You cannot adjust the Z motor position using the microscope controls or change objectives while continuous focus is on 8 Click Acquire 9 When the image acquisition is complete click Stop Continuous Focus When continuous focusing is off you can control the Z motor using the knobs on the microscope To use the ZDC or ZDC2 for a simple time lapse acquisition without a Z series or stage series continuous foc
111. e the Zeiss Definite Focus for a multidimensional image acquisition without a stage series 1 118 Configure the settings for the multidimensional acquisition at least those that affect Z motor position and illumination such as the channels and if applicable image autofocus anda Z series If your acquisition includes more than one channel on the Mode Multidimensional tab select the first channel in the channel series Note The focus stabilization system uses the first channel to calculate the focus adjustment value Subsequent channels are offset from the first channel In Multidimensional Acquisition click Configure to open the Acquisition dialog box In the Acquisition dialog box select the Focus Stabilizer check box to indicate that the acquisition should use the focus stabilizer hardware and then click Focus Stabilizer to display the related fields 5011685 B 10 11 12 13 MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Inthe Autofocus Frequency field Select To have the Zeiss focus at every time point focusing at every time point may slow down acquisition First at only the first time point Every Nth Point at specific time intervals specified in the next step for example you could focus at every third time point Every Point If you selected EveryNthPoint in the previous step in the Autofocus Rate field type the time point interval at which conti
112. e the stage and Z motors until the X Y and Z positions return to zero You set the point of origin in the Stage Motor Designations dialog box This dialog box also contains options for designating Z and stage motors for acquisition if more than one motor is installed See Designating Stage and Z Motors for Acquisition Prerequisites You must install and configure the appropriate hardware device drivers for your Z motors or stages as described in Installing and Loading Hardware Device Drivers To set a point of origin 1 On the Hardware Setup tab in Stage click the dialog box launcher in the lower right corner of the group 2 Move the Z motor to the point of origin the Z motor position is shown in the Z field on the Hardware Setup tab and then in the Stage Motor Designations dialog box click Set Origin for the Z motor The position shown in the Z field on the Hardware Setup tab changes to 0 zero to indicate that the current position of the Z motor is now the point of origin 3 If you are using a second Z motor move it to the point of origin the second Z motor position is 5011685 B 93 Chapter 7 Acquiring Images shown in the Z2 field on the Hardware Setup tab and click Set Origin for the second Z motor The Z2 position displayed in the field on the Hardware Setup tab changes to 0 zero to indicate that the current position of the Z2 motor is now the point of origin Move the stage to the point of origin the X and Y
113. e your changes without closing the dialog box if you want to install and configure other devices or click OK to save your changes and close the dialog box If you are using a communications port other than COM1 or COM2 confirm the communications settings for the device using the Microsoft Windows Device Manager Start gt Control Panel gt Device Manager 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Loading Device Drivers You must be able to access the Hardware Setup tab to perform the tasks described in this section See your system administrator for more information To acquire images and to create unique hardware and illumination settings you must load the appropriate hardware device drivers When you load a device the device and component names and settings that were defined during installation are loaded as the default settings Component names and settings may be changed again as they are loaded as described in this section Prerequisites Before you can load a device and its related components the device driver must be installed See Installing Device Drivers To load a device driver 1 On the Hardware Setup tab click Load Devices 2 Inthe User Settings for Default hardware configuration dialog box that appears in the list of available devices select the device that you want to load and click Add LarSes E Available Devices Claimed Devices Lud MAC5000
114. easurements Count The total number of nuclei in the image Minimum The minimum value of the measurement that was made Maximum The maximum value of the measurement that was made Total The sum of the values for a specific measurement For example the total value for the area measurement is the sum of area measurements for all nuclei in the image Average The sum of all measurements divided by the number of measurements The average is calculated by the formula Jaat n where n number of measurements Standard deviation A measure of the variability of a set of measurements around their average The standard deviation is defined as the positive square root of the variance Displaying Measurement Results in a Table Each time you measure objects or regions in an image or images the MetaMorph NX Software stores the results in a measurement set The Measure tab lists all of the measurement sets that belong to the open and selected dataset You can view the measurement results in a data table or graph and you can configure the results to display only the data that meet your specific criteria A measurement set has one data table The table contains the following types of data all of which can be included excluded or sorted e the settings or inputs used to make the measurement for example the low and high threshold values e the measurement results for example intensity perimeter and area e measurement su
115. ecececeeeeeeees 16 To minimize the ribbon 22 0 cee cee cee ce eeeeeeeeeeeeeeeeneeseeeees 16 To re display the ribbon 22 2 2 eee cece cece eee cece cece ceeeeeeeeeeteneeeeeeeeeees 16 Chapter 2 Viewing Images 0 00 0000 e eee rannan 17 Arranging and Displaying Images 0 00002 e cece nannan 17 To display a specific dataset 0000 0000000 e ee eeeeeeee 17 To display images acquired at a stage position Z position or time point 18 To navigate between images in the image grid 2222 000 e cece cece ee eee 19 To use the view modes to display and arrange multiple images or display a single image oe rae fos arts one eset eles ete Seceie dealt aoe aia Ses semi alee tosses teens 20 To change the display color of achannel 22 22222220002 e eee eee eee 23 To turn the display of a channel on or Off 22 2002 2 22222 e cece cece eee cece cece ee cece 24 Rearranging Dataset Views 0 0 22222 e cece cece cece cece cc ccc cceceeeeeees 24 To move or group tabbed windows 2 2 2 c cece ee eeeeeeeeeeee 25 To Move WINdOWS l 2 2222 ooo cece eee ccc cece ce ce cece cece cece eeeeeeeeeeeeenees 25 To hide a WindOW 2 222222 ee cece eee aaor eeeeeeeeeeeeenes 26 To auto hide a dialog box 2 2 2 26 To close a tabbed window or close all tabbed windows except the current one 26 Scaling IMageS oo eee cece cece cece eee ceceeeeececcececca
116. eeee 51 Filter Wheel Settings 00000000 ccc c cece eee ce eee eee cece cece cece cece eee eeeeeeeeeees 51 Objective SOUINGS 22 26 bashes Pee suis sails Seals O32 Nee EERE ET Eae IRINE EE EN eden eek EESE 52 Shutter Settings 2 2 2 2 cece eee eee cece cece eee eee eecececcecseceeeeees 52 Creating and Modifying Hardware Settings 00 0000 e cece eeeeeeee 53 PrereQuiSites ss tee cee ate hog Sarai ie Bek eisai eee 54 To create or modify a hardware setting 2 222000 54 Creating and Modifying Illumination Settings 0000000 0c eee cece cece eee eeeee 55 Prerequisites 2 23 20 22 ee ee estes on tee hi eat ete nee 55 To create or modify an illumination setting 0 0 00000002 ccc c cece cece e cece cece eee 55 Creating and Modifying Magnification Settings 0000 00002222 e cece cece cece cece e eee 57 Prerequisite 222 2 ia cect ecenerecue reste ee vexduccaorecybeaveeeacevyeseeeveusuuecenedee 57 To create or modify a magnification setting 000 000022 c cece cece ccc eee eee 57 Configuring Camera Drivers 0 02 coe cece cece eee eec cece cc cccceccececceceeeeteeeeeeeeeeeeeeeeees 60 To install and configure a camera river 22 c cece eee cece cece ec eeeeeeeceeeeeeenees 60 Camera SettingS 00000 c ccc c cece cece eee eee eee ee eee bebe eee e bebe ee eeeeeeeeeeeeees 61 Basic Camera Settings 002 0022222 c cece cece cece cece ccc cee cece eee e
117. eeeeeeeeeeeeeees 79 Configuring Stage Positions for a Multidimensional Acquisition 2 22 222 83 Prerequisites 2 2 2 2 2 0 2 cece cece cece aa h da apo Eese ae aO eoat oh ade Saana 84 To configure a stage series eee 84 Configuring a Z Series for a Multidimensional Acquisition 00 0 00 20 2 ceee eee ee eee 86 Prerequisites 00 cc cccecc cece cccccccccccccececceceeceeeeeeeeeeeeetesceeenececesceeees 87 To configure a Z SeVieS oo e eee ee cece cece cee ccccccceccccccccccccccecccceeceeeee 88 Configuring a Time Series for a Multidimensional Acquisition 22 22 222 89 To configure a time SerieS 2 2 2 e ccc cece cece cece ceeeeeeeees 90 Designating Z Motors and Stages for Acquisition 0 00 000222 e cece eee ee ee 92 Prerequisites 2 2 2 2 occ cee eee cece eee e cece cence cece eee ceeeeeeeeeeececeeeeeeeeeees 92 To designate up to two Z and stage motors 2 222222 92 Setting a Point of Origin 2022222 c cece cece cece cece eeteeeeeees 93 Prerequisites 2 222 2 ccc c cece ccc cece cece cece cece ccc e eee ccceeeeeeeeeeeceeteeeeeeeees 93 To set a point of origin 2 2 c cece cece cece cccecccceeeeeees 93 Displaying a Live View 00000000 c cece ccc cece ce cece dadaa raora aniraa raina 94 To display alive view while setting up the microscope 22022002222220 cece ee eee 94 To display a live view whi
118. efine in the Channels section In the example below the position of the Z motor when the sample is in focus using Channel 1 is 500 The position of the Z motor when the sample is in focus using Channel 2 is 700 The difference between the two positions is 200 so the Z offset distance is 200 700 Z postion for Sample Channel 2 Focal plane gt lt gt 500 Z position for Channel 1 Z Axis 5011685 B 81 Chapter 7 Acquiring Images 16 17 Inthe Acquisition Frequency field select the time points at which images should be acquired using the channel Select To acquire images using the channel Every Point at every time point First at only the first time point Every Nth Point at specific time intervals specified in the next step starting at the first time point for example you could acquire images using the channel at every third time point First and Last at the first time point and the last time point If the acquisition does not include a time series images are acquired at the first time point If you selected Every Nth Point in the previous step then in the Acquisition Rate field type the time point interval at which you want to acquire images with the channel For example if you want to use the channel only every third time point type 3 in this field Keep in mind that the first time point in a time series is always zero 0 The next steps that you take depend on whet
119. efix Prefix or base name of new datasets Opening an Experiment Only one experiment may be open at a time If an experiment is open when you open a second experiment the first experiment automatically closes and the second experiment opens To open an experiment 1 From the Application menu select Open Experiment 2 Inthe Open dialog box select the experiment mexp file and then click Open Ge The Application menu lists experiments that were recently open Select an experiment in the list to open it You can change the folder that the MetaMorph NX Software opens to look for experiments by modifying the default path in the administration settings See Creating a New Experiment Linking an Image to an Experiment To view an image that is not part of an experiment you can link the image to the experiment If an experiment is not open when you link an image the MetaMorph NX Software creates a new experiment to contain the image in this case the name of the new experiment that is created is based on experiment name prefix that is specified in the experiment configuration settings See the MetaMorph NX Microscopy Automation amp Image Analysis Software System Administrator Guide for information about how to change the name prefix that is used for new experiments Each image that you link to an experiment is displayed as a separate dataset in the filmstrip However the image file is not moved to or saved in the experiment folder
120. eld To show grid lines or a legend select the Show Grid Lines or Show Legend check box Select a background color for the graph Select the color style and size of the marker used for data points Click X Axis and in the Display field select the position of the axis on the graph Oo N QA Click Range and either select the Auto Scale check box to include all values for the measurement on the axis or clear the check box and type the minimum and maximum values for the axis 10 Click Axis Label and to display an axis label select the Show Label check box then select 156 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide either a Column Name as the axis label or select Custom and type a label in the Text field 11 To display tick labels on the axis select the Show Tick Labels check box 12 Click Y Axis and repeat Steps 8 through 11 for the Y axis The last graph style that you used before closing an experiment becomes the default style for the first graph that you create when you open the same experiment again or when you open another experiment To change the graph type 1 Select the open graph that you want to convert 2 On the Graph tab in Graph Types click Convert to Line Graph or Convert to Scatter Plot Note Line graphs always display time measurements along the X axis if the X axis on a scatter plot is not a time measurement the X axis will change to a time measureme
121. ensional Tab Acquisition Dialog Box Channel Settings Channel Settings Illumination setting Illumination setting Exposure time Exposure time All settings that you selected to Auto expose settings if applicable define per channel in the Acquisition dialog box Camera specific settings such as gain Center Z image acquisition if applicable Z offset distance between channels Frequency and rate of channel acquisition Prerequisites Before you configure a channel for an acquisition determine if you plan to use an illumination setting See Creating Illumination Settings To configure a channel 1 On the Acquisition Mode tab select Multidimensional select the camera channel and optionally select a hardware setting if one is needed to set the microscope devices to pre defined positions You will select magnification and illumination settings as described below 2 Onthe Mode Multidimensional tab in Magnification select the magnification you want to use for the acquisition 3 Onthe Mode Multidimensional tab click Configure to open the dialog box Gp You can also add remove and configure channels using the options that appear when you select a channel in the Channel Series group on the Mode Multidimensional tab All settings that you select to define per channel in Step 19 also appear in the options in the Channel Series group Changes that you make to channel settings on the Mode 5011685 B 79 Chapter 7
122. er to display the related fields Ge You can also use the options in the Focus group on the Mode Interactive tab to configure 5011685 B 10 MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide continuous focus for the Nikon PFS The changes that you make on the tab are reflected in the Acquisition dialog box and the changes that you make in the dialog box are reflected on the tab Click Start Continuous Focus Optionally slightly adjust the focus by using the knob on the Nikon PFS controller The Focus Adjustment field displays the surface to focal plane distance sometimes called the offset Note You cannot adjust the Z motor position using the microscope controls or change objectives while continuous focus is on Once you have configured all of the settings for the interactive acquisition click Acquire The Nikon PFS maintains focus of the image throughout the image acquisition Click Stop Continuous Focus to turn off continuous focusing When continuous focusing is off you can control the Z motor using the knobs on the microscope To use the Nikon PFS for a multidimensional acquisition without a stage series 1 Configure the settings for the multidimensional acquisition at least those that affect Z motor position and illumination such as the channels and if applicable image autofocus and a Z series If your acquisition includes more than one channel on the Mode Multidimensional t
123. eries for acquisition 5 Inthe Stage X and Stage Y fields select the Stage X and Stage Y motors Note Although you must designate a stage motor to move along the X axis and another stage motor to move along the Y axis in almost all cases the X and Y motors belong to the same stage that is typical stage configurations do not include an X stage motor from one manufacturer and a Y motor from a different manufacturer Setting a Point of Origin You must be able to access the Hardware Setup tab to perform the tasks described in this section See your system administrator for more information A point of origin sets a specific X Y and Z position as a reference point When you set the point of origin the current X Y and Z positions are set to zero As you move the stage and Z motors you can use the point of origin to gauge the position of the motors relative to the point of origin If the stage you are using has a hardware specific Home position you should set the point of origin to that position Example If you set the point of origin at the center of a slide the X Y and Z positions at the center of the slide are set to zero Then suppose you move the stage so that the X and Y positions are 4000 and 5000 Using the point of origin as a reference you will know that the stage is 4 millimeters to the right of the center of the slide and 5 millimeters below the center of the slide To find the center of the slide again you can simply mov
124. es a flattened object and a value of 1 indicates a perfect circle Shape Factor 47A p Where P Perimeter and A Area Shape Factor Elliptical Form Factor The ratio of the object s breadth to its length Length Elliptical Form Factor Breadth Note Due to changes in the way that Length is measured this measurement is also calculated slightly differently in the MetaMorph Software version 7 7 and earlier See the description of Length above Intensity measurements Measurement Definition The average of the pixel intensity values contained in the object Integrated Intensity The sum of the intensity values for all pixels contained in the 136 5011685 B Average Intensity MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Measurement Definition Intensity Center X and Y The X and Y coordinates of the intensity weighted centroid of the object Centroid Geometric 7 center of Intensity object center of object The standard deviation around the mean of the average intensity value in the object Intensity Standard Deviation Note This measurement is calculated differently in the MetaMorph Software version 7 7 and earlier The MetaMorph NX Software uses a slightly more comprehensive calculation Maximum The maximum pixel intensity value in the object Minimum The minimum pixel intensity value in the object Statistical Su
125. et is used for a stage position Define the stage label Set the focus adjustment value for the stage position if you are using focus stabilization requires specific hardware Prerequisites Install and configure the appropriate hardware device driver for your stage as described in Installing and Loading Hardware Device Drivers Set the X and Y points of origin and if more than one stage is installed specify which stage should be used for image acquisition as described in Designating Z Motors and Stages for Acquisition To configure a stage series 1 On the Acquisition Mode tab select Multidimensional select the camera channel and optionally select a hardware setting if one is needed to set the microscope devices to pre defined positions 84 5011685 B 10 11 12 13 MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide On the Mode Multidimensional tab in Magnification select the magnification you want to use for the acquisition On the Mode Multidimensional tab click Configure to open the Acquisition dialog box Inthe Acquisition dialog box select the Stage Series check box to indicate that the acquisition will include a stage series and then click Stage Series to expand the list of settings If aZ motor is installed and you want to make sure that the objective has adequate clearance during stage movements in the Travel Offset field type the distance that the motor should
126. evel Note Image autofocus is different than focus stabilization Image autofocus helps compensate for sample drift by focusing directly on the sample Focus stabilization helps compensate for stage drift by maintaining the specified distance focus adjustment value between the slide and the focal plane If the sample drifts the focus adjustment value may not be accurate In this case image autofocus can re establish focus If you have focus stabilization hardware you can use both focus stabilization and image autofocus during an image acquisition Image autofocus finds the best focus image with as few test image acquisitions as possible When configuring image autofocus you specify the total distance range along the Z axis within which the Z motor should move while focusing on the sample You also specify the step size which is initially used to locate the sample and the accuracy distance which is the smallest step that the MetaMorph NX Software will use when narrowing its search along the Z axis for the best focus position The step size that you set will vary depending on the objective that you are using The larger the depth of field the larger the step Typically the accuracy smallest distance is approximately 10 25 of the step size For example 98 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Depth of field Then the accuracy fine distance in microns may be
127. example you could focus at every third time point If you selected EveryNthPoint in the previous step in the Autofocus Rate field type the time point interval at which focusing should occur for example type 3 to focus at every third time point Keep in mind that the first time point in a time series is always zero 0 Inthe Continuous Frequency field type the interval in seconds at which the Zeiss Definite Focus will re establish focus Click Start Continuous Focus Note You cannot adjust the Z motor position using the microscope controls or change objectives while continuous focus is on Once you have configured all of the settings for the multidimensional acquisition click Acquire The Zeiss Definite Focus re establishes focus of the image at the intervals you specified 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide If the acquisition includes a Z series MetaMorph NX turns off continuous focusing immediately before acquiring the Z series After the Z series is acquired MetaMorph NX turns continuous focusing back on 15 Click Stop Continuous Focus to tum off continuous focusing When continuous focusing is off you can control the Z motor using the knobs on the microscope Pausing or Stopping an Acquisition Pausing a multidimensional acquisition temporarily suspends the acquisition until you resume it For example you may want to pause the acquisition so that you can
128. fthe camera Open the Acquisition to use to acquire images tothe full chip area ofthe cameraor dialog boxto configure and adjust exposure tothe area displayed inthe Latest additional settings for time if needed Image tab set the area to full chip and the acquisition then zoom in on the image to display justthe area that you want to acquire 12 m ie Sagen 100 hutter Snap T Bec Live 7 clo 2 ke Save acquired 9 Save the acquired Specify the prefix Confi gure the settings for image autofocus and focus stabilization Acquire images according to the settings on the Acquire tab and in the Acquisition dialog box or Pause the acquisition once it begins Specify the dataset Save the acquired thatwill contain Clear the images images into a the images that displayed in the new dataset you save Latest Image tab Save as Individuals Savingto lt New gt images images into the accordingto current dataset the settings in the Save group New Clear Display Create anew to use for the dataset that datasetthat will will contain the contain images next images that you save that you save 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Measure Tab Open the dialog Select the measurement box for configuring setthat contains data Open a line graph the selected you want to configure Display the data ofthe to graph data of Export the data table Selecta measureme
129. g acquisition and set a point full chip and then zoom in onthe image to display justthe area that you wantto acquire 14 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Creating Shortcuts to Options Using the Quick Access Toolbar With the Quick Access Toolbar located above the Home tab you can quickly create and access shortcuts to the options on the ribbon that you use frequently You can add almost any ribbon item to the toolbar For example the toolbar below contains shortcuts to the Live Display Mouse Wheel Control Export to PowerPoint and View Mode options If an option on the ribbon provides a drop down list of choices the list is also available from the Quick Access Toolbar In the example above the down arrows next to the Mouse Wheel Control and View Mode options indicate that the list of choices associated with those options is available from the Quick Access Toolbar When you click on the shortcut in the toolbar the MetaMorph NX Software carries out the option as though you had selected the option on the ribbon For example if you add a shortcut for the Snap Image option to the toolbar when you click on the shortcut the MetaMorph NX Software snaps an image Note Ribbon items that are specific to an experiment for example channels will not appear on the toolbar when you open another experiment You may have to re create shortcuts each time you open an experiment dependi
130. ge to the original range of intensity values and adds 5 percent of the range below the lowest value and 5 percent of the range above the highest value To turn off the display of a channel in the image grid click Show Channel Then to turn on the display of the channel click Show Channel again To change the display color of a channel click the name of the channel color and select a new color from the list The color changes for display purposes only the MetaMorph NX Software does not save the new color with the image or change any information about the wavelength that was used to acquire the image Note When you change channel display colors only the channels in the current selected dataset change colors The changes do not affect the display of channels in other open datasets 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Zooming in on Images You can use the wheel on your mouse to zoom in on or out of images in the image grid snapped images the live display or the filmstrip You can use the zoom options on the Home tab to change the zoom level of images displayed in the selected tab the zoom options on the Home tab do not control the zoom level of thumbnail images in the filmstrip To use the wheel on your mouse place the mouse cursor anywhere in the image grid Latest Image tab Live Display tab or filmstrip and scroll the wheel up to zoom in on images then scroll the wheel
131. guration 5011685 B 37 Chapter 4 Configuring a Keypad Keyboard or Mouse 10 11 12 13 14 If the input accessory is not included in the list make sure that the input accessory is connected properly to your computer and click Refresh to update the list Click OK Inthe Input Accessory Setup dialog box the status for the accessory that you selected changes from Unavailable to AWailable If you are configuring a keypad or keyboard click Edit Mode If you are configuring a mouse proceed to Step 13 On the keyboard or keypad press any key that you want to assign a command to A new row for the key that you pressed appears in the Keypad Button Commands section of the dialog box Inthe Command column select a command for the key If an additional parameter is needed for the command enter the appropriate value in the field that appears in the Parameters column For example if you assign the Select Channel command to a key the Channel field appears in the Parameters column Note While in Edit Mode you cannot use the input accessory to carry out the assigned commands To use an assigned command click the Edit Mode button to exit Edit Mode Then after you have used the command click the Edit Mode button again to continue configuring the input accessory Repeat Steps 8 10 for all keys that you want to assign commands to for the selected input accessory Click Edit Mode to exit Edit Mode which enables th
132. he following a dataset If the dataset exists does not yet exist Then in the Saving to field select the dataset that you want to save the image to If the number of channels binning camera area or size of the image is different than the images already in the selected dataset a new dataset will be automatically created for the image that you saved The MetaMorph NX Software names the new dataset by appending a number to the prefix displayed in the Name Prefix field click New Dataset and in the Name Prefix field type the prefix to use for the dataset that will be created for the image The MetaMorph NX Software appends a number to the prefix and increments the number by one each time you save an image For example if the name prefix is DatasetFLUOR the dataset that is created when you first save an image will be DatasetFLUOR1 and then when you save another image if you do not change the prefix the dataset that is created will be DatasetFLUOR2 and so on Once you create a dataset you can add images to it by selecting the dataset in the Saving to field as described above In the filmstrip double click the image or images that you want to save and then click Save Click Clear Display on the Mode Interactive tab to clear the images in the Latest Image tab and remove the orange border around selected images This option is especially useful if you have already selected multiple images in the films
133. he measurement set as needed You may want to resize or move the graph to another place in the Dataset View so that it takes up less space or so that you can compare it to the data table or another graph See Rearranging Dataset Views To filter data for a graph 1 On the Measure tab select the measurement set that contains the data that you want to graph 2 Onthe Measure tab click Configure Results 3 Inthe Configure Results dialog box click Filters 4 Inthe Filters section click Add Filter 5 Inthe Filter Type field Select To MinFilter include all values of the measurement that are more than or above the minimum value that you specify MaxFilter include all values of the measurement that are less than or below the maximum value that you specify RangeFilter include all values of the measurement between the minimum and maximum values that you specify MatchFilter include all values of the measurement that are the same as the value that you specify 6 Select the Use Inclusive check box to include the values that you specified in the filter criteria 5011685 B 155 Chapter 8 Measuring and Analyzing Images in Step 5 For example if you select this check box and type 30 in the Min Value field and 50 in the Max Value field the filter includes all values between 30 and 50 as well as the values 30 and 50 if you do not select this check box the filter includes all values between 30 and 50 but excludes the values 30 a
134. her you want to use the displayed value of a camera setting for all channels or if you want to set a specific camera setting for each channel For example you may want to use the same shutter state for all channels but use a different gain for each channel If you are using just one channel for your acquisition you can leave all of the check boxes cleared and type or select the values in this list in this case there is no need to set the values for each channel 18 19 82 Click Camera Settings to expand the list of camera settings The units of measurement for example ms or sec that are shown for each camera setting reflect the range that the camera supports Note This list of camera settings shows only the settings that can be changed and they are specific to the camera that is active and the acquisition that you are configuring See Camera Settings For each camera setting decide if you want to set a value for all channels or set a specific value for each channel To use Then leave the check box next to the camera setting cleared and type or select a value in the field next to the camera setting one value for all channels a specific value in each channel select the check box next to the camera setting a field for the camera setting that you selected is added to each channel in the list of channel settings Note The binning value applies to all channels and cannot be set per channel 5011685 B
135. ibed in this section See your system administrator for more information The MetaMorph NX Software provides control of automated microscopes motorized filter wheels shutters monochromators Z motors motorized stages digital and serial input output to communicate with unique devices and robotic devices from major manufacturers Setting up the MetaMorph NX Software so that it can be used with connected hardware devices involves two required tasks and a few optional tasks First you must install the device drivers to make them available for use with the MetaMorph NX Software Then you load the device drivers and related components that you want to use As you carry out these two required tasks you can also configure the device drivers and components For example you may want to rename a device driver or modify the settings of a component After you have installed and loaded the hardware device drivers you can create hardware settings illumination settings and magnification settings The settings that you create appear on the acquisition tabs where they can be selected for use with image acquisitions 45 Chapter 6 Installing and Configuring Hardware The flowchart below shows the general sequence of tasks for installing and configuring hardware device drivers and related components Required Preparing to Install Hardware Device Drivers You must be able to access the Hardware Setup tab to perform the tasks
136. ically saved as you work when you close an experiment it is automatically saved To create a new experiment e Tocreate anew experiment in the default location using the default base name from the Application menu select New and then select New again Anempty Dataset View appears and a folder for the new experiment is created in the default location For example if the experiment base name is Experiment the new experiment folder may be named Experiment1 To change the experiment default settings 1 To change the location where new experiments are saved and optionally change the base name for new experiment folders from the Application menu select New and then select Set Experiment Defaults 2 Change the locations and the prefixes for the experiment and dataset folders as needed 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Field Description Experiment Load Path Folder that opens when you select Open on the Application menu Experiment Save Path Location where new experiment folders are saved including all of the image data for images in the experiment be sure that this location has adequate disk space Molecular Devices recommends that you save experiments on a data drive rather than the C drive Experiment Name Prefix Prefix or base name of new experiments Image Link Path Folder that opens when you select Link Image on the Application menu Dataset Name Pr
137. ication settings as you need The components that are available depend on what hardware devices are connected and configured for use with the MetaMorph NX Software In the Magnification Settings dialog box the components and settings listed first are those that the MetaMorph NX Software considers most relevant to magnification At the bottom of the list are other components the settings for which can also be defined and saved as part of the magnification setting Prerequisites Before you can create a magnification setting the appropriate hardware devices and components must be installed and loaded See Installing and Loading Hardware Device Drivers To create or modify a magnification setting 1 On the Hardware Setup tab in Manage Settings click Magnification Settings 2 Inthe Magnification Settings dialog box do one of the following 5011685 B 57 Chapter 6 Installing and Configuring Hardware e Tocreate anew setting click Add New Setting and type a name for the setting in the blank field that appears in the list of settings e Tomodify asetting click the name of the setting e Torename a setting double click the name of the setting and type a new name 3 Ifthe objectives are controlled manually select the Manual Objective check box If the objectives are controlled manually you must supply the numerical aperture and refractive index values as described in Steps 4 and 5 If the Software controls the objectives then the Me
138. ice Drivers A CAUTION Be aware of the distance between the microscope objective and your slide Specifying a large Z axis range may result in damage to your specimen and microscope In the Autofocus Accuracy field type the smallest distance that the Z motor should move to refine its search for the best focus image This value is usually based on the depth of field and the numerical aperture of the objective Inthe Autofocus Step Size field type the step size a larger distance than the accuracy distance between the initial focus test images that will be acquired The step size is based on the field of depth of the objective and sample signal strength and is used to locate the strongest intensity signal from the sample If the step size is too small extra images will be collected during autofocus and signal noise may be misinterpreted as the focus peak If the step size is too large the focus position may be inaccurate Generally you should set a step size that may be too small rather than too large if the step size is too large the autofocus algorithm may not be able to locate the sample 5011685 B 101 Chapter 7 Acquiring Images 102 10 11 In the Autofocus Algorithm field select an algorithm Algorithm Description Brenner Standard Recommended for most imaging conditions and works well for a number of different microscopy techniques Measures contrast in the image Directional Average Recommended generally
139. ich the image was StagePositionY acquired StageLabel A label that stores stage position information at the time the image was acquired AbsoluteZPosition The exact position of the Z motor at the time the image was acquired If no Z motor was used during acquisition this row states not recorded Wavelength The value of the wavelength used to acquire the image CameraBinningX The number of pixels along the X axis that were binned when the image was acquired CameraBinningY The number of pixels along the Y axis that were binned when the image was acquired CameraChipOffsetX The X coordinate of the upper left corner of the image at the time it was acquired CameraChipOffsetY The Y coordinate of the upper left corner of the image at the time it was acquired Keep in mind that the MetaMorph NX Software starts numbering at zero 0 5011685 B 129 Chapter 8 Measuring and Analyzing Images Measuring Morphometry of Objects Measure Using Threshold The Measure Using Threshold measurement function measures properties of objects in an image of any bit depth Measurements are made using a single channel The first step in object analysis is to threshold the image to separate or segment the objects from their background Because this method requires that the intensity values of pixels in an object fall within a discrete range certain types of images are not suitable for automatic object analysis such as those acquired
140. icular channels only occasionally In this case the display fills in missing images by duplicating an existing image from the same channel The duplicate images appear in the image grid and are included in all measurements The number of columns and rows shown next to the View Mode on the Home tab determines the number of images that appear in the image grid The numbers that you specify reflect the number of images in the viewable area of the image grid you may need to scroll to view images that are outside of the image grid For example if the columns are set to 3 and the rows are set to 3 but your dataset contains more than 9 images you must scroll to view all of the images Some view modes may not be useful if your dataset does not contain one of the dimensions in the mode For example if your dataset does not contain a Z series the Time vs Z Channel vs Z and the Multi Z modes are not as useful as the other modes 22 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide To change the display color of a channel When viewing images you can choose the color that represents a specific channel in a dataset On the Home tab in the Channel Display group the color that represents a specific channel is shown in the icon for the channel For example EN E E E DIC Redi Green2 Channel Display Channel Display To assign a new color to a channel click the channel and then click the arrow next
141. ightly depending on the type of hardware that you are using Description Setting Objective Labels for each objective Refraction Medium Index The refraction medium for each objective The Index field displays the index value for the selected medium Air 1 0 Water 1 33 Oil 1 515 Num Aperture Labels for the numerical aperture of each objective Open Control Dialog Opens the Control Objective dialog box which provides the following options for testing the movement of the objective Position Controls Moves the device to the selected position lt lt Moves the device backward by one position gt gt Moves the device forward by one position Shutter Settings The shutter and lamp settings that are available may vary slightly depending on the type of hardware that you are using Setting Descripton Delays Open Delay Delay in milliseconds between the time the MetaMorph NX Software sends the shutter the command to open and the time exposure starts and the image is acquired This 52 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Setting Descripton delay is needed to ensure that the shutter has time to open before acquisition See illustration below Note When using an automated microscope the transmitted light may not be shuttered but may be turned on or off to simulate a shutter In this case the Open Delay setting must b
142. illumination settings see Creating Illumination Settings Touse auto expose for the channel select the Auto Expose check box and follow Steps 9 13 If you are not using auto expose clear the check box and proceed to Step 14 Note If your sample is light sensitive you may want to minimize the number of auto exposures Inthe Auto Expose Frequency field Select To auto expose Every Point at every time point auto exposing at every time point will slow down acquisition and will increase the sample s exposure to light First at only the first time point auto exposing at the first time point will ensure consistent exposure times for the channel throughout the time lapse Every Nth Point at specific time intervals Specified in the next step for 5011685 B 10 11 12 13 14 15 MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide To auto expose example you could auto expose at every tenth time point to accommodate photo bleaching If the acquisition does not include a time series auto expose will occur at the first time point If you selected EveryNthPoint in the previous step in the Auto Expose Rate field type the time point interval at which auto exposing should occur for example type 3 to auto expose at every third time point Keep in mind that the first time point in a time series is always 0 zero In the Target Intensity field type the maximum
143. ime you scroll the mouse wheel For example if the spin unit for the Z motor is 2 the Z motor position will move 2 units at a time when you scroll the mouse wheel for example from 10 to 12 from 12 to 14 from 14 to 16 and so on To use the wheel control 1 If you have access to the Hardware Setup tab in Mouse Wheel click Wheel Control to display the list of devices that can be moved with the mouse wheel Or if the Quick Access Toolbar contains a shortcut to the wheel control click the down arrow next to the mouse icon Select the device that you want to move with the mouse wheel On the Hardware Setup tab or in the Quick Access Toolbar click the mouse icon to turn on the wheel control The mouse icon tums yellow to indicate that the wheel control is on Scroll the mouse wheel to move the device that you selected in Step 2 While the wheel control is on you cannot use the mouse wheel to zoom in on or zoom out of images in the image grid You can however perform other tasks in the MetaMorph NX Software Click the mouse icon again to turn off the wheel control Configuring an Input Accessory Such as a Keypad Keyboard or Mouse You can assign MetaMorph NX Software functions commands to keys wheels or buttons on a device that Microsoft Windows recognizes as a keyboard keypad or a mouse For example you could assign the Snap command to the S key on a keyboard so that every time you press the S key animage is s
144. ing one channel Zo oe OCS OC Zo aa aE Saas Oe lt EH _ EH Keep in mind that in a time lapse experiment if you configure the acquisition to acquire additional images it will take longer to acquire a single time point As a result images may not be acquired at the specified interval While configuring the acquisition you can test your current settings by snapping a single image or by displaying a live view Snapped images are saved in the Scratch folder on the hard drive of your computer See Snapping an Image and Displaying a Live View When you start the multidimensional acquisition the MetaMorph NX Software saves the acquired images into a new dataset Unless you specify that images should not be displayed as they are acquired the Latest Image tab continuously updates the display of the most recently acquired image This display helps you gauge the quality of the images as they are acquired which can be especially 76 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide useful for lengthy time series acquisitions The Dataset tab updates the image display after images are acquired at each time point As images are being acquired you can scale the images to adjust the contrast of a channel or channels See Scaling Images Also at any time you can rearrange the display of the images in the Dataset tab See Viewing Images in a Dataset Note If an experiment is not open when
145. ingle of images in the Scale the channels using one of Zoominonoroutof image wavelengths image gid by three modes reset scaling to display Arrange the image images finduding are displayed as dimension for the original range of intensity values grid and dialog boxes Live Display composite ovenays example time points for each channel in the image inthe default layout Channel Display S Display imagesin Display images in Display images in Export the images Doubledick a channel to the image grid the image gid the image gidinthe exadiy as displayed show or hide the channel that belong to the that were seleded numberof inthe image gid to in the image display selected dataset acquired at the rows and colurns PowerPoint selected stage Acquisition Mode Tab Save the settings forthe current Import load acquisition to an Selectthe camera an aqquisition acquisition channel to use for Protocol file protocol file acquisition Hardware Setup Camera Channel Acquisition Mode Acquire images at Acquire images at Select a hardware one or more time one time point setting and move points stage stage position and Z devices to the positions orZ step using one or positions spedfied steps using one or more channels inthe seting more channels 5011685 B 11 Chapter 1 Navigating the Workspace Mode Multidimensional Tab Display a live view of the image or snap a single image using the spedfied binning and selected channel
146. intensity value that auto exposure should attempt to attain for the brightest pixel in the image The default target intensity value is 75 of the maximum intensity value that the camera driver is able to obtain The target intensity will determine the exposure time during auto expose In the Initial Exposure field type the length of the first exposure attempt Type an exposure time that is well below the typical exposure time required for this type of experiment generally the initial exposure time should be approximately one tenth of the typical exposure time If you do not know the required exposure time type 1 or 5 or use the default value Inthe Maximum Exposure field type the longest exposure time that you want to allow Be careful not to over expose your sample which may result in bleaching photo damage or photo toxicity Select Center Z Only if you have configured a Z series but you want to acquire only one image at the center Z position using this channel If you have configured a Z series and you want to acquire the entire Z series using the channel leave the Center Z Only check box unselected In the Z Offset field specify the difference between the focus position of the Z motor in this channel and the focus position of the Z motor in the first channel If the acquisition includes a Z series the entire Z series will be shifted by the Z offset distance Note The Z Offset field is not available for the first channel that you d
147. ition Perimeter The distance around the edge of the region measuring from the mid points of each pixel that defines its border If the region is a line the perimeter is the Line Length multiplied by two 5011685 B 143 Chapter 8 Measuring and Analyzing Images Measurement Definition Centroid X and Y in calibrated units The X and Y coordinates of the centroid of the region The centroid is the center of mass of the region ignoring pixel intensities Depending on the shape of the region the centroid may not be inside the region for example if the region is concave Centroid X and Y in pixel units The X and Y coordinates of the centroid of the region expressed in pixels If the calibrated unit is 1 this measurement is the same as the Centroid X and Y in calibrated units measurement 144 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Measurement Definition The angle between the longest chord through the region and the horizontal axis The range is 90 to 90 degrees Orientation Angles are measured from the three o clock East position and range from 0 to 180 degrees Downward angles are expressed as positive numbers and upward angles are expressed as negative numbers 90 Note In the MetaMorph Software version 7 7 and earlier in the Integrated Morphometry Analysis application module down
148. ition Using the top and bottom Z positions calculate the center Z position You can also use image autofocus to determine the center Z position When images are being acquired the value of the Current Z Position field will update to reflect the current position of the Z motor Once the acquisition is complete the Z motor will return to its initial position Note If the acquisition includes a stage series the Z position that is defined for each stage position is used as the center Z position for the Z series The center Z position may vary between stage positions See Configuring Stage Positions for Acquisition Select the Loop Within Channel check box to acquire a Z series for one channel at a time or clear the check box to acquire all channels at a specific Z position before all channels are acquired at the next Z position In the Z Step Size field type the distance along the Z axis between each image that will be acquired The step size is usually based on the depth of field of the objective Note The units of measurement for the Z axis positions are based on the Z motor configuration For information about device configuration see Installing and Loading Hardware Device Drivers In the Z Range field type the total distance along the Z axis within which all of the Z images will be acquired The Z range value always equals the number of steps minus one multiplied by the step size amp You do not necessarily need to know th
149. le Channel 2 Focal plane gt lt gt 500 Z position for Channel 1 Z AXIS The next steps that you take depend on whether you want to use the displayed value of a camera setting for all channels or if you want to set a specific camera setting for each channel For example you may want to use the same shutter state for all channels but use a different gain for each channel If you are using just one channel for your acquisition you can leave all of the check boxes cleared and type or select the values in this list in this case there is no need to set the values for each channel 13 Click Camera Settings to expand the list of camera settings The units of measurement that are shown for each camera setting reflect the range that the camera supports 5011685 B 71 Chapter 7 Acquiring Images Note This list of camera settings shows only the settings that can be changed and they are specific to the camera that is active and the acquisition that you are configuring See Camera Settings 14 For each camera setting decide if you want to set a value for all channels or set a specific value for each channel To use Then leave the check box next to the camera setting cleared and type or select a value in the field next to the camera setting one value for all channels select the check box next to the camera setting a field for the camera setting that you selected is added to each channel in the
150. le configuring an image acquisition 95 Snapping an Image 22 2 e cece cece cece eee eeeeeeeeeteeeeeees 95 To snap an image while configuring hardware settings 220222222 22200000008 96 To snap an image while in Multidimensional acquisition mode 96 Using Auto Expose 2 22000 ose ee ecnetdeonl lool ills EAN SANESA ieee lcs 2d dceeaseegentelidscs 97 To use auto expose before acquiring animage 222222222 eee eee 97 Configuring Image Autofocus 2 22 22 222 ee 98 PrereguisiteS 2 ace es ate eee sie Oe ce eee a eee eae es 99 To configure image autofocus before acquiring an image 222222 2 22 100 To use image autofocus in a multidimensional acquisition 2222222222 2 22 101 To use image autofocus in an interactive acquisition 0 20022222222 22222 eee eee 103 Using Focus Stabilization 00 eee cece cece ee cecceeccececcccceccceccccecceceeeeeee 105 Nikon Perfect FOCUS 2 22 2 22 c cece e cece cece cece cece cece ccc e cece cece ee eeeeeeeeeeenes 106 PPROPOQUISINOS pcan eels ata eee ae ee eee ee BS eee ee 106 To use the Nikon PFS for an interactive acquisition 2222222 222222 106 To use the Nikon PFS for a multidimensional acquisition without a stage series 107 To use the Nikon PFS for a multidimensional acquisition with a stage series 108
151. le of the Dataset View or workspace Place the window on an arrow or in the part of the target that points to the side of the Dataset View or workspace that you want to dock the window to To hide a window Some windows such as the filmstrip can be hidden and then displayed again later 1 Right click on the tab name or title bar of the window and select Hide 2 Todisplay the window again right click and select Add window name To auto hide a dialog box The Auto Hide feature minimizes a dialog box and then provides easy access to it when you move your cursor over the minimized dialog box label Once the dialog box is open again it automatically minimizes when you move your cursor outside of the dialog box 1 Right click on the title bar of the dialog box and select Auto Hide The dialog box closes and a vertical minimized dialog box label appears on the left or right side of the workspace To display the dialog box again move the cursor over the minimized dialog box label The dialog box opens and the minimized dialog box label remains on the left or right side of the workspace To hide the dialog box again move the cursor anywhere outside of the dialog box Ge If the dialog box does not hide click the cursor anywhere outside of the dialog box To close a tabbed window or close all tabbed windows except the current one 26 e Right click on the tab name and select Close or Close All But This 5011685 B Me
152. lenge during time lapse experiments especially when using objectives with a narrow depth of focus or when the acquisition involves a complex sequence of stage and Z positions Focus stabilization is a laser based method for ensuring that focus is maintained throughout image acquisition Because no test images are acquired focus stabilization does not cause bleaching or photo toxicity Although the design of each focus stabilization system is unique to its manufacturer all systems use a laser diode to find either the bottom or the top of the slide or dish Once you focus on the sample the system calculates the distance called the focus adjustment value from the slide or dish to the focal plane During image acquisition the focus stabilization system maintains that distance to help compensate for stage drift caused by vibration temperature changes and addition of media Note Focus stabilization is different than image autofocus Image autofocus helps compensate for sample drift by focusing directly on the sample Focus stabilization helps compensate for stage drift by maintaining the specified distance focus adjustment value between the slide and the focal plane If the sample drifts the position of the focal plane may change In this case image autofocus can re establish focus If you have focus stabilization hardware you can use both focus stabilization and image autofocus during an image acquisition The focus stabilization system tra
153. lick Measure The MetaMorph NX Software creates a segmentation of the image measures the objects in the image and stores the measurement results in anew measurement set The progress bar at the bottom of the workspace shows the status of the measurement Once the measurement is finished the measurement set appears at the bottom of the list of measurement sets on the Measure tab Note To cancel a measurement click anywhere in the progress bar at the bottom of the workspace and then click the Cancel button that appears The data that was calculated before you canceled the measurement is stored ina new measurement set 5011685 B 131 Chapter 8 Measuring and Analyzing Images 11 12 13 14 15 16 To display the segmentation of the image on the Measure tab select the measurement set To display the measurement results click Show Table See Displaying Measurement Results in a Table To display the statistical summary of all measurements in the measurement set click Show Summary If the data table is already open the summary appears at the bottom of the table If the data table is not open the data table opens and the summary appears at the bottom of the table To hide the statistical summary click Show Summary again To display the original source image on the Measure tab select Plane Information To delete the measurement set in the list of measurement sets click the X that is next to the measurement set If
154. lways 0 zero 13 Click Start Continuous Focus 14 Once you have configured all of the settings for the multidimensional acquisition click Acquire At each stage position the MetaMorph NX Software waits for the Nikon PFS to re establish focus before acquiring the image If the acquisition includes a Z series the MetaMorph NX Software tums off continuous focusing immediately before acquiring the Z series so that the Z motor can move to the center of the Z series After the Z series is acquired the MetaMorph NX Software turns continuous focusing back on 15 Click Stop Continuous Focus to tum off continuous focusing 5011685 B 109 Chapter 7 Acquiring Images When continuous focusing is off you can control the Z motor using the knobs on the microscope Olympus ZDC and ZDC2 The Olympus ZDC and ZDC2 focus stabilization systems provide both one shot focus and continuous focus options During a multidimensional acquisition the ZDC and ZDC2 use one shot focus cycles to maintain multiple focus positions for example acquisitions with either multiple stage positions or a Z series Continuous focus may be used during a long simple time lapse acquisition an acquisition with multiple time points but no stage series to maintain a single focus position Consult the Olympus documentation for information about the types of slides dishes and objectives that work best with the ZDC and ZDC2 Note The search range along the Z axis and s
155. m can find the dim site The step size may be too small in which case the autofocus system may not be able to accurately distinguish noise from intensity signals from the sample as a result the autofocus system may focus on something other than the sample The range may be too small You may need to increase the range so that the autofocus system can find the sample An exposure time may be too long causing the autofocus camera to become saturated and unable to differentiate intensity signals from the sample You may need to reduce the exposure time or change camera settings You may need to use the other autofocus algorithm To use image autofocus in a multidimensional acquisition On the Mode Multidimensional tab click Configure In the Acquisition dialog box select the Autofocus check box to indicate that the acquisition should use autofocus and then click Autofocus to expand the list of autofocus settings If you are including both image autofocus and a Z series in the acquisition the MetaMorph NX Software will first autofocus using the center Z position and then acquire a Z series of images The new autofocus position will be used as the center of the Z series Inthe Autofocus Range field type the total distance range along the Z axis that the Z motor will move while acquiring focus test images The units of measurement for the Z axis positions are based on the Z motor configuration See Installing and Loading Hardware Dev
156. may be measured at a time Although you cannot measure across datasets you can make as many measurements as needed on the images in a dataset and you can make more than one measurement at a time that is you can start a measurement and then start another measurement while the first measurement is still running 125 Chapter 8 Measuring and Analyzing Images A CAUTION If you delete images from a dataset on the hard drive of your computer the MetaMorph NX Software will not be able to make measurements on that dataset Each time you make a measurement the MetaMorph NX Software stores the results as a measurement set and saves it with the dataset All of the measurement sets in a dataset are listed in the Measurement Set group on the Measure tab Note Measurement sets are saved with a dataset as part of the database file and may be viewed only when the dataset is open in the MetaMorph NX Software measurement sets are not saved as individual files in the dataset folder See About Experiment and Dataset Folders You can view measurement results e On a segmentation of the image that shows how the measurement settings identified objects e Ina table that shows the data for each measured object or that shows a statistical summary of the data collected from all of the objects e Ina line graph that shows how a region measurement such as intensity changed over time e Ina scatter plot graph that shows the relationship between any two measu
157. mes that images will be acquired in this time series 8 To add another time segment click Add and repeat Steps 6 and 7 9 Optionally configure other parts of the acquisition before acquiring images See Configuring Channels for Acquisition Configuring a Stage Series Acquisition Configuring Image Autofocus Using Focus Stabilization or Configuring a Z Series for Acquisition 10 Select the Do Not Update Display check box to indicate that images should not be displayed as they are acquired in this case the new dataset is not added to the experiment until the acquisition is complete or clear the check box to indicate that the Latest Image tab should display the most recently acquired image and that the images should appear in a new Dataset tab as they are acquired 11 When you have configured the acquisition click Acquire If the Do Not Update Display check box is cleared the MetaMorph NX Software opens the Latest Image tab which displays the most recently acquired image This image helps you gauge the quality of the images as they are acquired which can be especially useful for lengthy time series acquisitions During acquisition the MetaMorph NX Software saves the acquired images into a new dataset You can review images as they are acquired by displaying the Dataset tab See Viewing Images in a Dataset for more information Note If an experiment is not open when you start an acquisition the MetaMorph NX Software will create o
158. mmary measurements Measurement Definition The total number of objects measured Minimum The minimum value of the measurement that was made Maximum The maximum value of the measurement that was made Total The sum of the measurements for a specific parameter selected to be measured For example if you are measuring width the total value is the sum of width measurements for all objects measured The sum of all measures divided by the number of measures The average is calculated by the formula Average 5011685 B 137 Chapter 8 Measuring and Analyzing Images gt x yee A where n number of measures Standard deviation A measure of the variability of a set of measurements around their average The standard deviation is defined as the positive square root of the variance Drawing and Measuring Regions Using the region drawing tools you can draw regions on images and then measure the area shape position dimension such as perimeter and length and intensity of the regions For example you may want to measure the intensity of a region on an image acquired with multiple channels over time then you can use the measurement results to create a line graph that compares the intensity values of the region across channels As you draw a region on an image the region is simultaneously drawn on all images For example You can measure the regions on just the images that are dis
159. mmary data for example total average and standard deviation e plane information for example image height and width plane information is also available as a separate measurement set If a graph for the measurement set is open when you click on a point in the graph the data table highlights the row that corresponds to the point and if the image is in view the image highlights the 5011685 B 149 Chapter 8 Measuring and Analyzing Images corresponding object Likewise when you select a row in the data table the graph highlights the point that corresponds to the row and the image highlights the corresponding object When you click on an object in an image the graph and table highlight the corresponding point and row To select more than one object row or point use lt CTRL gt click to select each item When you double click a point on a graph or a row in the data table the image display updates so that the corresponding object is in view This section describes how to e Open a data table for a measurement set e Sort data in a column by ascending or descending values in the data table e Include or exclude a column in the data table e Filter dataso that data is excluded from the data table Note The Configure Results option on the Measure tab provides options for configuring filter criteria for both the data table and graphs The data table and graphs associated with a measurement set share the same data and the same filter
160. mouse cursor anywhere in the filmstrip and use the wheel on your mouse to zoom in to and out of thumbnail images in the filmstrip You can also resize the filmstrip by dragging the top of the filmstrip window When the filmstrip is larger you can zoom in further on the thumbnail images in the filmstrip If you close the filmstrip you can display it again by right clicking anywhere in the image grid and selecting Add Filmstrip 18 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide To navigate between images in the image grid If a dataset contains images acquired at more than one time point or Z position scroll horizontally to navigate through time points and scroll vertically to navigate through Z positions Earlier time points are displayed on the left and later time points are displayed on the right Even if a dataset contains multiple channels or stage positions the image grid displays images acquired at time points from left to right and Z positions from top to bottom the only case when this is not true is when images are displayed using the Multi Z view mode as described in the next section m Time 0 2 of 0 3 Z02004 m Scroll horizontally to view time points v Scroll vertically to view Z positions 5011685 B 19 Chapter 2 Viewing Images To use the view modes to display and arrange multiple images or display a single image The view modes on the Home tab provide a quick way to
161. n click the Help icon and select Check for Updates Using this option you do not need to log in with your System ID to view the MetaMorph NX Software updates page e Visit www meta moleculardevices com updatesNX Using this option you must first log in with your System ID before you can view the MetaMorph NX Software updates page 165 Chapter 12 Microscopy and Image Processing Resources Web sites www microscopy fsu edu www modemmicroscopy com Journals Imaging amp Microscopy published by Wiley InterScience Journal of Microscopy published by Wiley Blackwell Journal of Microscopy and Analysis published by Wiley Books 3D Images of Material Structures Processing and Analysis Joachim Ohser and Katja Schladitz Wiley VCH 2009 Digital Image Processing A Practical Primer Gregory A Baxes Cascade Press 1988 Digital Image Processing Kenneth R Castleman Prentice Hall 1995 Digital Image Processing Third Edition Rafael C Gonzalez and Richard E Woods Prentice Hall 2007 Digital Microscopy Greenfield Sluder and David E Wolf Academic Press 2004 Fluorescence Microscopy Brian Herman and H J Tanke Springer Publishing 1998 Handbook of Biological Confocal Microscopy Second Edition James B Pawley Springer Publishing 2006 The Image Processing Handbook Sixth Edition John C Russ CRC Press 2006 167 Chapter 12 Microscopy and Image Processing Resources Light and Video Mi
162. n PFS controller The Focus Adjustment field displays the surface to focal plane distance sometimes called the offset Note You cannot adjust the Z motor position using the microscope controls or change objectives while continuous focus is on Once you have configured all of the settings for the multidimensional acquisition click Acquire The Nikon PFS maintains focus of the image throughout the image acquisition If the acquisition includes a Z series the MetaMorph NX Software tums off continuous focusing immediately before acquiring the Z series After the Z series is acquired the MetaMorph NX Software turns continuous focusing back on Click Stop Continuous Focus to turn off continuous focusing When continuous focusing is off you can control the Z motor using the knobs on the microscope To use the Nikon PFS for a multidimensional acquisition with a stage series 108 1 Configure the settings for the multidimensional acquisition including the stage positions and channels and if applicable image autofocus and a Z series If your acquisition includes more than one channel on the Mode Multidimensional tab select the first channel in the channel series 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Note The focus stabilization system uses the first channel to calculate the focus adjustment value Subsequent channels are offset from the first channel Click Live to
163. n a time period shorter than the interval for example if the time required for exposure and collecting a Z series is longer than the interval specified the acquisition will move to the next time point as soon as the current time point is completed That is the MetaMorph NX Software will acquire all required images at a time point even if the specified interval is not achieved As a result your dataset may not contain as many time points as you expected 7 Inthe Duration field type the total amount of time in which images should be acquired A change in this value automatically updates the number of time points The Duration field is unavailable if the interval is set to 0 Note The acquisition ends as soon as the duration ends even if the acquisition has not acquired images at all time points To extend the duration of an acquisition while images are being acquired pause the acquisition extend the duration and then resume the acquisition See Pausing or Stopping an Acquisition As you type values in the time series fields the MetaMorph NX Software uses those values to calculate other fields in the time series For example if you type 10 in the Interval field and then 40 in the Duration field the Number of Points field will display 5 Duration is calculated with a starting time point of zero 0 The duration equals the number of points minus one multiplied by the interval The Number of Points field indicates the total number of ti
164. n acquisition or when testing a microscope setup Note The shutter setting determines the state of the shutter when images are acquired The shutter setting also determines whether you can use the Shutter button on the ribbon to toggle the shutter open and closed To open or close a shutter using the Shutter button on the ribbon the shutter state in the selected illumination setting must be set to active Active The shutter will open only during acquisition or when the Shutter button on the ribbon is used to open and close all active shutters in the illumination setting Open The shutter will open when the illumination setting is selected and will stay open until the illumination setting is changed Use this setting if you are using the eye pieces Closed The shutter will remain closed during acquisition and while the illumination setting is selected use this setting if you are using transmitted light and do not want any fluorescent excitation or fluorescent light and do not want any transmitted light Best Practice Change a setting for a component only if needed Often an illumination setting does not need to control the position of certain components For example a fluorescent setting does not require that the lamp voltage be adjusted for transmitted light illumination Setting the lamp voltage value in this case would serve no purpose and would waste acquisition time as the lamp adjusted To avoid this situation you would
165. n conditions channels Channels can accommodate brightfield fluorescence and other microscopy techniques such as differential interference contrast DIC and phase contrast For each channel you can set a variety of specific camera settings Images are acquired with each channel using auto expose or the exposure time that you specify You can configure as many channels as you need Note Interactive acquisition mode supports acquisition of images using multiple channels but it does not support automatic acquisition at multiple stage positions time points or Z steps To acquire images using one or more channels at multiple stage positions time points or Z steps use the Multidimensional acquisition mode Images acquired using the Interactive acquisition mode are not automatically saved You choose which images should be saved either individually or together in a dataset Datasets are saved in the open experiment Images that are snapped or acquired in Multidimensional acquisition mode cannot be saved in the same dataset as images that are acquired in Interactive acquisition mode The filmstrip displays a vertical green line between images that cannot be saved to the same dataset Plane information is available for an image that was acquired using the Interactive acquisition mode only after you have saved the image into a dataset After images are acquired you can view them as separate images using the individual channels or as composite im
166. name the installed device or a component in the list of installed devices select the device or component and type a new name To change the settings of the installed device or a component of a device in the list of installed devices select the device or component and click Settings In the Settings dialog box make changes and click OK See Device and Component Settings for specific settings information for the device or component Note You can change the settings for a device and remove a component from or add a component to a device only in this dialog box These are settings that connect the MetaMorph NX Software to the hardware through a serial port or manufacturer s driver Device settings cannot be changed and components cannot be removed or added when the device is loaded using the User Settings for Default hardware configuration dialog box However component settings can be changed in both this dialog box and in the User Default hardware configuration dialog box as described in Loading Device Drivers Installed Devices gee Mad City Labs Micropositioner fp MCL MicroDrive Xx Stage git MCL MicroDrive Y Stage fpr Olympus Filter Cube ith Olympus Excitation ait Olympus Emission ite Olympus ND ite Olympus Objective ite Olympus Condenser fit Olympus Top Lens ite Olympus Camera Port atin Olympus Confocal Disk Components Ww Inthe Install System Devices dialog box click Apply to sav
167. napped Note One keypad keyboard and one mouse can be configured as input accessories Do not use your primary system keyboard or your primary mouse as an input accessory If you do so you will not be able to use the keys or buttons to perform common tasks For example you may not be able to use the number keys to type a value such as a time series interval or exposure time If you accidentally configure a primary keyboard or mouse as an input accessory press the F12 key to change the status of the input accessory to Unavailable which will allow you to use the input accessory for commands other than the ones assigned to it 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide The Input Accessory Setup option is located on the Hardware Setup tab You may need a password to access the Hardware Setup tab or you may not be allowed to access it If you cannot access the Hardware Setup tab your system administrator may have placed a shortcut to the Input Accessory Setup option on the Quick Access Toolbar for your use A CAUTION If you have assigned commands to keys or buttons that you will use in another application be sure to close the MetaMorph NX Software before working in the other application While the MetaMorph NX Software is open commands that you have assigned to an input accessory will function even when you are working in another application For example suppose the Snap command is assigned to th
168. nd channels and if applicable image autofocus and a Z series If your acquisition includes more than one channel on the Mode Multidimensional tab select the first channel in the channel series 5011685 B 119 Chapter 7 Acquiring Images 120 10 11 12 13 14 Note The focus stabilization system uses the first channel to calculate the focus adjustment value Subsequent channels are offset from the first channel Click Live to display a live view of the image In Multidimensional Acquisition click Configure to open the Acquisition dialog box Inthe Acquisition dialog box select the Focus Stabilizer check box to indicate that the acquisition should use the focus stabilizer hardware The focus stabilizer options appear in the Stage Series section Inthe Stage Series section click Go To to move the stage to a stage position Use the focusing knobs on the microscope to bring the image into focus Inthe Stage Series section in the row that corresponds to the current stage position click Set Focus The Memorized field for the stage position displays Yes Repeat steps 6 8 for each stage position Inthe Focus Stabilizer section in the Autofocus Frequency field Select To have the Zeiss continuously focus Every Point at every time point focusing at every time point may slow down acquisition First at only the first time point Every Nth Point at specific time intervals Specified in the next step for
169. nd 50 To configure the graph style and range of data Use the options on the Graph tab to quickly change many settings of the open and selected graph such as the layout marker style and range of the X and Y axes data Use the Graph Settings dialog box to configure additional settings Graph Tab Options Graph Settings Dialog Box Options X and Y axes data and axes range for example auto scale or a defined range zoom in on and out of the range of data displayed or display full range of data and scroll the graph left right up or down X and Y axes data and axes range for example auto scale or a defined range Layout include or exclude grid lines Include or exclude grid lines title legend axes axes legend axes axes labels title labels and tick labels Marker shape size and color Additional marker shapes sizes and colors Options for the range of data displayed Background color Text for title and axes labels 1 Select the open graph that you want to configure 2 Onthe Graph tab in Graph Types click the dialog box launcher 8 in the lower right corner of the group G Right click anywhere on a graph and select Graph Settings to open the Graph Settings dialog box 3 Inthe Graph Settings dialog box confirm or select the measurement data to plot on the X and Y axes 4 Click Data Display and to display a title on the graph select the Show Title check box and type a title in the Title fi
170. ne to contain the new dataset 5011685 B 91 Chapter 7 Acquiring Images 92 A separate window within the Latest Image tab displays the status of the acquisition as well as a histogram for each channel The MetaMorph NX Software reports the number of images acquired as well as the current time point in a time series if applicable stage position if applicable channel and Z position if applicable Other statistics may also be reported to help you monitor progress Designating Z Motors and Stages for Acquisition Note If only one Z motor and one stage motor are installed you do not need to follow the procedure described in this section By default the MetaMorph NX Software will use the installed Z motor and stage motor You must be able to access the Hardware Setup tab to perform the tasks described in this section See your system administrator for more information If more than one Z motor is installed you must designate which Z motor should be used for image acquisition The MetaMorph NX Software supports the use of up to two Z motors for acquisition If you plan to use two Z motors you must designate which motor should be used for acquiring a Z series image autofocusing and focus stabilization and the travel offset distance Best Practice Designate the same Z motor for both autofocusing and acquiring a Z series If you must use different motors designate the motor that can travel the greatest range along the Z axis typicall
171. neral Workflow Before you measure images decide if you want to measure all or just some of the images in a dataset Only the images that are displayed in the image grid are measured unless you select one or more of the check boxes on the Measure tab to measure images acquired at all time points Z steps or stage positions The flowchart below shows the general sequence of tasks for measuring images Viewing Plane Information Every dataset has a Plane Information measurement set which contains basic data about the images in the dataset The Plane Information measurement set appears at the top of the list of measurement sets on the Measure tab As with any measurement set you can sort and filter the plane information to display only certain columns and data that meet your criteria When you measure the images in a dataset the plane information is included in the measurement data table along with the measurement results You can export plane information to a Microsoft Office Excel spreadsheet and you can include plane information data in a graph 5011685 B 127 Chapter 8 Measuring and Analyzing Images Note Plane information is available for images that have been saved into a dataset In Multidimensional acquisition mode images are automatically saved into a dataset In Interactive acquisition mode because images are not automatically saved plane information is available for an image only after you save the image into a dataset
172. nfo Dataset Each dataset has one Plane Information measurement set which contains basic The Info Dataset file can be opened image data such as the image width and e using the MetaMorph NX height camera bit depth and so on Software using version 7 7 of the MetaMorph Software Measurement sets if Measurements db When you measure the images ina applicable dataset using a measurement function The Measurements db file can be the results are automatically saved ina opened only using the MetaMorph new measurement set All measurement NX Software sets that belong to a dataset are stored in the Measurements db file Acquisition settings AcquisitionProtocol xml When you acquire images in used to acquire the Multidimensional acquisition mode images in the dataset The acquisition protocol file canbe acquisition settings are saved if applicable opened only using the MetaMorph automatically In Interactive acquisition NX Software mode you can manually save acquisition settings You can move or copy an acquisition protocol file to another computer running the MetaMorph NX Software and then acquire images using the protocol on that computer You can give this file any name as long as you keep the xml extension and the name is valid does not contain any special characters such as exclamation points By default the Scratch folder stores snapped images and images that were acquired using the Interactive acquisition mode
173. ng on the items that are in the toolbar This section describes how to e Add an item to the toolbar e Remove an item from the toolbar e Move the toolbar above or below the ribbon To add an item to the toolbar 1 On the ribbon right click the option or item that you want to add to the toolbar 2 Select Add to Quick Access Toolbar A small icon that represents the ribbon option or item appears in the toolbar To remove an item from the toolbar 1 On the Quick Access Toolbar right click the shortcut that you want to remove 2 Select Remove from Quick Access Toolbar 5011685 B 15 Chapter 1 Navigating the Workspace To move the toolbar above or below the ribbon 1 Click the down arrow on the Quick Access Toolbar to display its menu options 2 Select Show Below the Ribbon or Show Above the Ribbon Minimizing the Ribbon To create more space for a Dataset View you can minimize hide the ribbon so that just the tab names appear across the top of the MetaMorph NX Software workspace While the ribbon is minimized you can select a tab to display its options and you can navigate between tabs and options Then when you click inside the Dataset View the ribbon will minimize again This section describes how to e Minimize the ribbon e Re display the ribbon To minimize the ribbon 1 On the Quick Access Toolbar located just above the Home tab click the down arrow to display its menu options 2 Select Minimize
174. nification settings on the Hardware Setup tab in the Select Setting group The magnification setting is also included in the list of magnification settings available to use for image acquisition in the Magnification group on the Mode Multidimensional tab and Mode Interactive tab 15 To move the devices to the positions defined in the magnification setting on the Hardware Setup tab in Select Setting select the magnification setting Configuring Camera Drivers You must be able to access the Hardware Setup tab to perform the tasks described in this section See your system administrator for more information To use the MetaMorph NX Software with a connected digital camera or video camera you must first install the camera driver Software Then activate the camera driver as described in this section and optionally verify or change the camera settings Once a camera is activated the settings that the MetaMorph NX Software can control for that camera are available when you configure image acquisitions Camera settings can be set the same for all channels in an acquisition or set individually for each channel See Configuring Channels fora Multidimensional Acquisition or Acquiring Images in Interactive Mode Note Select the camera channel that you want to use for acquisition on the Acquisition Mode tab To install and configure a camera driver 1 On the Hardware Setup tab in Install and Configure Hardware click Camera Setup 2
175. nt if you convert the scatter plot to a line graph Adaptive Background Correction The Adaptive Background Correction system automatically corrects uneven image backgrounds throughout the image by adapting to local content This allows for more robust segmentation and analysis repeatability Uneven background levels that vary between images or within a single image are a common problem in automated fluorescence image analysis For example the following image has a significantly brighter background in the lower right quadrant than in the upper left quadrant 5011685 B 157 Chapter 8 Measuring and Analyzing Images As aresult of uneven background intensity thresholding cannot extract the cells for counting and measuring Too high a threshold misses cells in the dark background regions while too low a threshold incorrectly detects the bright background region as a large object In the following example the thresholding result is shown as a red graphic overlay on the original image The Adaptive Background Correction system automatically estimates and compensates for uneven background fluctuations resulting in better object segmentation for more accurate counting and measurements For example in the following image the segmentation result is shown as a red graphic overlay on the original image with far superior detection than the non adaptive thresholding method 158 5011685 B Chapter 9 Frequently Asked Questions
176. nt measurement viewin a table or plot selected measurement the selected as displayed to Excel function fundion ona graph setin a table measurement set and open Excel Script Hardware Setup er ane Information Ce conga Results Mode Interactive pe D All Time BE Daz sd i _ All Stage eee Show Addline Add Scatter Exportto Export to Summary Graph Pio Excel File M sasurement Display oos eae ie Selecta region Selecta check box to measure Measure the images Configure the data Display the Opena scatter plot Export the data table drawing tool images acquired at all time points using the selected of the selected summary data of to graph data of the as displayed to an all Z steps or all stage positions if measurement measurement set the selected selected Excel ortext file all check boxes are dear only the fundion measurement set measurement set images that are displayed in the in the data table image gid are measured Graph Tab The Graph tab appears only when you add a graph to a Dataset View Selecta graph type forthe current graph Acquisition Mode Mode Interactrve Measure script Hardware Setup X Avis Data ObjectID Y Axis Data Area am EJ re a lt 5 Minimum F Auto Scale Minimum WY Auto Scale e H H H e ConvertTo C tT i i PA Une Graph ttar Ding Maimum d P Maximum 1 YV z
177. nuous focusing should occur for example type 3 to focus at every third time point Keep in mind that the first time point in a time series is always 0 zero Inthe Continuous Frequency field type the interval in seconds at which the Zeiss Definite Focus will re establish focus On the Mode Multidimensional tab click Live to display a live view of the image Use the focusing knobs on the microscope to bring the image into focus In the Acquisition dialog box in the Focus Stabilizer section click Memorize Position Click Start Continuous Focus Note You cannot adjust the Z motor position using the microscope controls or change objectives while continuous focus is on Once you have configured all of the settings for the multidimensional acquisition click Acquire The Zeiss Definite Focus re establishes focus of the image at the intervals you specified If the acquisition includes a Z series the MetaMorph NX Software turns off continuous focusing immediately before acquiring the Z series After the Z series is acquired the MetaMorph NX Software turns continuous focusing back on Click Stop Continuous Focus to turn off continuous focusing When continuous focusing is off you can control the Z motor using the knobs on the microscope To use the Zeiss Definite Focus for a multidimensional image acquisition with a stage series 1 Configure the settings for the multidimensional acquisition including the stage positions a
178. of 43 Devices Installing 47 Loading 49 Moving with mouse wheel 35 Directional average algorithm 102 104 Displaying Images 17 24 32 Live View 94 Measurement results 149 153 Metadata on images 32 Docking windows 17 24 Duplicate images in image grid 159 Duration of time series 89 E Elliptical Form Factor measurement 136 145 Experiments Contents datasets images 39 169 Index 170 Creating 42 Linking an image to 43 Opening 43 Storage of 39 Exporting images and data 33 Exposure 78 95 97 F Fiber Breadth measurement 130 138 Fiber Length measurement 130 138 Filmstrip 9 Fixed scaling 27 Focus 92 98 105 106 110 117 Frequently Asked Questions 159 Full chip camera area 78 G Graph type converting 153 Graphs 153 H Hardware Configuring settings 53 55 57 Installing 47 Loading 49 Height measurement 130 138 Hole Area measurement 130 138 Illumination settings Configuring 55 Using in an acquisition 66 78 Image Information 32 127 Images Acquiring 66 Exporting 33 Linking 43 Measuring 125 Opening MetaMorph 7 7 images 43 Scaling 27 Snapping 95 Storage of 39 Viewing 17 Zooming 31 Importing linking an image 43 Input Accessory Setup 37 Integrated Intensity measurement 130 138 147 Intensity Above Local Background 147 Intensity Standard Deviation 130 138 Intensity values 130 138 147 Intensity target 78 97 Interactive image acquisition 69 Interval for time series 89 K Ke
179. olumn When specifying a defective column the X Y coordinates indicate the starting point for the column All pixels in the column from the X Y coordinate to the bottom of the chip are included Chip Location Indicates the X Y coordinates of the defective pixel or column s of pixels Defect Type Indicates the type of defect associated with the camera The defect type can be either for a single pixel or an entire column Column locations run from the specified pixel location to the bottom of the chip Width Indicates the number of columns affected by the column defect Remove Defect Deletes the selected defect from the list Remove All Deletes all defect information from the list Enable defect correction When selected pixel information for identified defective pixels is derived from adjacent good pixel information Demonstration Camera Driver The Demonstration camera driver emulates image acquisition using digital and video cameras This driver is useful for testing or teaching users how to acquire images when no camera is attached to the system For example you may want to test the functionality of a script before carrying out an experiment You can emulate two separate channels using the Channel 1 and Channel 2 tabs on the dialog box 5011685 B 63 Chapter 6 Installing and Configuring Hardware Setting Description Channels 1 and 2 Image Content Wedge defined by frame size and bit Select this o
180. on your computer instead the location of the image from where it was linked is referenced in the plane information Info Dataset file 5011685 B 43 Chapter 5 Creating and Managing Experiments You can link the following types of image files e tif e stk e nd The calibration of an image does not change when it is linked to an experiment To link an image 1 To link the image to the open experiment from the Application menu select Link Image and then Link Image into Current or to link the image into a new experiment select Link Image and then Link Image into New 2 Inthe Open dialog box select the image and click Open The image appears as a new dataset in the open experiment or in the newly created experiment Note You can change the folder that opens by default when you link an image See Creating a New Experiment 44 5011685 B Chapter 6 Installing and Configuring Hardware This chapter includes the following topics e General Workflow for Installing and Loading Hardware Device Drivers e Preparing to Install Hardware Device Drivers e Installing Device Drivers e Loading Device Drivers e Device and Component Settings e Creating and Modifying Hardware Settings e Creating and Modifying Illumination Settings e Creating and Modifying Magnification Settings e Configuring Camera Drivers e Camera Settings General Workflow You must be able to access the Hardware Setup tab to perform the tasks descr
181. only if the Standard algorithm produces slightly different results than expected Most accurate when using objectives with magnifications of 20X or greater Inthe Autofocus Frequency field Select To autofocus Every Point at every time point First at only the first time point Every Nth Point at specific time point intervals specified in the next step for example you could autofocus at every third time point If the acquisition does not include a time series autofocusing will occur at the first time point If you selected EveryNthPoint in the previous step in the Autofocus Rate field enter the time point interval at which autofocusing should occur for example enter 3 to autofocus at every third time point Keep in mind that the first time point in a time series is always zero 0 Note The new autofocus position will be used at all subsequent time points for the current stage position unless another autofocus occurs Optionally configure other parts of the acquisition before acquiring images See Acquiring Images in Multidimensional Mode Select the Do Not Update Display check box to indicate that images should not be displayed as they are acquired in this case the new dataset is not added to the experiment until the acquisition is complete or clear the check box to indicate that the Latest Image tab should display the most recently acquired image and that the images should appear in a new Datase
182. organize the display of images In the list of view modes select a mode to change the display of images in the image grid You may want to adjust the number of columns and rows in the image grid when you change the view mode You can click the icon on the top of the View Mode button to view the selected mode This button is particularly useful if you want to alternate between Single View mode and another view mode View Mode Description Single View Displays a single image comprised of one or more channels Channels are displayed ina composite overlay on the image While in Single View mode you can turn the display of each channel on or off as described in the next section When switching from another view mode the Z step and time point of the single image is determined by the middle image in the image grid Time vs Z Displays images acquired at successive time points from left to right and displays images acquired at successive Z steps from top to bottom Images acquired earlier are to the left of the currently displayed image and images acquired later are to the right Scroll left or right to see images acquired at other time points Images acquired at higher Z steps are above the currently displayed image and images acquired at lower Z steps are below Scroll up or down to see images acquired at other Z steps Time vs Channel Displays images acquired at successive time points f
183. orrection capability of the camera Compute exposure and Computes exposure and gain whenever you display alive gain on live startup view Quality Speed Helps achieve an ideal balance between quality and speed when continuously updating the live view The better the quality the slower the speed or the faster the speed the lesser the quality Image Type Specifies the type of illumination Brightfield or Darkfield Show Focus Indicator Displays a value on the live image display that reflects focus accuracy where the highest value equals the most accurate focus Reset Resets certain camera settings to default values Defect Correction The settings that are available will vary depending on the camera that you are using Description Camera The active camera List of CCD chip defects All CCD chip defects that will be corrected with the defect correction option Add Defect and Edit Defect Opens the Add Defect dialog box If the defect that you are entering is a single 62 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Setting Description pixel defect type the location X Y coordinates of the defective pixel in the X Column and Y Row fields and select Pixel Defect as the defect type If the defect you are entering includes part or all of one or more adjacent columns select Column Defect and type the X Y coordinates of the starting location in the defective c
184. ou set the camera area a snapped image of the area appears in the Latest Image tab and a thumbnail of the image appears in the filmstrip Typically you begin by selecting the full chip area of the camera Then if you want to acquire a smaller area you can zoom in until just the area that you want to acquire is displayed and set it as the camera area After you have defined the camera area to use for acquisition you can configure other parts of the acquisition such as stage positions a Z series channels or a time series To set the camera area 1 On the Mode Interactive tab or Mode Multidimensional tab in Camera click the Full Chip button If you want to acquire the full chip area you do not need to follow the remaining steps in this procedure The MetaMorph NX Software snaps an image of the full chip area of the camera using the selected channel 2 Use the wheel on your mouse to zoom in on the snapped image until just the area that you want to acquire is displayed in the image grid 3 Onthe Mode Interactive tab or Mode Multidimensional tab in Camera click the Current View button Ed The MetaMorph NX Software snaps an image of the area displayed in the Latest Image tab using the selected channel resizing the image to the area that will be used for acquisition Configuring Channels for a Multidimensional Acquisition You can acquire images using one or more optical or illumination conditions channels Channels can
185. played in the image grid or you can measure the regions on the images in the dataset that were acquired at all time points all Z steps or all stage positions When regions are measured they are converted to objects Region measurements are the same as the measurements obtained with the Measure Using Threshold measurement function which also uses threshold values to define objects in an image For regions the MetaMorph NX Software provides an additional measurement line length as well as intensity data for the selected channel or all channels 138 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide After regions are measured they are saved as a measurement set and are stored with the dataset This section describes e How to draw and measure regions e Region measurement definitions To draw and measure regions 1 Open the dataset that contains the images 2 Doone of the following e To measure regions on just one image or the images that can fit in the image grid on the Home tab use the view modes to display just the images that you want to measure e To measure regions on the images that were acquired at all time points Z positions or stage positions on the Measure tab select the All Time All Z or All Stage check box you can select more than one check box If you select one or more of these options it does not matter which images are displayed in the image grid Note Mea
186. preadsheet e Export data to an Excel file while Microsoft Excel is closed or not installed e Export measurement data to a text file CSV format To export data to an open Excel spreadsheet If Microsoft Excel is not open before you export data the MetaMorph NX Software will open a new spreadsheet for the exported data If a Microsoft Excel spreadsheet is open each time you export data that data appears in a new worksheet in the open spreadsheet 1 Configure the measurement results so that the data table contains only the data that you want to export 2 Onthe Measure tab click Export to Excel Microsoft Excel opens and displays the data that you exported in a new Microsoft Excel spreadsheet 3 To export another set of data into a new worksheet in the same spreadsheet keep the spreadsheet open and repeat Steps 1 2 To export data to an Excel file while Microsoft Excel is closed or not installed While keeping Microsoft Excel closed you can export data directly to a Microsoft Excel file that you specify Even if Microsoft Excel is not installed on your computer you can export data to an Excel file which can then be opened on another computer that has Excel installed CAUTION If you export data to an existing spreadsheet the exported data overwrites all existing data in the spreadsheet a new worksheet is not created in the spreadsheet 152 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide
187. ption to choose the width depth height and depth of the emulated image or stream The emulated stream is a diagonal display of the minimum and maximum pixel range as defined in the Bit Depth field Images opened from sample file Currently not available Wedge Image options in this section are available only if Wedge defined by frame size and bit depth is selected Width The width of the wedge image Height The height of the wedge image Bit Depth The bit depth of the wedge image Emulation Camera to Emulate The Camera to Emulate field lists the cameras that can be emulated The options that are available for emulation vary depending on the camera You can select Custom and select the features to emulate or you can modify one of the existing camera settings 64 5011685 B Chapter 7 Acquiring Images This chapter includes the following topics e Overview of Acquiring Images e General Workflow for Acquiring Images e Selecting an Acquisition Mode e Setting the Camera Area for Acquisition e Acquiring Images in Interactive Mode e Acquiring Images in Multidimensional Mode e Designating Z Motors and Stages for Acquisition e Setting a Point of Origin e Displaying a Live View e Snapping an Image e Using Auto Expose e Configuring Image Autofocus e Using Focus Stabilization e Pausing or Stopping an Acquisition e Saving and Loading Acquisition Settings Protocols 65 Chapter 7 Acquiring Images Ove
188. quisition includes both autofocus and a Z series the MetaMorph NX Software first performs the autofocus and then acquires the Z series of images The Z series illustrated below contains 5 Z steps a Z range of 4 anda Z step size of 1 86 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Z Step 5 Step Size 1 Z Step 4 Z Step 3 ZRange 4 Center lnitial Z Position Z Step 2 Z Step 1 Use the Mode Multidimensional tab to define the basic settings for a Z series Use the Acquisition dialog box to define basic and advanced settings for a Z series By default all channels are acquired at a specific Z position before all channels are acquired at the next Z position To acquire a Z series for one channel at a time loop the Z series within the channel change the setting in the Acquisition dialog box Mode Multidimensional Tab Z Acquisition Dialog Box Z Series Settings Series Settings Current Z position Current Z position Z step size Z step size Z range Z range Number of Z steps Looping whether all channels are acquired at a specific Z position before all channels are acquired at the next Z position or whether the Z series is acquired using a channel before acquiring the Z series using another channel Prerequisites A Z motor must be installed and configured See Installing and Loading Hardware Device Drivers If more than one Z motor is installed or you
189. re replaced with black As you increase this number you increase the number of pixels that are replaced with black 29 Chapter 2 Viewing Images 3 30 Method Description Pixels that are equal to and above the high value are replaced with the maximum intensity of the channel display color As you decrease this number you increase the number of pixels that are replaced with the maximum intensity of the channel display color If you selected Normalize or Fixed scaling you can change the range of intensity values for a channel by dragging the lower or upper limit bar on the histogram to the left or to the right or by typing values in the lower or upper limit fields that are below the histogram as shown below When you move the limit bars the values in the fields below the histogram update accordingly and as you type new values in the fields below the histogram the limit bars move accordingly Channel Display 4x e Fixed Normalize Camera E oc Lower limit of Upper limit of range of intensity N range of intensity values displayed values displayed e 0 To reset the range of pixels displayed to the full range of pixels for the channel click the Reset All Scaling button le For Normalize scaling the Reset All Scaling option resets the range so that it is from 0 zero to 100 percent of the original range of intensity values For Fixed scaling the Reset All Scaling option resets the ran
190. rements Tables and graphs can be configured to display only the data that meet your criteria You can export the data table to an Excel spreadsheet or file See Displaying Measurement Results in a Table Graphing Measurement Results and Exporting Measurement Data The following types of measurements are available e Plane Information provides image metadata including size calibration date created and comments e Measure Using Threshold measures basic properties of objects in an image including area width height centroid position intensity statistics perimeter length orientation and so on e Measure Regions converts regions to objects and measures properties including intensity values for one or all channels e Count Nuclei counts nuclei and measures intensity of nuclei The following table lists some of the main differences between the measurement functions Capability Measure Measure Using Count Regions Threshold Nuclei Detects objects automatically S Works with objects of all shapes and f f f sizes Works with dark objects on a bright f X background Works with uneven backgrounds S X A 126 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Capability Measure Measure Using Count Regions Threshold Nuclei Works with touching objects Pa Identifies objects even when they move X oY oF r Supports the capability X Does not support the capability Ge
191. rom left to right and displays a separate channel in each row Images acquired earlier are to the left of the currently displayed image and images acquired later are to the right Scroll left or right to see images acquired at other time points Channel The number of rows is determined by the number of channels selected to be displayed 20 5011685 B View Mode Channel vs Z Multi Time 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Channel Description those channels that are not hidden See the next section for information about how to tum on or off the display of each channel Displays a separate channel in each column and displays images acquired at successive Z steps from top to bottom The number of columns is determined by the number of channels selected to be displayed those channels that are not hidden See the next section for information about how to turn on or off the display of each channel Images acquired at higher Z steps are above the currently displayed image and images acquired at lower Z steps are below Scroll up or down to see images acquired at other Z steps Displays a different time point in each grid position The image acquired earliest is displayed in the upper left grid position The image that was acquired next is displayed in the grid position just below the first image The order of images starts over at the top of
192. roximate maximum nuclei width in calibrated units to include in the count This value is used to differentiate nuclei from non nuclear material background fluctuations or shading errors to separate touching cells and to exclude nuclei greater than the specified size from the segmentation Do one of the following e Tocount the nuclei in just one image or the images that can fit in the image grid on the Home tab use the view modes to display just the images that you want to measure e Tocount the nuclei in the images that were acquired at all time points Z positions or stage positions on the Measure tab select the All Time All Z or All Stage check box you can select more than one check box Note Nuclei are counted in either the images displayed in the image grid or the images acquired at all time points Z steps or stage positions If you want to count nuclei ina range of images that cannot fit into the image grid count the nuclei in all of the images and then filter the measurement results On the Measure tab click Measure The MetaMorph NX Software creates a segmentation of the image counts the nuclei in the image and stores the results in a new measurement set The progress bar at the bottom of the workspace shows the status of the measurement Once the measurement is finished the new measurement set appears at the bottom of the list of measurement sets on the Measure tab Note To cancel a measurement click anywher
193. rview The MetaMorph NX Software provides many options for acquiring images of the samples and processes that occur in your experiment Image acquisition can be as simple as a single image or as complex as multiple sets of images acquired at various sites at certain times with a variety of illumination conditions You control when where and how images are acquired The goals of your experiment determine the complexity of the image acquisition The following types of acquisitions are available e Interactive image acquisition acquire an image with one or more channels at one stage position one Z position and one time point select individual images and save them into a dataset e Multidimensional image acquisition combine some or all of the series acquisitions described below to create a multifaceted acquisition that can include almost any combination of channels stage positions Z positions and time points automatically save all acquired images into a dataset e Time series acquisition acquire images at specific time intervals for a certain amount of time e Stage series acquisition acquire images at multiple sites in your sample e Zseries acquisition acquire multiple images along the Z axis e Channel series acquisition acquire images using one or more illumination conditions which may include brightfield fluorescence and other microscopy techniques set exposure and camera settings for each channel or use the
194. s enabled both the system mouse and input accessory mouse wheels will perform the command that is assigned in the Mouse Wheel Control option When the Mouse Wheel Control option is disabled the input accessory mouse will perform the input accessory commands The Mouse Wheel Control option is located on the Hardware Setup tab You may need a password to access the Hardware Setup tab or you may not be allowed to access it If you cannot access the Hardware Setup tab your system administrator may have placed a shortcut to the mouse wheel control on the Quick Access Toolbar so that you can use it All of the mouse wheel control settings that are available on the Hardware Setup tab are also available in the shortcut on the Quick Access Toolbar This section describes how to e Configure the wheel control e Use the wheel control To configure the wheel control 1 If you have access to the Hardware Setup tab in Mouse Wheel click Wheel Control to display the list of devices that can be moved with the mouse wheel Or if the Quick 35 Chapter 4 Configuring a Keypad Keyboard or Mouse 36 Access Toolbar contains a shortcut to the mouse wheel control click the down arrow next to the mouse icon 0 Inthe Spin Unit fields that appear type the increments by which the devices will move when you scroll the wheel The spin unit is not a specific unit of measurement but is a value that reflects the amount that the device will move each t
195. s are listed in the stage series The Stage Series group on the Mode Multidimensional tab shows a map of the stage positions that are defined for the current acquisition When you add or remove stage positions the map updates 5011685 B 83 Chapter 7 Acquiring Images accordingly For example the map below shows that the acquisition is configured to acquire images at three stage positions Mo N O X The MetaMorph NX Software automatically provides a label that stores stage position information when an image is acquired You can use this label or provide your own when you configure the stage position Label information is available in the Plane Information measurement set A CAUTION Be aware of the position of the stage relative to both the objective and the condenser of the microscope A motorized stage may collide into either of these microscope components possibly causing damage Use the options on the Mode Multidimensional tab to define the basic settings for a stage series Use the Acquisition dialog box to define basic and advanced settings for a stage series Mode Multidimensional Tab Acquisition Dialog Box Stage Series Settings Stage Series Settings Update selected stage position to Update selected stage position to use the current stage position use the current stage position Add remove or go to stage Add remove or go to stage positions positions Define the travel offset distance and whether travel offs
196. s than the value defined as the standard area If an object has a standard area count less than 1 0 it will count as asingle object standard area value o 1 0 units 3 75 units 1 0 units rounded to 4 0 objects 5011685 B 133 Chapter 8 Measuring and Analyzing Images Dimension measurements Measurement Definition Width The horizontal dimension of the object The vertical dimension of the object The span of the longest chord through the object VS Note This measurement is calculated slightly differently in the MetaMorph Software version 7 7 and earlier The MetaMorph NX Software uses a more comprehensive system for determining maximum possible length Breadth The caliper width of the object perpendicular to the longest chord g Note Due to changes in the way that Length is measured this measurement is also calculated slightly differently in the MetaMorph Software version 7 7 and earlier See the description of Length above Fiber Length The length of an object assuming that it is a fiber Fiber Length A P P 16A Where P Perimeter and A Area C 134 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Measurement Definition Fiber Breadth The width of an object assuming that it is a fiber Fiber Breadth P P 16A Where P Perimeter and A Area Shape position and orientation measurements
197. s the channels based on the bit depth of the camera that was used to acquire the image If the camera bit depth is unknown the scaling range is 0 4095 Camera Bit Depth Intensity Value Range Scales the channels based on a percentage of the original range of intensity values for the channel The scaling range is from 0 to 100 Step 3 describes how to specify a percentage of the original range Normalize scales each image based on the pixel data in the image Normalize makes it easy to see the data in every image but does not allow you to compare two images and determine the differences in intensity just by looking Pixels that are in the percentage of intensity values equal to and below the low value are replaced with black As you increase the low value you increase the number of pixels that are replaced with black Pixels that are in the percentage of intensity values equal to and above the high value are replaced with the maximum intensity of the channel display color As you decrease the high value you increase the number of pixels that are replaced with the maximum intensity of the channel display color Scales the channels based on a subset of the original range of intensity values The scaling range is the actual lowest and highest original intensity values of the channel in the images displayed Step 3 describes how to specify a subset of the original range Pixels that are equal to and below the low value a
198. same camera settings for all channels 66 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide General Workflow Accurate image acquisition depends on a proper microscope setup which your MetaMorph NX representative may have already done for you and which most likely needs to be updated only occasionally After the hardware is configured in most cases you should be able to configure acquisitions using just the options on the tab for the selected acquisition mode The flowchart below shows the general sequence of tasks for acquiring images Interactive Acquisition Mode Multidimensional Acquisition Mode Acquire image or images Acquire multidimensional image Requires specific hardware 5011685 B 67 Chapter 7 Acquiring Images Selecting an Acquisition Mode The first step in configuring an image acquisition is to select an acquisition mode The mode that you select determines the options and settings that appear on the ribbon only the options that are needed for the selected mode are displayed Two acquisition modes are available Interactive and Multidimensional Use the following table to determine which acquisition mode will best meet your needs Use this mode If you Interactive have a microscope with little to no automation and you plan to ih manually move the stage to various sites want to acquire a single image for publication purposes want to acquire single images of a si
199. surements are made on the regions in either the images displayed in the image grid or the images acquired at all time points Z positions or stage positions If you want to measure a range of images that cannot fit into the image grid measure all the images and then filter the measurement results 3 Onthe Measure tab in Regions select one of the drawing tools Use this tool To create closed rectangular regions in which all of the pixels within the boundaries of the region outline are measured must be at least two pixels by two pixels closed circular regions in which all of the pixels within the boundaries of the region outline are measured must be at least two pixels by two pixels hand traced closed regions in which all of the pixels within the boundaries of the region outline are measured must be at least two pixels by two pixels line regions that are one pixel wide and at least two pixels long all measurements will be made on only pixels that are under the line multiple point lines that are one pixel wide and at least two pixels long all measurements will be made on only those pixels that are under the line e freehand line regions that are one pixel wide and at least two pixels long all measurements will be made on only those pixels that are under the line 5011685 B 139 Chapter 8 Measuring and Analyzing Images 10 140 Left click the mouse and drag it to draw a region on an image As you draw a region
200. t tab as they are acquired When you have configured the acquisition click Acquire If the Do Not Update Display check box is cleared the MetaMorph NX Software opens the Latest Image tab which displays the most recently acquired image This image helps you gauge the quality of the images as they are acquired which can be especially useful for lengthy time series acquisitions During a multidimensional acquisition the MetaMorph NX Software saves the acquired images into a new dataset You can review images as they are acquired by displaying the Dataset tab See Viewing Images in a Dataset for more information 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Note If an experiment is not open when you start an acquisition the MetaMorph NX Software will create one to contain the new dataset A separate window within the Latest Image tab displays the status of the acquisition as well as a histogram for each channel The MetaMorph NX Software reports the number of images acquired as well as the current time point in a time series if applicable stage position if applicable channel and Z position if applicable Other statistics may also be reported to help you monitor progress If autofocusing does not produce in focus images e Anexposure time may be too short especially if the sample is dim in which case the autofocus system may not be able to find the sample You may need to increase the exposur
201. t 43 Table 149 Opening MetaMorph 7 7 images 43 Target intensity 70 80 97 Orientation measurement 132 141 Thresholding images 130 Origin point of 93 Time points Channel acquisition frequency 78 P Configuring for acquisition 89 Time vs Channel View Mode 20 Pausing an acquisition 121 Time vs Z View Mode 20 Perimeter measurement 130 138 Time Z Stage measure option 125 130 138 Plane Information 127 147 Point of origin 93 Total Area measurement 130 138 Travel offset 84 R U Reading recommendations 167 Rearranging Dataset Views 24 Updates checking for software 165 Reference point for motors 93 Regions clearing 138 5011685 B 171 Index 172 V Viewing Images Live Display Measurement results Metadata on images W Width measurement Workspace Rearranging Tour of Z Z motor Designating for acquisition In autofocus InZ series Z Offset Z series Z step In autofocus In Z series Zeiss Definite Focus System Zooming in on images 17 149 32 137 146 79 66 75 86 98 86 105 117 31 5011685 B
202. t from the first channel Click Live to display a live view of the image Use the focusing knobs on the microscope to bring the image into focus In Acquisition click Configure to open the Acquisition dialog box Inthe Acquisition dialog box select the Focus Stabilizer check box to indicate that the acquisition should use the focus stabilizer hardware Olympus ZDC or ZDC2 and then click Focus Stabilizer to display the related fields D You can also use the options in the Focus group on the Mode Interactive tab to set the focus and to re establish focus In the One Shot Range field type the total distance above and below the current Z motor 5011685 B 111 Chapter 7 Acquiring Images 10 11 position that the Z motor is allowed to move while the ZDC or ZDC2 is locating the surface of the dish or slide For example if you type 100 the Z motor will be allowed to move 100 microns above and 100 microns below its current position This distance should not let the Z motor go above the near limit position specified in the objective component settings Best Practice Use the default one shot range If you set a very large range the ZDC or ZDC2 may locate something other than the surface and focusing will not be possible With the image still in focus click Set Focus to set the focus at the current focal plane The Focus Adjustment field displays the surface to focal plane distance in microns and the Olympus ZDC or ZDC2 beeps on
203. taMorph NX Microscopy Automation amp Image Analysis Software User Guide Scaling Images Scaling an image redistributes the range of intensity values used to display channels based on lower and upper limits that you specify The result is an increase or decrease in the brightness and contrast of the channels in the image By default the MetaMorph NX Software scales channels based on the bit depth of the camera You can scale channels in images that are of any bit depth You can scale channels in any images that are displayed in the image grid the Latest Image tab or the Live View tab and you can scale channels while acquiring images Note Scaling is for display purposes only the MetaMorph NX Software does not save scaling data with the image or change any information about the intensity values in the image A 12 bit image for example consists of 4095 possible intensity values However especially in low light experiments much of the important image information may be contained within a narrow range toward the lower end of the intensity values By scaling an image to increase the contrast of displayed channels you can more easily see variations in brightness that might otherwise be impossible to detect visually Before scaling 5011685 B 27 Chapter 2 Viewing Images The illustration below shows that the Fixed scaling method will redistribute intensity values using only the pixels with intensity values between 100 and 2
204. taMorph NX Software will automatically use the numerical aperture and refractive index values based on the objective values defined in the hardware configuration See Device and Component Settings for more information 4 Inthe Numerical Aperture field type the numerical aperture of the objective Inthe Refractive Index field type the refractive index of the objective 6 Select Display Message When Setting Is Selected to have the MetaMorph NX Software display a message when you select this magnification setting When you select this magnification setting the Z motor moves to its escape position defined in Step 11 and a message appears that asks you to confirm that the Z motor should move back to its original position For example a Move the objective turret to the 40X position and then click OK The message gives you time for example to apply oil to the lens of an oil immersion objective before the objective is moved back to its original position e 7 Inthe X Offset and Y Offset fields type the distance to move the X and Y positions of the stage to keep the point of origin in its original position when using this magnification setting When the objective changes the point of origin may shift slightly in the field of view The X and Y offset distances specify how far the stage should move along the X and Y axes to maintain the point of origin The example below illustrates the process for calculating the offset distan
205. taset A separate window within the Latest Image tab displays the status of the acquisition as well as a histogram for each channel The MetaMorph NX Software reports the number of images acquired as well as the current time point in a time series if applicable stage position if applicable channel and Z position if applicable Other statistics may also be reported to help you monitor progress Configuring a Z Series for a Multidimensional Acquisition A Z series acquires images at multiple Z positions These images are sometimes referred to as the optical sections that are perpendicular to the Z axis A collection of optical sections provides a through focus view of the sample A Z series can be used alone to obtain a through focus view of one location at one time point using a single channel Or a Z series may be part of a multidimensional acquisition that acquires images over time at multiple locations using multiple channels When you configure a Z series you specify the number of Z steps at which images will be acquired as well as where along the Z axis the images should be acquired If your acquisition includes more than one channel you can specify whether the Z series is acquired with all channels in the acquisition or with specific channels When the Z series acquisition begins the Z motor moves the objective to the lowest Z position that you have defined and then images are acquired as the objective moves up the Z axis If your ac
206. te for a simple measurement for example if you plan to manually count objects or manually calculate simple statistics for all collected data do not want to acquire a Z series stage series or a time lapse of images want to choose which individually acquired images should be saved into a dataset Multidimensional want to use an automated stage or Z motor p want to acquire a Z series stage series or a time lapse of images using one or more channels want to automatically save all acquired images into a dataset want to automatically measure multiple images in a dataset for example region intensity over time The following table lists the major differences between the acquisition modes Feature Interactive Mode Multidimensional Mode Live Display Snap Images Auto Expose Image Autofocus Focus Stabilization Channel Series Stage Series 68 5011685 B KITA ERE RTS SK ee MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Feature Interactive Mode Multidimensional Mode Z Series vo Time Series xX S Automatically saves all X vd images acquired Automatically saves f acquisition settings for future use Allows manual save of f xX individual images into a dataset Y Available X Not available Requires specific hardware Acquiring Images in Interactive Mode Using the Interactive acquisition mode you can acquire individual images with one or more optical or illuminatio
207. that you want to measure 2 On the Measure tab in Measure select Measure Using Threshold e3 The options for configuring the measurement appear in a popup area You can configure the measurement in the popup area or if you prefer you can click Configure and open a dialog box that contains the same options 3 Inthe Threshold Image field select the channel to use to create the segmentation for measuring or select Plane if no channels are available 4 Inthe Standard Area Value field type the size of a typical object for example a cell or 130 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide nucleus in square microns Counting objects is often complicated by the fact that objects overlap or clump together when thresholded resulting in counts that are lower than the actual number of objects The Standard Area Value helps compensate for overlapping or clustered objects The Standard Area Value determines the standard area count reported in the measurement results 5 Inthe Threshold Low and Threshold High fields type the minimum and maximum intensity values that should be included in the segmentation 6 Select the Inclusive check box to include in the segmentation only those pixels with intensity values that are between the Threshold Low and Threshold High intensity values Clear the check box to include in the segmentation only those pixels that have intensity values equal to or outside of
208. the Acquisition Mode tab select Multidimensional select the camera channel and optionally select a hardware setting if one is needed to set the microscope devices to pre defined positions 2 Onthe Mode Multidimensional tab in Magnification select the magnification you want to use for the acquisition On the Mode Multidimensional tab click Configure to open the Acquisition dialog box 4 Inthe Acquisition dialog box select the Time Series check box to indicate that the acquisition will include a time series and then click Time Series to expand the list of settings If your acquisition contains just one time segment you can adjust the intervals and duration for the time segment using the options on the Mode Multidimensional tab Or you can set these values in the Acquisition dialog box as described below Changes that you make to the time segment on the Mode Multidimensional tab are reflected in the dialog box and changes that you make in the dialog box are reflected on the tab 5 Select the Time Series check box to indicate that the acquisition will include a time series and then click Time Series to expand the list of settings 6 Inthe Interval field type the amount of time that should pass between image acquisitions Type 0 zero if you want to acquire images as fast as possible 90 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Note If the acquisition cannot be accomplished i
209. the measurement results for example intensity perimeter and area o measurement summary data for example total average and standard deviation o plane information for example image height and width e Line graph shows how a specific measurement of a region or regions changes over time While a dataset is open you can create as many graphs as needed for each measurement set Graphs are displayed in the Dataset View of the dataset that supplied the data for the graph so when 5011685 B 153 Chapter 8 Measuring and Analyzing Images multiple graphs and datasets are open it is clear which graph belongs to which measurement set and dataset Ge You can create a scatter plot using the data in the Plane Information measurement set without making any measurements For example you may want to graph time points and Z steps Note The Configure Results option on the Measure tab provides options for configuring filter criteria for both the data table and graphs The data table and graphs associated with a measurement set share the same data and the same filter criteria If you create a filter for a graph that excludes certain data that data will also be excluded from the data table Likewise when you create a filter for a data table that excludes certain data that data cannot be included on a graph When configuring filter criteria for a line graph do not exclude time measurements line graphs always plot time measurements on the X axis
210. tings Position The current position of the stage Move Click the or buttons or type a new value to move the stage Go to Origin Moves the stage to its origin position Discrete Component Position Settings The settings that are available for discrete components for example lenses disks wheels lamps and mirrors may vary slightly depending on the type of hardware that you are using Settings Description Position Labels Position Labels for each position on the component Discrete Component Parameters Open Control Opens the Control Device dialog box which provides the following options for Dialog testing the movement of the device Position Controls Moves the device to the selected position lt lt Moves the device backward by one position gt gt Moves the device forward by one position Filter Wheel Settings The filter wheel settings that are available may vary slightly depending on the type of hardware that you are using 5011685 B 51 Chapter 6 Installing and Configuring Hardware Setting Description Filter Labels Filter Labels for each filter on the wheel Filter Visiting Status Normal The wheel will stop at this filter position as needed No Stop The wheel will pass through but not stop at this filter position as needed No Visit The wheel will not be allowed to pass through this filter position Objective Settings The objective settings that are available may vary sl
211. to configure additional channels An icon for the channel appears in the Channel Series group on the Mode Interactive tab The illumination setting that you select in Step 7 will be used as the channel name In the Exposure field If you Then do not plan to use auto expose for type the exposure time to be used for the channel this channel plan to use auto expose for this type an exposure time that will be used if auto expose channel fails to achieve the target intensity Note The Shutter button 2 on the ribbon opens and closes all shutters that are set to Active in the illumination setting that is used with the selected channel See Creating Illumination Settings for more information In the Illumination Setting field select a setting from the list Illumination settings contain pre defined information such as the wavelength and positions of various components For information about illumination settings see Creating Illumination Settings 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide 8 To use auto expose for the channel select the Auto Expose check box and follow Steps 9 11 If you are not using auto expose clear the check box and proceed to Step 12 Note If your sample is light sensitive you may want to minimize the number of auto exposures 9 Inthe Target Intensity field type the maximum intensity value that auto exposure should attempt to attain
212. tofocus Accuracy field type the smallest distance that the Z motor should move to refine its search for the best focus image This value is usually based on the depth of field and the numerical aperture of the objective 5 Inthe Autofocus Step Size field type the step size a larger distance than the accuracy distance between the initial focus test images that will be acquired The step size is based on the field of depth of the objective and sample signal strength and is used to locate the strongest intensity signal from the sample If the step size is too small extra images will be collected during autofocus and signal noise 5011685 B 103 Chapter 7 Acquiring Images may be misinterpreted as the focus peak If the step size is too large the focus position may be inaccurate Generally you should set a step size that may be too small rather than too large if the step size is too large the autofocus algorithm may not be able to locate the sample 6 Inthe Autofocus Algorithm field select an algorithm Description Algorithm Recommended for most imaging conditions and works well for a number of different microscopy techniques Measures contrast in the image Brenner Standard Recommended generally only if the Standard algorithm produces slightly different results than expected Most accurate when using objectives with magnifications of 20X or greater Directional Average 7 Optionally config
213. ton on the Mode Interactive tab dimmed and not available to use Make sure you select the Latest Image tab by clicking anywhere inside the tab Once the tab is selected an orange border appears around the tab and the Save button will be available am trying to configure image autofocus the sample looks like it is in focus so why is autofocus not able to find the sample and autofocus the image If autofocusing does not produce in focus images e Anexposure time may be too short especially if the sample is dim in which case the autofocus system may not be able to find the sample You may need to increase the exposure time or change camera settings so that the autofocus system can find the dim site e The step size may be too small in which case the autofocus system may not be able to accurately distinguish noise from intensity signals from the sample as a result the autofocus system may focus on something other than the sample e The range may be too small You may need to increase the range so that the autofocus system can find the sample e Anexposure time may be too long causing the autofocus camera to become saturated and unable to differentiate intensity signals from the sample You may need to reduce the exposure time or change camera settings e You may need to use the other autofocus algorithm 160 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide See Configuring Image Autofocus
214. trip but want to save for example just one image acquired with one channel When you select more than one image to save the images are displayed as a composite image in the Latest Image tab For example if you select three images that were each acquired with a different channel the Latest Image tab overlays the three channels on one image 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide If a dataset was created when you saved the image a tab for the new dataset opens and displays the saved image If you saved an image into an existing dataset you may want to open the dataset and verify that it contains the image that you saved The next time you acquire all channels or snap an image the image grid clears the images that were just saved and displays the new image Acquiring Images in Multidimensional Mode By acquiring images of a fixed or living sample from various perspectives a multidimensional acquisition can provide a comprehensive view of overall cell morphology The collected data can be used to analyze both significant and subtle trends over time fluctuations in intensity changes in shape and position and so on Example You could configure an acquisition to acquire images at 5 stage positions using DAPI and FITC channels with a Z series that obtains images at 10 Z positions as well a single image at the center Z position that will be acquired with transmitted light Or you can acq
215. u can simply click Install Devices on the Hardware Setup tab select the driver and click Remove Device and Component Settings This section provides information about the settings that are common to most of the devices and components that the MetaMorph NX Software supports e Device Calibration Settings e Discrete Component Position Settings e Filter Wheel Settings e Objective Settings e Shutter Settings For information about settings that are specific to certain hardware and to view a list of currently supported hardware visit the MetaMorph NX Support web site at http support meta moleculardevices com hardware hardware php Device Calibration Settings Use the options described below to define the unit of measurement for device movements For example you could define the unit of measurement for X stage movements so that 4 microns equals 1 step 50 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide The settings that are available may vary slightly depending on the type of hardware that you are using Setting Description Unit Conversion User Units Number of units that comprise the unit of measurement User Units Label The measurement for the user unit Device Units Number of units that equal the number of user units For example 1 micron equals 1 step Device Units Label The unit of measurement as defined by the device For example step or 1 10 micron Position Set
216. u have configured the acquisition click Acquire If the Do Not Update Display check box is cleared the MetaMorph NX Software opens the Latest Image tab which displays the most recently acquired image This image helps you gauge the quality of the images as they are acquired which can be especially useful for lengthy time series acquisitions During acquisition MetaMorph NX saves the acquired images into a new dataset You can review images as they are acquired by displaying the Dataset tab See Viewing Images in a Dataset for more information Note If an experiment is not open when you start an acquisition the MetaMorph NX Software will create one to contain the new dataset A separate window within the Latest Image tab displays the status of the acquisition as well as a histogram for each channel The MetaMorph NX Software reports the number of images acquired as well as the current time point in a time series if applicable stage position if applicable channel and Z position if applicable Other statistics may also be reported to help you monitor progress Configuring a Time Series for a Multidimensional Acquisition A time series acquisition captures processes and events in your experiment as they happen When you configure a time series acquisition you specify that images will be acquired at specific intervals of time for as long as you indicate A time series can consist of one or more time segments For example the time series shown
217. uire an image every 5 seconds at a particular site using an exposure of 80 milliseconds and then every 2 seconds at another site using an exposure of 100 milliseconds You can acquire some channels more frequently than others Although the MetaMorph NX Software provides advanced settings for configuring a multidimensional image in many cases you can configure an acquisition using just the options on the ribbon The Time Series group Z Series group and Stage Series group on the Mode Multidimensional tab each contain a check box that you can use to include or exclude the series from the acquisition For example Interval Duration 10 sec 7 40 sec E 5 ms v 50 l ms a You do not need to configure the acquisition series that comprise a multidimensional acquisition in any particular order However during a multidimensional acquisition that contains a time series stage series Z series and channel series the MetaMorph NX Software acquires images in the following order 1 Time point 2 Stage position 3 Center Z position or Z series 4 Channel or 1 Time point 2 Stage position 5011685 B 75 Chapter 7 Acquiring Images 3 Channel 4 Center Z position or Z series In the following illustration images are acquired every 10 milliseconds at 3 stage positions At all time points a Z series of images is acquired using 2 channels At the first and last time points an image at the center Z position is acquired us
218. ure other parts of the acquisition before acquiring images See Acquiring Images in Interactive Mode 8 After you have configured the acquisition do one of the following To acquire an image On the Mode Interactive tab double click the channel or select the channel and click Snap using all channels click Acquire All Channels If autofocusing does not produce in focus images using one channel e Anexposure time may be too short especially if the sample is dim in which case the autofocus system may not be able to find the sample You may need to increase the exposure time or change camera settings so that the autofocus system can find the dim site e The step size may be too small in which case the autofocus system may not be able to accurately distinguish noise from intensity signals from the sample as a result the autofocus system may focus on something other than the sample e The range may be too small You may need to increase the range so that the autofocus system can find the sample e Anexposure time may be too long causing the autofocus camera to become saturated and unable to differentiate intensity signals from the sample You may need to reduce the exposure time or change camera settings e You may need to use the other autofocus algorithm 104 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Using Focus Stabilization Keeping a sample in focus can be a chal
219. urement results e Filter data for a graph e Configure the graph style and range of data e Change the graph type To create a graph of measurement results Decide whether you want to create a line graph or a scatter plot Line graphs can be used only with measurement sets that contain region data and are typically used for comparing intensity values in regions over time 154 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide 1 Open the dataset that has the measurement set containing data that you want to plot ona graph 2 Onthe Measure tab select the measurement set and then in Measurement Display click Add Graph to create a line graph or click Add Scatter Plot to create a scatter plot graph The graph appears in the Dataset View and a Graph tab appears on the ribbon Note Line graphs show how a region measurement changes over time Line graphs always display time measurements along the X axis 3 Onthe graph in the X Axis field and Y Axis field select the measurements to plot on the X and Y axes You can also select the measurements for the X and Y axes on the Graph tab while you are configuring the style of and range of data for a graph See To configure the graph style and range of data Ge When you double click a point on a graph or double click a row in the data table the image grid shifts the images to bring the corresponding object into view You can create as many graphs for t
220. urface location are different with each objective As aresult the focus adjustment value which is the surface to focal plane distance some manufacturers call this the offset will not be the same for all objectives The sample must be brought into focus using each objective to find the focus adjustment value This section describes how to e Configure the ZDC or ZDC2 for an interactive acquisition one shot focus e Configure the ZDC or ZDC2 for an interactive acquisition continuous focus e Configure the ZDC or ZDC2 for a simple time lapse acquisition continuous focus e Configure the ZDC or ZDC2 for a time lapse multidimensional acquisition without a stage series e Configure the ZDC or ZDC2 for a time lapse multidimensional acquisition with a stage series Prerequisites You must be able to access the Hardware Setup tab to perform these prerequisite tasks See your system administrator for more information if you are not sure if these tasks have been performed Follow the steps below before using the Olympus ZDC or ZDC2 for image acquisition 1 Install and configure the Olympus IX2 BSW Software Consult the IX2 online help or documentation for information on how to use the IX2 Software 2 Inthe MetaMorph NX Software when configuring the objective component settings set the Z motor near limits and the focus jog sensitivity In the Objective Settings dialog box click Configure Parameters and then click Read to automatically set
221. us 1 Configure the channels that you plan to use for the time lapse acquisition and if applicable image autofocus 2 If your acquisition includes more than one channel on the Mode Multidimensional tab select the first channel in the channel series Note The focus stabilization system uses the first channel to calculate the focus adjustment value Subsequent channels are offset from the first channel Click Live to display a live view of the image 4 Use the focusing knobs on the microscope to bring the image into focus 5 If you are using the Olympus ZDC2 on the Mode Multidimensional tab in Focus click Set Continuous Focus If you are using the Olympus ZDC skip this step and proceed to Step 6 The Olympus ZDC or ZDC2 displays the surface to focal plane distance in the Focus Adjustment field and beeps once to indicate that it is configured correctly Note The Olympus ZDC or ZDC2 emits three beeps if it did not successfully calculate 5011685 B 113 Chapter 7 Acquiring Images the lens focus adjustment value Try to set the focus again If you do not hear any beeps the ZDC or ZDC2 is not configured correctly Make sure that the observation method for the objective is set to something other than None check that you are using the correct lens and check that the IX2 Software is configured correctly Optionally slightly adjust the focus by clicking the buttons or by typing a value in the Focus Adjustment field
222. using polarized light or using phase contrast the phase halo expands the outlines of the objects resulting in inaccurate measurements Because objects are separated based on differences of intensity values the location of the outline of an object may vary depending on the threshold levels that are set Carefully select the upper and lower limits for the threshold range You may want to preview a measurement several times using different threshold ranges to be sure that your measurements will be based on an accurate segmentation Also keep in mind that when you measure discrete items such as objects and pixels with continuous functions for example length or perimeter the precision of the measurement is based on the clarity of the discrete item If one image is much sharper than another the measurement of the object in the sharper image will be more precise than the measurement of the object that is in the image that is less sharp because the details of the object are not as visible and therefore cannot be measured as precisely Note If you are measuring more than one image the MetaMorph NX Software uses the center image to display thresholding To verify that the thresholding is accurate you may want to display a different image using the Single View mode This section describes e How to measure morphometry e Morphometry measurement definitions To measure morphometry using thresholding 1 Open the dataset that contains the images
223. ward angles were expressed as negative numbers and upward angles were expressed as positive numbers A value from 0 zero to 1 representing how closely the region represents a circle A value near 0 indicates a flattened object and a value of 1 indicates a perfect circle Shape Factor 47A p Where P Perimeter and A Area Shape Factor Elliptical Form Factor The ratio of the breadth of the region to its length Length Elliptical Form Factor Breadth Note Due to changes in the way that Length is measured this measurement is calculated slightly differently in the MetaMorph Software version 7 7 and earlier See the description of Length above 5011685 B 145 Chapter 8 Measuring and Analyzing Images Intensity measurements Measurement Definition Average Intensity The average of the pixel intensity values contained in the region Integrated Intensity The sum of the intensity values for all pixels contained in the region Intensity Center X and Y The X and Y coordinates of the intensity weighted centroid of the region Centroid Geometric i center of Intensity object center of object Intensity Standard Deviation The standard deviation around the mean of the average intensity value in the region Note This measurement is calculated differently in the MetaMorph Software version 7 7 and earlier The MetaMorph NX Software uses a slightly more comprehensive
224. y the larger and slower motor for autofocusing A fast motor is more likely to drift out of the autofocus range while a slower motor is better able to stay within the autofocus range If more than one stage is installed you must designate which stage moves along the X axis and which stage moves along the Y axis The MetaMorph NX Software supports the use of up to two stages for acquisition The Stage Motor Designations dialog box also contains options for setting a point of origin See Setting a Point of Origin Prerequisites You must install and configure the appropriate hardware device drivers for your Z motors or stages as described in Installing and Loading Hardware Device Drivers To designate up to two Z and stage motors 1 On the Hardware Setup tab in Stage click the dialog box launcher i in the lower right corner of the group 2 Inthe Stage Motor Designations dialog box in the Z Motor field select the Z motor 3 If more than one Z motor is installed but you plan to use only one Z motor for image acquisition inthe Z2 Motor field select None otherwise select the Z2 motor 4 If you plan to use two Z motors for image acquisition designate the Z motor to use for acquiring a Z series the Z motor to use for image autofocus and focus stabilization and the Z 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide motor that will move the travel offset distance as defined in the stage s
225. yboard or keypad configuring 36 L Length measurement 130 138 Line length region measurement 138 Linking an image 43 Live Display 94 Location of experiments datasets images39 Loop within channel option 86 M Magnification settings 57 Maximum Intensity Value 130 138 Measure Regions 138 Measure Using Threshold 130 Measurements 125 127 Cancelling 130 138 147 Configuring and displaying results 149 Count Nuclei 147 Exporting 152 Graphing 153 Morphometry 130 5011685 B MetaMorph NX Microscopy Automation amp Image Analysis Software User Guide Plane Information 127 d Regions drawing and measuring 138 Regions 138 o Relative Hole Area measurement 130 138 Minimum Intensity Value 130 138 f Reset All Scaling option 30 Mode Acquisition 66 68 Single View 20 S View 17 Mouse wheel control of devices 35 36 Scaling images 27 Multi Channel View Mode 22 Scatter plot 153 Multi Time View Mode 21 Shape Factor measurement 130 138 Multi Z View Mode 22 Shutter opening and closing 55 94 Multidimensional acquisition 66 75 79 83 Single View mode 20 50 82 Snapping an image 95 97 Stage positions 9 83 92 93 N Comparing side by side 159 Statistical measurements 132 141 149 Nikon Perfect Focus System 105 106 Stopping an acquisition 121 Normalize scaling 27 Storage of experiments and datasets 39 Nuclei counting tar Summary measurements 130 138 147 Support obtaining 163 O Olympus ZDC and ZDC2 105 110 T Opening an experimen

MetaMorph NX User Guide

Contents

Download Pdf Manuals

Related Search

Related Contents