Axiovision Take Off Guide
Contents
1. By default AxioVision will store your images data and settings in the My Documents area of your computer This has the advantage that different users can log on using their own log on names and work independently However you may want to make some files available to everyone e g scaling so Microsoft Windows provides an additional My Documents area for All Users which is accessible to everyone However you can decide to store your images and other data anywhere on your computer General User data Layout Use standard profile Folder Folder C Use selected folder User templates Use standard profile Folder C Use selected folder C Documents and Settings russell hobson My Doci Workgroup data Use standard profile Folder Use selected folder To find out where the default folders are click on Options in the Tools menu and select the Folder tab You will see that folders are defined for user data and templates usually the users My Documents folder and also for Workgroup data and Workgroup templates usually All Users My Documents Workgroup templates Use standard profile Folder C Use selected folder Documentsic A Folder info To change folders or to see the full path click on or click on Folder info You need to back up the files in these folders they contain scaling calibration files and microscope setting files OK Cancel Apply2. 4 Image Display and Adjustments In this section we will look at various image display modes and adjustments Image Size Load an image from your images folder Normal View 1 1 42 Auto Zoom E Right click the mouse in the image to see the context menu Try Zoom In the various image sizing functions to see the effect R ag Zoom In Zoom Out also F7 and F8 2 Zoom a EB Auto Zoom display the entire image inside the display window Player E Normal View displays the image at its true resolution no E Full Screen E zoom i e one image pixel per screen pixel l Full Screen Press the Esc key to exit this view Ruler view Annotations 4 Tb Show bounding box gt Player Panel CiriH Display Panel E Properties Alt Enter Lo Navigator Try using the Navigator window This is particularly useful when viewing large images e g those which do not fit on the display area You will probably want to drag the Navigator window out to make it a bit bigger and then move the red square around inside it i Navigator 10 Image Display Properties You may want to adjust the contrast and brightness of the displayed image This is particularly useful when viewing high bit depth images i e images with more gray values as obtained from the AxioCam camera range r Teo ohne i Lau ven ante view Tree view ery won SA 2H ACSA Ria elf Right click in the image to see the context m
3. In addition to backing up your images and calibration files you may want to keep a copy of your customization features i e Workflows short cut keys screen layout You can do this by exporting your configuration FETTE x Click on Import Export Configuration in the Please select the components you want to import or export Too Ss m e n u i Select Export in the first screen Now check all the boxes except the last and click Next oO TERRES Enter a file name Image Capture O Save all user documents to ZIP File To import a set up use the same function but select Import on the first screen Note The last option Save to Zip file creates a zipped up configuration file This is useful if you need to e mail your configuration to a colleague or Back m gt a to support staff Select all Unselect all 21 10 Automation If you have a microscope from Carl Zeiss with some encoded or motorized features you can read or control the settings from within AxioVision however a detailed discussion of these features is beyond the scope of this guide You will need to configure the microscope module using the Microscope Configuration program before you can use these features usually this will have been done by the installation engineer To control the microscope you have various options Common Refie Trans St E ee Click on Microscope in the Workarea and use the control panel Close
4. For further information please contact your local Carl Zeiss representation Contents 1 Introduction to the AxioVision Environment cccccccececeececeececesceececeenes 1 image Types near 2 2 Image Acquisition using the AxioCam Camera u 2u022002000n0n nun en 3 2 1 Getting Started nassen 3 EXPOSTO nee ee Re ae E eee ee ery ee 4 Tg 1 8 cetera eg ee ert ene Pe SER te a IEE PC WESER OE oP EY WEN REE CSO ORE RST MED TER CE 4 White Balance applies only to color CAMELAS ceeccceeeeeeeeceeeeeeeaeeeeeesaeeeeeeaeneeeesaaes 5 ACGUIFING AN Mage 2 a E a ES 5 2 2 AXioCam Featurfes an ee ee A r AAS 6 RESOLUHON Es ren a Renee 6 EE A f a GENERALE NERO OL EOS RT 6 Monochrome or Color cccccececececececececececcceccuacauaeacaeaeneneneneneneneneueneceueteteuenenenenenenenens 6 Black REfEreNCe ccccccccecececececececececececccuauacauauauaeaeneneaenenenenensneneneneaeaenenenenenenenenenenens 7 1 AFe 18 Ale MGS a X21 WO ee a De rer ee ME 7 AN ACO see EREIRRR EN a cote tcc ENTER FRERARTENEEERDERERNEEENERSFRENLERSEREERRHRE E E 8 S DA DE ee eS MR SERED es Oe Erne Ee 8 3 Saving and Archiving Images 2u0220 200200 000n0nnunanunnun ann nnnn nun nun nun nenn 8 Image Display and Adjustments 2 02002200000 000n0nnnn ann nnun anna nun 10 Image Size nee are 10 Image Display Properlles u ae 11 Image Splitter Display Window 22uu022202000220nnn nennen
5. Form Frame grabber Gallery Camera which produces an analog video signal Graphics e g text and arrows drawn on top of the image Technique used to increase the sensitivity of the camera by adding together groups of adjacent pixels Number of intensity values resolved e g 8 bits 256 levels 12 bits 4096 levels 14 bits 16384 levels A board inside the computer which reads the image in from a digital camera Charge coupled device solid state technology used for camera Sensors Settings for image capture of a specific fluorochrome or contrast technique A Microsoft Windows facility used by many applications to exchange data A menu specific to a particular window usually activated by clicking with the right mouse button inside the window Technique used to improve color image quality by moving the CCD chip Inherent background signal from camera when no light is reaching the sensor An image restoration technique in which the blurred information from planes above and below the plane imaged is removed The window associated with a function used to set up its parameters Camera which directly produces a digital image Click on an object with the left mouse button hold the button down and move to a new location drag then release the button to drop the object in its new location List of options associated with a menu item accessed by clicking on a down arrow to the right of the entry line An
6. dialog box If you have drawn several scale bars or other graphics you can select any one and change its attributes independently Automatic scaling The size of the pixels in a digital microscope image is determined by the following microscope components and camera parameters e Magnification of the objective used e Inthe case of an upright and inverted microscope magnification factor of the optovar tube lens used or if applicable magnification factor of an optovar cube in the reflector turret e Inthe case of a stereomicroscope zoom factor e Magnification factor of the camera adapter s used e The physical size of the pixels on the digital microscope camera s CCD sensor e Binning mode used on the digital microscope camera AxioVision and the active MTB2004 configuration allow you to take advantage of the benefits of automatic scaling If you are using a motorized or encoded microscope with a correct MTB2004 and camera configuration AxioVision will know which components have been used and the automatic scaling calculates the appropriate scale for you You do not need to do manual scaling Automatic measurements are very convenient and generally sufficient for most applications Manual scaling can still be done for all microscope types and in contrast to the theoretical automatic scaling a measured scaling takes very small manufacturing tolerances into account If you require highly precise measurements we recommend wor
7. If you do not use a clear field to set the Shading Correction any dark objects or features in the reference image will be seen as bright areas in all subsequent images as AxioVision will try to correct the apparent non uniformity This can give the impression that there is some dust in the microscope or on the camera surface Gain Factor If you have low light levels and find that your exposure time is unacceptably long you may use binning as described previously however this sacrifices resolution An alternative is to apply gain to the signal using the controls at the top of the General tab This will increase the sensitivity of the camera at all resolutions but in this case as previously discussed you will also increase the noise in the acquired image Sharpening By checking the Enable box you can apply sharpening to your image automatically upon acquisition The slider can be adjusted to determine the strength You can also choose to use the same sharpening tool as a post processing function from the Processing menu Tip Sharpening will alter the data contained within your image If you are subsequently going to analyze your images we recommend that you do not use this as a post acquisition function The post processing sharpening may be useful to make images look good for publication purposes Note Upon acquisition sharpening is not available for the AxioCam ICc range of cameras from Carl Zeiss 3 Saving and Archiving Images If
8. TRTIC channels are viewed as the original monochrome image the fourth image is the original images pseudo colored and merged By selecting Channel Color the individual channels can be pseudo colored i e the DAPI image will be displayed as blue A Flat AA Ai a WE Gia w a g apasa This example is a three channel Z Stack image and by selecting Z Stack it is possible to view the merged images at all z positions If the example image were a time lapse then selecting Time Lapse would allow all time points to be viewed simultaneously By holding down the Shift button and scrolling the wheel button of the mouse all images are zoomed in out If an image is selected it becomes identified by having a white spot in the centre of the image Once selected you can hold TEL Ce Ina Coleen down Ctrl and scroll the wheel TT button of the mouse to zoom in on each individual image at the same time 13 It is possible to use Gallery View to edit the Cowen image dimensions see Chapter 14 In the Men Gallery View a single image can be selected by ae clicking on the image a white spot is Bw superimposed on the image Additional images row can be added to the selection by holding down tt the Ctrl button and clicking on the required a images Complete Z Stacks can be generated by clicking on the first z position you want and whilst an holding down Shift clicking in the last z position ERA of interest In
9. image data sets for several fluorochromes through a focus range and for a series of time intervals They are then saved as a single file in ZVI format see Chapter 5 Note There is a further multidimensional acquisition option called Mark amp Find This is used with a motorized microscope stage to move to a series of pre defined positions prior to image capture However a description of this module is also outside the scope of this guide Workarea x ApoTome A no Click on Multidimensional Acquisition in the Workarea the example DE below shows all of the options however you may only have some of XK Multidimensional Acquisition them available Multidimensional Acquisition a Sanita The Experiment tab gives access to the general settings and the C Z 3 Experiment Z 1 and T tabs contain settings for Multichannel Z Stack and Time Lapse o s Goa respectively maSin eA ae The first thing to do is to enter an image name In this example the first z Sean en image set acquired will be called Experiment_1 a second image will be 3 called Experiment_2 and so on Experiment Before we look at the other experiment controls we will see how to set up Name zZ k Image name Experiment each of the Multichannel Z Stack and Time Lapse modules Show preview at Start Settings for Experiment Before experiment v After experiment v Go 26 12 1 Multichannel Even if you are not actual
10. images in another image format If you just want to export a single image then you can simply use Save As in the File menu and select the file format However if you want to export multidimensional images it becomes more complicated as each image in the ZVI file must be exported as a separate image In this case you need to use the function described below First ensure that the image to be exported is the active document i e click on the bar at the top then use Export in the File menu Refer to the online help for a detailed description of this function we will just cover the main points 14 perime nt Base name Experiment_14 C Documents and Settings russell hobson Desktop 506 Save in IV Create project Folder RE Mode Channel selection CA e 3 Z Stack at T Time CZ J Generate merged imagefs C Channels Use color for channel images Displayed image Use channel names Merged images only File type rs JPEG Standard ba Gray scale Compression s a Yo V Apply display mappings V Burn in annotations Set as default Output Files Name Status Experiment_14_c1 JPG Experiment_14_c2 JPG Experiment_14_c3 JPG Experiment_14_ c1 c2 c3 JPG Close Start Insert aBase name and a Save in location all images contained within the ZVI will be exported to a file with a name starting with the base name If you click Create project folder a folder will be created with the Base name
11. mode In this case you should move the microscope to the top and bottom of the range and then click on the Start and Stop buttons These have deliberately not been called top and bottom as is doesn t matter whether you enter the top position as Start and the bottom position as Stop or vice versa Generally you do not need to use this button as it may result in too many slices being acquired but it is not worthwhile sampling more finely than this setting However you should use this setting if you subsequently want to use the AxioVision Deconvolution option to improve the resolution of your images since double oversampling is a requirement condition for the algorithm See Saving Your Experiment and Acquiring the Images in the Multichannel section to capture the images 12 3 Time Lapse Click on the T tab to see the Time Lapse settings Note You do not need microscope automation to use this option x Experi B E Tou ax Bn IV Time Lapse In our example shown here we have chosen to acquire images from 10 time points one every 15 seconds Therefore the total duration of the experiment will be 135 seconds the first time point is at t 0 Interval Settings Maximal Speed 15 000 al Second zal r Duration Settings id Mn Maximal Duration Cycles 4 Calculate C Seco Duration Interval Alternatively we could click on Cycles in which case we would enter the interval and total du
12. these will use a specific interface called FireWire All systems supplied by Carl Zeiss will have appropriate connection for the Carl Zeiss range of cameras Some older camera types will requires specific capture board to be installed in your computer Some use a TWAIN interface which is a piece of software supplied with the camera and is used to capture the image You can start the TWAIN interface inside AxioVision and import the captured image The first thing to do is to check that AxioVision is configured for use with the camera you are using In the Acquisition menu select the camera eile Processing Annotations Measure Archiv Select amera j xio amHR AxiolamMr Cameras Basic adjustments j Sf Live 4 Snap Fz The AxioCam camera range are very flexible high performance digital cameras You can use them for bright field and low light level fluorescence imaging to acquire images at a range of resolutions A detailed explanation of the camera technology is beyond the scope of this guide Note Most modern cameras communicate with the computer via a FireWire cable however older cameras such as the AxioCam HRc and HRm Rev 1 and 2 AxioCams communicate with the computer via a fiber optic cable to reduce electromagnetic noise effects and therefore have a separate power supply You should take care not to kink this cable sharply If you need to disconnect the cable at either end then cover the exposed cable ends and sockets with th
13. you have worked through the previous chapters you can now capture some images to use as examples when working through this section If not example images are on the DVD delivered with your system You can open them using the Open Image option in the File menu In principle any image can be used you will see that all common image file formats are supported Capture an image from your camera then click on Info View at the bottom of the image window F athe eth Apolori E Amageststandand mageso Samples w and wa 2x with ApoTome m 2803 2006 10 51 44 Eavan Aam rages EC Plan Meofuar 2000 50 Ph 2 Mar 44 DAP BAP Dakfeld Apotomvel sm Exposure time ms 171 Acquisition date 07022005 13 21 20 Image format 16 bit gray bevele Irma Sir 1383 a 1080 Fisel Scaling Dar Mecrormolor Piel Scallng v 037 Micrometer 4 Pixel Salir 7 It Fixel J Pinal ap wew info view Tree view Galery view AAR AC anaw s The Info View shows information associated with your image If you have a motorized or encoded microscope some of the details will have been automatically entered You can enter further details as required including specific comments relating to your experiment for example Click on Save or Save As in the File menu The default folder will be the last folder used to save an image and is likely to be in the My Documents area of your computer Create a new folder in the
14. C TRITCitimeO HeLa_FITCS tif Not Ch Move Up E C Zeiss Images HeLa FITC TRITC timeO HeLa_FITC6 tif Not Ch Move Down _ 16 C Zeiss Images HeLa FITC TRITC timeO HeLa_FITC tif Not Ch C Zeiss Images HeLa FITC TRITC timeO HeLa_FITCS tiF Not Ch Remove 8 C Zeiss Images HeLa FITC TRITC timeO HeLa_FITCS tif Not Ch Po C Zeiss Images HeLa FITC TRITC timeO HeLa_FITC10 tif Not Ch Remove All Select Finish at the bottom to import the images Once the images have been imported they can be saved as ZVI files via File Save or Save as 32 14 Image Editor Change Image Dimensions This command allows you to edit a Multichannel Z Stack or Time Lapse image should some images not be required for instance a Z Stack may have extremities with little or no data Image Editor is opened from the Edit menu and Change Image Dimensions or by right clicking on an image and selecting Change Image Dimensions It is also possible to edit the image dimensions from the Image Gallery View see Chapter 4 Bi Step 1 Choose action selects whether selected images are to be kept or removed by ticking the relevant box V Remove selected images Keep selected images Select Channels Channel 1 Channel 2 r Select Resolution i a 1 2 wt i 2 z V Lock aspect ratio r Select Z positions IV Enable this action From 3 lt Get player position To 5 lt Get player position Every 1 Im
15. Counter Yalue e g 0001 Current hour 0 24 Current minute 0 59 Current second 0 59 Current year yyyy e g 2004 Current month 1 12 Current day 1 31 GUID Globally Unique Identifier System locale specific date representation e Additional token if available e g Grabber r Windows User Name e g admin Current document only available while repe E Image storage Folder for Auto save C Imagesi xiovision Abe Settings for acquired images Keep original Convert to 8 bit O Convert to 16 bit Snap C Auto save Save as type Carl Zeiss Vision Image zvi Multidimensional image acquisition C Auto save Ka Save as type Carl Zeiss Vision Image zvi C No archive for position list _ Use Piezofocus for small Z Stacks C Disable focus knob joystick during acquisition C Allow Focus Presteps for Z Stacks with small interval _ image processing callbacks can use hardware devices C Disable Default Handling of Definite Focus MosaiX acquisition Max MosaiX image size 8192 pixel C Prevent overview display during MosaiX acquisition There is also a built in short cut to save an image to disk with an incremental name In the Tools menu select Options and then the Naming tab Enter an image name prefix Select the number of digits for the incremental number e g 2 01 99 Choose additional designations from th
16. My Documents area called AxioVision Images and save your image here It will be saved by default in ZVI format As well as the optimized ZVI format AxioVision allows the image to be saved as a Tagged Image File tif a Microsoft Windows Bitmap bmp or as a JPEG compressed file jpg you can choose the level of compression required in the JPEG here You can also choose to compress the ZVI and TIFF image formats using the compression factor setting in the Save As window Very high values generate very small files However in this case image quality may be significantly reduced There is no compression available for the BMP file format Note TIFF compression is loss free and the file size is barely affected In the three other file formats TIFF Bitmap and JPEG you can choose to either Burn in annotations Apply display mappings and Convert to 8 bit This will be discussed further in Chapter 5 Note The advantage of using My Documents is that it is specific to the user logged on to the computer and therefore reduces the risk of confusion between users as you won t see another user s My Documents folder Once you have saved your image you can close the image window A Repeat this to acquire and save several images in the AxioVision images folder Now we Browser can use the browser to review these saved images Click the Browser button and navigate to AxioVision images The AxioVision browser will create small thumbnail images
17. R SFERENEFENEERSEEFEEDENECENORENDCENICENDEREDEENPEEECEERUEEREREREREREREFEREEN 34 1 Introduction to the AxioVision Environment AxioVision is a powerful and flexible software package for capturing archiving and preparing images for publication Whilst AxioVision can perform many complex tasks it can be customized so that you only see the information and controls you need Important information in Chapter 8 will explain how you can set up your system for easy efficient work flow For the purposes of this tutorial we need to set up a standard configuration Start AxioVision by double clicking on the AxioVision icon on your desktop and compare your screen with the picture below Note If you do not see this icon on the desktop then click on the Microsoft Windows Start button and look in the Carl Zeiss Vision program group 1 Agena Shor F r Fat View Micrasenpe Acqgen Pransseung Annatabera Mansur 30 Mesure Applications Evolve Archive Commander T iD B aA Ra Hr a ce Mew Open Dpi image Brows lt n Pant Gut Cay Pate lant Snag Sree har est Mmagatar Werle Withers t Wat j E Mcroscope ed Cameras T Axiocammics eS Incubaben Em Apes Tee 44 Srabag AL Wie field multichannel Linmonng Cammen Arheeied Transmed vz alalalalal Microscope Manager er Enable Parfneal Coneehon Rib oe Fe i If your AxioVision window does not look similar to the EE picture above select Options in the Tools menu
18. Takeoff Guide TATP ET Bre RE ye ae et FR A r ty PHP re fi LER i Tutorial to get started with the AxioVision Imaging System Based on Release 4 7 2 December 2008 This Takeoff Guide has been prepared for Carl Zeiss by Imaging Associates Ltd www imas co uk Copyright 2009 by Carl Zeiss Carl Zeiss Microlmaging GmbH 07740 Jena Deutschland BioSciences Industrial Standort Gottingen Postfach 4041 37030 Gottingen Deutschland Telefon 49 0 551 5060 660 Telefax 49 0 551 5060 464 E Mail micro zeiss de www zeiss de axiovision How to Use This Tutorial This tutorial has been prepared to help you get started with your AxioVision imaging system You are invited to work through the topics step by step Some of the functionality of AxioVision has been deliberately left out of the guide the idea is to explain the main trunk and branches of the program from an application point of view You are left to explore the twigs on your own For a description of the full functionality please refer to the manual or help file accompanying the system AxioVision can support various cameras each with their own characteristics however in software versions later than AxioVision 4 6 analog cameras are not supported In this guide only the AxioCam interface is described in detail Tips shown in italic text will give hints and short cuts Notes shown in italic text will give additional information and explanations
19. You can then open any image by double clicking it or by clicking on the Load button Change View le Tage File growser Desktop 4 My Computer H Control Panel E J Mobile Device EI See System ZH z See Images D H 4 ApoTome DVD a 4 ApoTome Images ee 29 axiovision takeoff images al 45 CZ Movies g JO FRET Images au of materials ApoTome E JO Standard Images Es GA DVD RW Drive Ei E S vision on aphrodite data U Se company on aphrodite see Composed Drosophila A Drosophila C Experiment Experiment You can view the information associated with each image by clicking on one of the Change View buttons try all three of these to see what they do use the arrow buttons to change image You can change the information associated with images here Images can be deleted using the red cross Tip If you save new images in this folder the thumbnails will not automatically be updated use the Refresh button to update all thumbnails eytology zwi 1K pearite zvi Leaf zvi You can open several images and view them in separate windows alternatively maximize one of the image windows and use the tabs to switch between images bon Use the macs button on the toolbar to open a gallery of open images bon Gallery Lay a Tip Hit F6 to swap quickly between open images cytology zwi zvi TREE Leaf zwvi _
20. age Step 2 Channel Selection allows you to choose which channels you want to remove or keep Step 3 Reduce pixel resolution allows you to dramatically reduce the volume of data when you are working with very large images without necessarily losing information that you need From the drop down list box for X select the desired reduction factor Step 4 Z Stack selection allows you to remove selected images from your Z Stack If you wish to skip over this step you can deselect the Enable this action check box To select the desired z plane images either type in the From and To fields the positions of the relevant images or to view images click on Get player position and select image s using the slider It is possible to select every nth image in the selected range meaning that every nth image will be kept removed Step 5 Time Point Selection allows you to remove certain time points Again this step can be skipped by deselecting the Enable this action button The method of time point selection is similar to the Z Stack selection Select Preview and or the Finish button The images generated can be subsequently saved via File Save or Save as 33 15 Glossary Analog Camera Annotation Binning Bit Depth Camera Interface CCD Channel Clipboard Context Sensitive Menu Co site Sampling Dark Current Deconvolution Dialog Box Digital Camera Drag and Drop Drop Down Menu Encoded Extended Focus Fluorochrome
21. and 7 Use Standard Menu and Toolbars Reset Window Layout then restart AxioVision Reset Window Layout Controls IV Enable docking J Small caption Commander Fr EJ lll MTB2004 Configuration Microscope Configuration Program MTB2004 AxioVision allows you to control and read the settings from motorized ZEISS microscopes Before you can do this you need to tell AxioVision about your microscope using MTB2004 This has probably been done for you when the system was installed and is outside the scope of this guide If you are not using a motorized or encoded microscope set the microscope to other Note MTB2004 has a Simulate Hardware check box You can use this to simulate a motorized microscope i e AxioVision will behave as if a motorized microscope was connected Referring to the picture on the previous page the areas you see in the AxioVision window have the following functions Toolbar these contain groups of functions belonging together e g annotation functions You can switch toolbars on and off by right clicking in a toolbar and you can drag them around the screen to dock them at the edges or leave them floating To keep things simple we have switched most of the toolbars off Workflow these are groups of functions put together to perform a particular task e g capture annotate and save an image You can make up your own workflows to make repetitive tasks easier to perform This is explained in C
22. and hence the color of the resulting image will also vary as the lamp intensity is changed AxioCam can automatically correct for this as follows AxioCamMR5 White Balance show Chern Move to a region of your sample which contains an area with no color i e white or light gray Click on Interactive As you move the mouse pointer over the image it will change to a dropper Now click on the area which should be white or light gray This will adjust the camera color balance to show the true colors of the sample These settings will remain in effect for subsequently acquired images however you may need to repeat the white balance procedure if you change microscope settings Alternatively click on Automatic in which case AxioVision will search for an area which is close to white and use it for correction The 3200K button will set the camera color balance for use with illumination of this color temperature This can also be considered as a default setting and should be used if it is not possible to do a white balance e g in the case of fluorescence imaging where there will not be a white area Tip You can manually adjust the color offset by using the slider thus making the image more yellow warm or blue cold Acquiring an Image AxioCamHR Dis Ad BE Gen Navig i tac con Nevo Click on the Frame tab on the camera control panel AxioCamHR Camera Mode OBiw RGB 1388 x 1040 standard color Set the res
23. aphics will be saved with the image when you close it If you re open the image you can view the measurement data by right clicking in the image and selecting Properties and then selecting the Measurement tab 25 12 Multidimensional Acquisition optional AxioVision is a very powerful image acquisition tool allowing multidimensional images to be captured Three optional multidimensional acquisition modules are available Multichannel Up to 32 images can be captured and overlaid with a separate pseudo color assigned to each channel This is generally used to capture and combine images of different fluorochromes or to combine fluorescence and bright field typically phase contrast images Z Stack Images are acquired for a range of equally spaced focus positions This allows imaging through a thick section or of a rough surface Often these images are further processed using Deconvolution to improve the resolution prior to 3D reconstruction or using extended focus techniques to create a single image containing the most in focus data from the Z Stack series Note Deconvolution 3D Reconstruction Inside4D and Extended Focus are optional modules available for AxioVision and a description of their use is outside the scope of this guide Time Lapse Images are acquired at equally spaced time intervals allowing dynamic processes to be observed and recorded These multidimensional acquisition modes can be combined so that we can acquire
24. ar in the image window otherwise select it from the gallery or load it from a file Once you have selected your image click Next Now select the Single distance calibration method Enter the calibration distance and units Draw a line on the image between two points corresponding to the calibration distance entered Finally enter a name for your scale and it will be entered in the scale list Note The calibration assigned is stored with the image Click on the scale bar button on the toolbar and drag a line on the image to draw a scale bar any length in x or y Use this to draw a scale bar on your image of a scale and check its accuracy remember to apply your new scale to the image first 16 Try loading another image from your archive apply this calibration and draw a scale bar lf you want to make your scales available to all users then copy the files from your user data folder to the workgroup data folder see chapter 9 for more information about this Note You can calibrate the system independently in x and y directions although this is often not necessary since the calibration factors will be similar in both directions However you should confirm this for yourself on your imaging system Changing the scale bar color Right click in the image and select the Properties option and the Attributes tab Click on Scale bar and change the text and line color attributes using the buttons at the bottom of the
25. capture Experiment Mame User DAPI FITL Go back to the Experiment tab Click on Save and enter a name for your experiment e g DAPI Image name Experiment FITC M Show preview at Start Now you can click on the Load button to reload this image acquisition setup Note ReUse allows you to use the experiment settings from an image captured previously Acquiring the Images It seems like we have put a lot of work in so far and still not acquired an image but it will all be worth it If you have a motorized microscope and have used the Hardware Settings to assign microscope configurations to each channel then all you have to do is to hit the Start button and your automated multichannel image acquisition will begin 28 Experiment ac ne If you have a manual microscope then select the first channel e g My DAPI set up your microscope for DAPI and click on the Snap button Repeat this for each channel Tip You can repeat and overwrite channels if necessary You can continue to acquire images and they will be automatically named for you Exposure Viewing Complex Images If you have acquired multidimensional images you will see additional display controls at the bottom of the image window HR RA R O O2 at e In this example we have a three channel Z o flee KDAT fe Stack image All three channels are overlaid z Drm and we are showing the first image in the Z Stack You can vi
26. channel right click on the tab again Extended Parameters We have now set up the basic channel parameters however there are more parameters available Click on Extended parameters to open the Extended Channel Parameters window shown below Enable Name My _DAPI My _FITC Here you can select more settings for the My DAPI channel we have just made For example we have an additional exposure setting which will start a series posure Time ms 40 on aoe Channel settings n with an automatic exposure and then use it Maximum pixel intensity 75 100 unchanged for subsequent images Shading Correction Use camera setting Use camera setting v Shading Correction Name a We might want to use these channels again in another Setting during acquistion User DAPI user DAP experiment so before you close this window click on SE son gt Save to pool to add each channel to the Channel pool 7 Dye FITC v ious re are m If you now click on Channel pool you will open the Focussing Current focus position Current focus positic w Channel pool and you can add any channel from the _ d pool into your current experiment by clicking on Add to Experiment E Channel pool la Save to pool Duplicate x Remove Fit to window Start Saving Your Experiment Once you have set up your channels you should save your experiment settings so that you can always repeat this type of image
27. d Di r Objective 5 5 A J E r Microscope Manager MV Enable Parfocal Correction V Enable Light Manager xi AA Use the options in the Microscope menu or Microscope toolbar 8 B 8 8 8 8 8 these can be left out on the desktop for easy access x D The potential of microscope remote control is realized when we start to save and recall microscope configurations This means that we can then construct image acquisition sequences using different microscope settings e g settings for various fluorochromes To save a microscope setting settings tr Activate the Settings Editor in Storad harinar settings T a oe m the Tools menu mew Rename Soe wka ch MD 7 Gonos er Poe a The example highlights a pe ee BER Setting for FITC amp User TITE nhs ig Reflected Light Piker L 1W0 100 Bprateted ugk von Click New to make a new tn setting Select microscope components from the right side and add them to the group of selected settings W nseroscone ul Reflector 049 ME Disktedfiteflechor is Dessen is Reflected ligt Rutter Open engen El Thumination Flucresarerece Eu Tragik ed Lag Sw Diwad tki emasa ni a Click on the Edit LJ button for each selected component to set it x x In this case the reflector is set to the TRITC block and the internal reflected light shutter is open Tip Be careful about the components you select e g w
28. e Format ID list e g U will add the Microsoft Windows user name In the Tools menu select Options and then the Storage tab The folder for Autosave can be typed in or selected via Microsoft Windows tree by clicking on In the Tools menu select Options and then the Acquisition tab It is possible to keep the settings for the acquired image or alter the bit depth lf Auto save is checked captured images will automatically be saved in the place identified in the Storage tab with the name identified in the Naming tab We recommend that you save the images as Carl Zeiss Vision Image zvi files but you can automatically export the images to different file formats If you assign a keyboard short cut to the Snap function you can acquire and save an image with a single key stroke 20 9 Backing up your data As with any computer system you need to make adequate back up copies of your data This consists of settings data and images You need to know where these are so that you can copy them to your back up media e g CD ROM ZIP disk or network folder As long as the images are stored in ZVI format they will contain all of the additional information and scaling information within the image file Backing them up is simply a matter of copying the image files to your back up media then you can either copy them back again to restore the data or use the browser to open an image directly from the back up media Options x
29. e caps provided to keep dust out 2 1 Getting Started Set up a sample under the microscope and ensure that the light is directed to the camera Activate the camera controls by clicking on the relevant camera button in the acquire menu or by selecting it from the work area In this instance we will be using an AxioCamHR Workarea 8 Microscope B Cameras Eu AxioCamHR 4xioCanMr 423 Scalings Processing Annotations Measure Archive Multidimensional Acquisition Select Camera j asf a gt Fast Acquisition pa H E Deconvolution cameras Cameras amare WA Widefield Multichannel Unmixing d BP Live Panorama Snap Fo Mosais a Image Processing F Lwe Next click on the live image icon on the toolbar to open the live image window You may or may not see an image in the live window Next we need to set exposure focus and white balance For convenience many of the routine controls such as measuring the exposure and automatic white reference are accessible from buttons on the toolbar in the live window or click on the live image a properties icon ZS to do the camera adjustments AxioCamMR5 Live W Dis Adjust Fr Gen Na AxioCamMR 5 Exposure 250us 60s 100 0 me sa a gt 5 120 100 m gt _ Auto Snap _ Auto Live AxioCamMR5 White Balance show chame Exposure The AxioCam range of cameras can adjust to various illumination levels by varying the ex
30. e would not usually want to save the focus motor position as this would always be used when we activated the setting Generally select the minimum number of settings required When using fluorescence you may find it useful to make a setting which closes the reflected light shutter to prevent bleaching e g called shutter closed In this case you only need the Internal Shutter in the closed position in the group of selected settings If you want to check your setting click the Apply button or Tools Settings User to activate it The microscope will then be adjusted according to the setting 22 Microscope settings can be added to the Workflow see Chapter 8 for ease of use Hude u 11 Interactive Measurement There are various advanced measurement options for AxioVision This chapter only describes the standard interactive measurement functions found in the Measure menu We will use the Interactive Measurement Wizard tte 30 Measure Applications Evi be Select Alt F1 a2 show Values Keep tool gt Magnetic cursor Open an image a Click Interactive Measurement from the Measure menu and Calipers then select Configure Tools Curve gt Outine AxioVision provides you with various tools for defining an area igs to be measured whether the radius of a circle the area of a angie circle or the distance between two points for example The lg Configure Tools dialog allows you to select measurement A Features fo
31. encoded device e g objective nosepiece can tell the computer which position it is in as distinct from motorized in which case the computer can also move the device Software technique in which the sharpest most in focus features from a Z Stack are combined into a single image Marker dye which fluorescence Template used to display information from the archive on screen Device usually a single board which digitizes a video image and stores it in computer memory Collection of miniature images which show the contents of all the image stores 34 Gamma Graphics Graphics plane Histogram Kohler Illumination Live Image Merge Microscanning Multichannel Nyquist Theorem Overlay Pixel Pseudo color Scroll Bars Shading Correction Sync Thumbnail Toolbar White Balance White Reference Defines the relationship between the intensity values in the image and the intensity of the display If Gamma is set to 1 0 then the relationship is linear Often a Gamma value less than 1 0 is used e g 0 5 Drawings and annotation drawn on top of an image Place where the graphics are drawn Graph of the number of pixels y axis against intensity value x axis Method for setting up the microscope condenser Direct display of the image from the camera Copy contents of the graphics plane into an image Technique used to acquire very high resolution images by shifting the camera chip Several sepa
32. ennnnnne nennen nnnnnne nennen nennen nnnnnnennn 12 Image Gallery MEW ae Reha 13 5 Image File Types and Exporting and Importing 4 20200202 14 6 Scaling and Scale Bars na 15 Changing the scale Dar Color ccc neh ne nn BE 17 AUlOMallc SCANING 2 2 eines 17 7 ANMOLALON SS ve een een 18 8 Workflow Customization amp Short Cuts 22u 0040 0 0 ann nenn ann nn nn ann nn 19 9 BACKING Up your Gala 21 10 AOMA UO a e A 22 11 Interactive Measurement u uz 00 0000annannannannun nun ann ann ann ann an nun nunnnnnnn 23 12 Multidimensional Acquisition optional 2022022002002000n0nn0n 26 12 1 Multichannel 2 3es een esevitext ee 27 1 0 6 Jo ee ee N ee ee 27 PAPO ARS SELUII OS een ee ee ee Res 27 Channel Actions c ccecececececcceccccccccccececececacauauuauauceaucecenenecenececenenenenensueneeaunenes 28 Extended Parameters cccccccececececcccccucececececeneauaceneneauauacateneneneanersteneneneanseansneneaears 28 Saving KOUrEXPEIIMEN ee ee nee 28 CO BIENEN SHINE ae iknnge 28 Viewing Complex lmages u A aoesdaaesacidess 29 12 2 2 Stack E EA a BS eae AT 29 12 3 AIG AD SC elie es a a a r E 30 12 4 General ReMmarks 2 Au a EE te ee eb ue 31 13 Importing Multidimensional Images from another File Format 31 14 Image Editor Change Image Dimensions 2 22202020n002n002 33 19 GOSS A PBEERAREIEEREIED RE
33. enu Select Properties to open the Properties window and select the Display tab You use this window to modify the way in which your image is displayed The histogram shows the number of pixels at each intensity level for each color RGB Contrast brightness and gamma can be controlled via the sliders or by dragging the curve on the histogram ki Click on Linear for the default display curve a Try selecting Min Max to stretch the contrast to the histogram maximum and minimum gt Tip Best Fit is similar to Min Max but it will stretch the contrast a little further since it ignores the extreme tails of the histogram This is set at 5 by default but can be changed Close the Properties window when you have finished adjusting the image Note images acquired with a monochrome camera will only have one histogram line relating to the grey values as there is no RGB data If you have modified the display properties of an image you will be asked if you want to save the changes when you close it but don t worry you haven t really changed the image data The updated display properties are saved with the image so that it will be displayed in the same way if it is reopened but the image data has not been changed and the display properties can still be put back to linear Image Window Toolbar The toolbar at the bottom of the image window provides shortcut buttons for zooming and display adjustment Note You can set the display properties for t
34. ew different slices using the Z scroll bar or the player buttons to the right To view a selection of the channels click on the corresponding colored button e g click on button 3 to switch it off To change the pseudo color for any channel right click on the corresponding button and choose your color eo if you click on this button you can view each channel separately without pseudo color You must switch to this mode before you can adjust the display settings as described in Chapter 4 12 2 Z Stack Click on the Z tab to see the Z Stack settings Note You must have a motorized focus on your microscope to use this option X Experiment re Fz te T _ Z Stack Settings There are two ways to define the focus range in the example shown we Slices 3 have clicked on the Center mode Sedina L m Move the microscope focus to the centre of the focus range this is a matter for your judgment Mode Center Start Stop a Click on the Center button to enter the current position 0 000 am um Now decide how many slices you want to acquire and the spacing 2 Stack height 2 000 um between them and enter the values In our example we will acquire 15 all channels per Slice slices spaced at 0 5 um This means that the center slice will be number _ 2 Stack at current focus position 8 and we will acquire 7 above and 7 below this position Z Stack navigation a reed 5 a You can use the Z Stack navigation controls at the bo
35. hapter 8 Workarea this is a quick way to access frequently used functions and control panels e g microscope control or scaling Document area this is free space for images and information Workflow Workarea You can hide or show the Workflow and Workarea using the buttons on the toolbar Image Types AxioVision is a very powerful image acquisition environment Most of the features will be explained using standard images i e single color or monochrome images However AxioVision can acquire more complex image types as follows Multichannel multiple monochrome images which can be pseudo colored and superimposed usually acquired for various fluorochromes or contrast techniques Z Stack A series of images acquired at different focus positions Time Lapse A series of images acquired over a period of time MosaiX A series of overlapping tiled images acquired over a defined area Mark amp Find A series of images acquired at different pre defined positions on a sample These image types can also be combined e g as a multichannel z stack The acquisition of complex images is covered in Chapter 12 2 Image Acquisition using the AxioCam Camera The first step is to capture an image from the camera attached to your microscope If you are working on an AxioVision system which is off line i e not connected to a camera you may want to skip this section AxioVision can acquire images from a variety of cameras most of
36. he live mage window The acquired image will inherit these properties 11 Image Splitter Display Window ae You can also compare up to 12 images using the splitter window The Splitter button is on the standard toolbar or can be accessed via the View menu In the View menu select Windows and Splitter Display With your images already open click on the Gallery button to select the image for each pane in the splitter window Check Synchronize to adjust all of the images simultaneously Ed Sotho Dispdar 6 Images 8 Images By clicking on this button an additional window containing some Extended settings is opened From this window it is possible to create images containing the selected images side by side If the original images are Z Stack images then the entire Z Stacks can be xi viewed side by side by selecting Create Z eee TT pixel Createimage Stack image It is the same for time lapse es ae TENN a reste tinelepseimage Mages just select Create Time lapse image You can also control the spacing Interpolation for zoomed images cubicspline Create Z Stack image between the images and the color used to Burn in annotations IV Close Splitter Display after image creation Cancel sepa rate the ali g ned ima g es 12 Image Gallery View The Gallery view enables the individual images in a multidimensional image to be viewed simultaneously In the image below the individual DAPI FITC and
37. king with measured scalings Automatic scaling can be turned on and off by navigating to Measure on the Menu toolbar 17 7 Annotations B Select E rn Scale bar Text Line Aligned Rectangle Alt F2 Outline I Curve gt Ellipse Fl LUT Scale Frequent Annotations Burn in Annotations Draw Annotations View Annotations gt Show bounding box AIt F1 ee j 20 mm Info View Tree View Gallery View Solfi mm 55 We have already annotated an image by adding a scale bar You may want to add further annotations using the options in the Annotations menu or switch on the Annotations toolbar see Chapter 8 Select the drawing tool you require and then draw on the image Click the right mouse button to finish drawing a shape If you want to change or move the graphics then click simply left click on the item and move it The colors and fonts for each graphic object can be set independently by right clicking in the image and select the Properties option and the Attributes tab see Chapter 6 adding a scale bar Tip Use Undo and Redo in the Edit menu to correct mistakes Note If you store the image in ZVI format the graphics are stored as separate objects with the image i e they can be changed at a later date For further information on image file types see Chapter 5 Frequent Annotations Use this option to add annotations to image sequences e g time lapsed since star
38. l here The features chosen in the configuration step are now displayed Toolbar Interactive Measurement module Outline spline tool ita a ae SE ay ra 3 Line Length Calipers Distance Multi Calipers Multi Distance Point relative Points relative Curve Curve Spline i Select Circle Circle Out In Circle Points Alig Rectangle Rectangle Outline Outline spline 1 Outline 5 Area Pixel 6679 08 fe Tool Feature Value Draw around the features to be measured Click OK to close the measurement window The desired measurement data is now shown on the image The data can be viewed by right clicking in the image and selecting Properties and then selecting the Measurement tab as shown below Properties FluoCells_DCY_CI_res_201c3 JPG x ry Display A Attributes Le Colors L Info A Profile ta Histogram Measurement To Clipboard _ Tod Feature Value bi Outline Spline Area 6748 37 Create Table 2d View C All Slices Alt As Table Here you have the option to send the data to the clipboard for subsequent pasting into another application such as Excel Below are some typical region measurement descriptions and their meaning DensStrR Standard deviation of intensity of red color channel DensStrG Standard deviation of intensity of green color channel DensStrB Standard deviation of intensity of blue color channel The measurement values and gr
39. ly using more than one channel you should still review this section you will need to set up a single channel to be able to go on and use the Z Stack or Time Lapse modules Click on the C Channel tab to see the Multichannel settings Ig Experiment Qi z GT Note You don t need any microscope automation to use this option Coke m un If this is the first time the option has been used you will see a single ee channel 1 Snap T Entered parameters Exposure Time 40 a nu measure Hoda Mtn i Fie Camara Settings In this example we have selected DAPI from the list of dyes If your dye is not listed you can type any Dye name in this box ur The blue color is associated with DAPI but you can change it if you wish Oo Led Now we can enter a name in this case My_DAPI Very often the name will Er be the same as the dye but we may for example have two DAPI filter sets en and want to call them DAPI_Narrow and DAPI Wide id Swonpu K Gee Tip Make sure your AxioVision window is large enough to show all of the buttons as shown in the example image Saal Exposure This allows us to set the camera exposure time for this channel You have three options Auto the exposure is calculated automatically before the image is acquired This will always give you a well exposed image but the exposure time may be different for each image and a brief additional calculation exposure is performed before each acquisition
40. n and electronic gain rather than a long exposure when in live mode Thus the live image you see will be relatively low in resolution and noisy but will be updated frequently enough to allow focusing and stage movement The final acquired image will be at full resolution as set in the Frame tab of the camera driver interface see page 6 ee You can vary the update speed of the live image by clicking on the button on the toolbar at the bottom of the live image fast medium and slow options Live image quality will decrease as update speed increases a 8 a a d aA wka Oo amp Snap Slow Exposure White balance 4 Best Fit A Min Max Linear G 0 45 OverExp Properties Automatic v AxioCams also have a focus meter Right click in the live image window to toggle between spot meter focus bar on off As you change focus the green bar will move the red line shows the peak value gt PER reached The focus meter measures the Eo o sharpness of edges within the green box To Focus meter p get the best results you should position the box over an area containing some structure or detail the focus meter cannot work when the focus box is positioned over a uniform background area Tip This region is also used to set the illumination level for the exposure Measure function White Balance applies only to color cameras The color spectrum of the illuminating microscope lamp varies with voltage
41. ndow E Help E Acquisition Reset In the Tools menu click on My AxioVision Select the Workflow tab Click on New Workflow and enter Image Capture as the name Click on the Image Capture workflow on the left side of the dialog box Now we can add tools to the workflow by selecting them from the right hand side and clicking on Add In the example above we have added Live from the Acquisition group Snap from the Acquisition group Navigator from the View Windows group Scale bar from the Annotations group Save from the File group You can change the order of the tools on the Workflow using the Move Up Move Down buttons moge cepte Close the dialog box and you will see the Image Capture workflow added to the Workflow area Eo Workflows can be exported see Chapter 9 on backing up your Snap data Co Tip Further discussion of customization is beyond the scope of iii this guide However you may like to try assigning a shortcut Hri key to a function using the Keyboard tab in the My AxioVision oni dialog box A 19 General Layout Folder Storage Acquisition Naming Display ID Commander General Layout Folder Indexed Names Category Image processing output Image prefix IP Digits l2 z Format P Hoh ol Initial counter value 1 Save restore counter value Format IDs double click to add to format Info Prefix Current
42. ns each time You can temporarily disable the Z Stack or Time Lapse elements by un checking the boxes This is useful to grab a single image as a preliminary check We described how to attach microscope setting to a channel in the Multichannel section You can also attach a microscope setting before starting the experiment and after e g you might use a motorized binocular head to switch the light to the camera before the experiment and back to the eyepieces after the experiment 13 Importing Multidimensional Images from another File Format It may be necessary to import images into AxioVision If the images are single images in any of the file formats mentioned in Chapter 5 they can simply be opened using File and Open or Open Image However you may wish to import more complex Multidimensional images In the File menu select Import to ZVI This wizard allows you to import various file formats in any combination of Multichannel Fluorescence Z Stack and Time Lapse Step 1 Sebup Image Dimensions Step 2 Channel Setup Steg a stack parameters Step a T Parameter Setii Step 5 Cige ac Step 6 Preview Resu ER Step 1 Setup Image Dimensions identify the NEN es type of image to be imported Multichannel Fluorescence Z Stack or Time Lapse For the HF rac purposes of this description all import steps will be discussed In Scale lateral X box the number w of um pixel can be set It may be necessary to create a differen
43. olution to 1300x1030 and click on the p icon For convenience you can also find this on the toolbar 4xioCamHR Frame at the bottom of the live window You can now close the live window and you should see the high resolution acquired image Tip Right click in the live image and select Close after Snap to automatically close the live window after acquiring an image Now that you have successfully acquired an image we can Framestart 170 j 150 center take a look at some of the other features of the camera Framesize 1048 x 766 Fu Memory used 4 59 MB 2 2 AxioCam Features Resolution Click on the Frame tab on the camera control panel All AxioCams allow images to be acquired at a variety of resolutions i e number of pixels The AxioCam HR uses patented technology to achieve high resolution and color quality by scanning the CCD chip Choose a resolution to suit your requirements You may want to capture a very high resolution image for publication or you may want to save disk space and processing time by capturing a medium resolution image Although very high resolution images can be acquired many people find that 1300x1030 pixels are sufficient for every day requirements If you are working with very low emission level fluorescence samples you may find that exposure times become unacceptably long and sample bleaching becomes a problem In this case you can binning Binning is a method of alte
44. oved by correcting for the dark current i e the level of signal when no light is reaching the camera Close the light path to the camera it is essential that no light can get to the CCD camera and click on Black Reference Now the camera takes a few seconds to acquire an image at the maximum resolution which is then used to correct subsequently acquired images This is only necessary for exposures greater than approximately 2 seconds Re open the light path to the camera AxioCamMR5 Sharpening Cerati AxioCamMR 5 Shutter _ Enable for Snap _ Enable for Live Control Signal Low active fo m AxioCamMR 5 Filter Operations Acquisition Delay Shading Correction If you acquire an image from a clear field e g a clear glass slide you may find that the image is not uniform e g it may be slightly brighter in the centre of the field than at the edges This is due to optical aberrations and variations in illumination The Shading Correction can correct for this non uniformity eS e Move to a clear part of your Ben aeaa microscope slide and click on Shading Correction You may want to slightly defocus your microscope to remove any small specks When the Enable box is checked all images will be automatically corrected for shading non uniformity Note The Shading Correction is correcting for non uniformity of illumination shading across the image whereas White Balance is adjusting the color Tip
45. posure time Ensure that the Auto Snap box is not checked and then click on the Measure button to let the camera find the optimum exposure level You can further adjust the exposure manually using the top slider control the range can vary from 400us to 60s depending on the type of camera If you find that image brightness after using Measure is always too dark or too bright for your purposes you can adjust the level e g set to 90 for a darker image underexposed or 110 to overexpose using the lower slider control If you have Auto Snap box checked the exposure will be adjusted automatically before each single snap Checking the Auto Live box enables continuous exposure time adjustment in the live image AxioCamMR5 Color Saturation Saturation 9 9 0 10 gt een inn Note The region of the image used to measure the exposure is the same as the focus region see following section If the focus window is inactive then the whole image will be used Therefore you may want to position this over the regions of interest before clicking the Measure button to set the exposure Focus Focusing is often difficult when using high resolution or low light level cameras Because of the time required to transfer high resolution images to the computer or the long exposure times required for low light levels it is not possible to update the live image frequently enough To overcome this the AxioCam range of cameras use a standard resolutio
46. r each individual drawing tool You can also Points choose which measurement values are shown as a graphic overlay by choosing Draw Features from within the specific Online Measurement gt A Load Toots feature Once the settings have been edited they can be y ace Nesnenin Ye Sevvesseos saved for either individual users or for the workgroup FQ Append Table Automatic Measurement gt BE Measure Profile BE Measure Histogram Automatic Measurement Programs gt 423 Scalings Automatic Scaling 23 Drtinitten of the messurement base Inabures Douek a heel inet oy Gani then deine een aed drow baluti Seino mecca ot draw beatae ane ee Tee bo eld to tht cunendly ee bol mr ie i ESE res J Configure Tools To use a configuration select Measure Interactive Measurement Load Tools Load Tools Click on to browse the various saved configurations J Start Measurement and select the one you want to use Click OK J Configure Tools Click on select Measure Interactive Measurement Start BM oad Took Measurement This opens the active image in the interactive measurement window as shown on the following page start Measurement Toolbar AxioVision Basic Measurement Select the tool from the toolbar and perform the measurement Measurement results will be displayed in the list Hi Length Distancefum 24 Select a drawing tool we have indicated the Outline spline too
47. rate images usually fluorochromes captured from the same field within a sample using different filter sets and overlaid States that to measure a given frequency you must sample the waveform at twice that frequency in terms of imaging it means the pixel size should be half the size of the theoretical resolution See graphics plane Single element of a digital image The value of a pixel sets the intensity or color of the point in the image Artificial color applied to monochrome images Controls at the side of an image or window which change the window view point Correction of an image for illumination or background non uniformities Synchronization signal In the case of a monochrome signal this is usually added to the video signal composite sync In the case of a separated R G B color signal the sync signal may be added to the green channel or it may be separate Small copy of an image used in image galleries etc Collection of function buttons Process by which the image from a color camera is adjusted to give true colors Background image used for shading correction 35
48. ration and then the number of cycles would be automatically calculated Note The interval is measured from start of image acquisition to the start of acquisition of the subsequent image If you are acquiring multichannel Z Stack or long exposure images at each cycle you must ensure that the time 135 00C pe Settings before after Timepoint before time point after time point required to acquire the images does not exceed the interval between acquisitions See Saving Your Experiment and Acquiring the Images in the Multichannel section to capture the images 30 12 4 General Remarks Workarea ApoTome 423 Scalings Deconvolution R Widefield Multichannel Unmixing H HDR 3 4 Multidimensional Acquisition Multidimensional Acquisition Aa Smart Experiments Experiment QB C 2 B 1 Options g lt lt 1Channel Dzstak Z 3 1 000 um CS Tmelape T 1 1 000 sec 5 Experiment Name ie Image name Experiment Show preview at Start Settings for Experiment Before experiment J After experiment lt Returning to the Experiment tab there are just a couple more things worth pointing out Saving and reloading your experiments makes it very easy for a less experienced person to use the system This is particularly useful for multichannel acquisition for Z Stacks you will probably have to reset the Center or Start Stop positio
49. ring the camera resolution by combining square groups of pixels 2x2 3x3 etc and outputting them as one pixel in the image The result is a proportional increase in sensitivity with an associated reduction in resolution Binning has the added advantage of increasing sensitivity without increasing noise Frame You may want to capture a region of interest from the camera rather than the full frame Click on Refresh Overview to capture the full image into the window on the right then click on Select Frame and select a window size Now move the window around the full image to an area suitable for focus fine adjustment You can interactively resize the region of interest Monochrome or Color If you are using a color camera you can switch it to monochrome mode by clicking the B W button This is useful when working with fluorescence samples Note Switching a color camera to monochrome mode does not increase its sensitivity Monochrome cameras have several times more inherent sensitivity than color cameras as there is no color mask in front of the sensor chip Black Reference AxioCamMR5 Live AxioCamMR 5 Digital Gain FEST Index Gain factor 1 2 Index 0 a AxioCamMR5 Image Orientation Original v AxioCamMR5 Black Reference Black Reference AxioCamMR 5 Shading Correction Shading Correction Cam Auto _ Automatic Click on the General tab on the camera control panel Your image can be further impr
50. t followed by automatic scaling Select Scaling from the Workarea Here you can select an existing scale and apply it to the currently open image or activate it in which case it will be applied to all images subsequently captured 15 Scaling Available scalings Name Scaling Y Scaling ca i b rate A Pixel 1 00 Pixel 1 00 Pixel W _Automatic__ 1 00 Pixel 1 00 Pixel 10x EC Epipla 0 65 um 0 65 um 2 5x EC Epipla 2 53 um 2 53 um 20x EC Epipla 0 32 um 0 32 um 50x EC Epipla 0 13 um 0 13 um 5x EC Epiplan 1 30 um 1 30 um Pixel 1 00 Pixel 1 00 Pixel Apply to image Select methad Kg Fl Single distance r Distances in andr Single parallel lines 7 Parallel lines ines and r Rectangular grid HE Automatic line detection calengs Wizard E lol x Uhslarcns Pinan dia the les in the image ond argy che disiances AEI Drege z RAR Daeehnn N Diae Facini Era rea rie Miniometen il 0 10 20 30 _ o Back Finish Cancel Before we can make a new scale we must acquire an image of a micrometer scale using the objective lens we want to Note Scaling should be done using the standard camera resolution approx 1300x1000 pixels If you then switch the camera to a higher or lower resolution a new corresponding temporary scale factor will be created Click Advanced and then New to start the scaling wizard If your scale image was active it will appe
51. t of a time sequence 18 8 Workflow Customization amp Short Cuts Having mastered many of the AxioVision functions you may want to customize the interface to make repetitive tasks easier and to help out other users who have well defined requirements The best way to do this is to hide all unnecessary toolbars right click on a toolbar and make a workflow to suit your application It s very easy to do and may save a lot of time in the long run You can make as many workflows in AxioVision but note that they are specific to the person who is logged on to Microsoft Windows This has the advantage that each user can set up AxioVision to suit their needs If you want to make a Workflow for somebody else you will need to use their log in and then start AxioVision before you make the workflow My AxioVision board Workflow Dialogs Icons Microscope Workflow Available commands 2 TIC Measurement w Workflow Macros Partide Analysis Scripts start interactive 3 67 Standard 5 Scripts start automatic Q Measurement a Settings 6 Grains EE User Dialogs NMI Measurement 3 Menu 29 Graphite 3 File 9 Multiphase Edit 29 Online Measurement ES View 9 Image Capture Microscope a Live H EH Acquisition Snap Remove gt Processing Co Navigator Annotations a Scale bar Measure la Save 3D Measure E Applications E Evaluate E Archive 2 E3 Commander E Tools 4 ES Wi
52. t y scaling which can be achieved E E um by ticking the Different Y scaling box If the offer Y cag um pixel relationship is unknown the scaling foes me dialog box can be ignored but any subsequent measurements will be incorrect Click Next 31 Step 2 Channel Setup allows you to choose up to 8 different Fe Ss fee image channels in which you can select the dye assign a color and C O name the channel Additional channels can be added with Add es DEE D channel click Next when all channels have been identified Gr me SSS a Ss d PEE l channel Renee chants Bee Step 3 Z Stack parameters allows the number of slices and the ie distance between theses slices needs to be entered Alternatively e 10 the number of slices and the Z Stack height can be used It is 2 Stack height 10 00 4 um r Extended parameters possible to add Extended parameters including the magnification numerical aperture of the lens and the immersion medium Again if any of these parameters are unknown the import process can proceed but any future calculations which use these values will be affected Enter extended parameters Magnification fi x Numerical aperture 0 01 EF Immersion medium fo lt Doreen am a Step 4 T Parameter Setup enables you to enter the number of Cycles fo cycles and either the time interval between each image acquisition Duration m or the time dura
53. this example below all three channels in six z positions have been selected When Extract Selection is clicked the selected images will be extracted and inserted into a new image S Eee ie ee If you select Create Image a new image of the current view will be produced Zn ea bfo Oit Wie Tape iiem Galery vma 4 AAA SD a Ms om 2 5 5 Image File Types and Exporting and Importing So far we have saved and loaded our images as ZVI files Now it s time find out how to export the images in more common file formats which can be used by other imaging and document processing packages A number of different file types or formats have been developed for storing images to disk AxioVision supports all major formats However its default native format is a new image format ZVI Why develop yet another image format As imaging technology has advanced so have the requirements of users therefore the current formats are not able to offer the flexibility required For example if you annotate your image with a scale bar and text you need to burn this information into the image when it is stored as a TIFF file thus the annotations can t be removed or changed at a later date The new ZVI format keeps all graphic information separately so that it can always be changed and the underlying image is not destroyed The ZVI format can also handle the full range of bit depths multichannel images and image sequences How do I export my
54. tion of whole experiment Step 5 Compose Images allows you select the files to be imported Locate the position of your stored files and select the images to import Once all of the images have been selected click Get Selected The selected images now appear in the bottom half of the panel Click on Consistency to check that the images are compatible At this stage it is possible to check the images that will be imported by viewing them in Preview Select Images Current Drive Preview Image information 7 timed 7 t HeLa_FITC1 EHeLa_FITC10 ElHeLa_FITc2 PP HeLa_FITCS BIER ElHeLa_FITC i HeLa_TRITC2 HeLa_TRITC3 i HeLa_TRITC4 HeLa_TRITCS i HeLa_TRITC6 HeLa_TRITC HeLa_TRITC8 i HeLa_TRITC9 IV Display Preview My Documents abe FITCS Not available gt HeLa_TRITC1 ee i HeLa_TRITC10 Places Files OF Type All Picture Files bmp tif jpg j2k tga png psd cmp pct ras eps wmF m Y Selected Images To In t2 13 14 Is tz te r BB irc IL TRITC E Image Consist ac Get Selected Jo C Zeiss Images HeLa FITC TRITC timeO HeLa_FITC1 tif Not Ch ql C Zeiss Images HeLa FITC TRITC time0 HeLa_FITC2 tif Not Ch Consistency J2 C Zeiss Images HeLa FITC TRITC timeO HeLa_FITC3 tiF Not Ch E C Zeiss Images HeLa FITC TRITC timeO HeLa_FITC4 tif Not Ch 14 C Zeiss Images HeLa FIT
55. ttom of the 0 000 pm control panel to step the microscope to each position Z Index E Tip Click this button to see a live image for focusing Settings before after Z Stack before Z stack v Go J after Z stack 29 Mode Center Skart Stop oo bm L Ea i 2 Stack height Lin Tip If you are combining multichannel and Z Stack acquisition AxioVision will normally acquire all the z images for channel 1 then all the z images for channel 2 and so on This gives the fastest acquisition but may suffer from slight z registration problems However if you check All Channels per Slice AxioVision will acquire all channels for the first z position and then move on to the next z position and so on This will give exact z registration but usually increases acquisition time The Optimal distance button will set the ideal slice spacing to match the z resolution of the optics you are using This is also known as the Nyquist criteria as it takes into account the double over sampling required by this theorem It depends on the numerical aperture of the objective lens and the wavelength of the illumination Therefore you must have an encoded or motorized objective nosepiece which has been properly configured so that AxioVision knows which objective is being used You must also have entered all the dyes you are using in the multichannel setup The alternative way to define the focus range is Start Stop
56. which may increase sample bleaching Note You should not use this mode for Time Lapse if you want to make relative intensity measurements Also you should not use when capturing Z Stacks if you want to subsequently use Deconvolution Fixed the value in the Time box is used To find out what this value should be click on the measure button Camera the current exposure value set in the camera control dialog see Chapter 2 Hardware Settings This section only applies if you have a motorized microscope First you must set up microscope settings for the dyes you want to use see Chapter 10 Now you can attach a setting to be used for acquisition to this channel e g move filter block to DAPI and open the reflected light shutter and a setting to be used after acquisition e g close reflected light shutter If you have the Autofocus optional module installed you can perform an autofocus before each acquisition Otherwise the current focus position will be used Tip Click on the Go buttons to check the hardware setting 27 Channel Actions Once you have set the channel up you can save it to the channel pool Save to pool and subsequently load it Click on Duplicate to make a copy of My_DAPI You can change the settings here from now on for example to set up a FITC channel Tip You can temporarily inactivate a channel by right clicking on its colored tab The tab will gray out and have a cross through it To re activate the
57. which will contain all exported images Mode allows you to export some of the image e g a specified channel time z position or all images Generate merged images will combine multichannel images as overlaid color images File Type select the output file format Convert to 8 bit only seen when saving as a tif will convert a higher bit image down to an 8 bit TIFF Gray scale will convert a color image to a black and white image Apply display mappings will retain the current display property settings see Chapter 4 Burn in Annotations will merge any graphics into the image before it is saved Click Start to export the images Tip To convert more than one image select File Export and click on Batch Choose Add files type select the files to be exported then click Run batch How dol import an image from another format AxioVision will open all of the common image file formats mentioned in Chapter 3 simply by selecting a file using File and Open Image You can subsequently save the image in the ZVI format If you want to import a sequence of images such as a Z Stack then refer to Chapter 13 6 Scaling and Scale Bars There are two ways of creating scale factors in AxioVision Firstly you can manually define and store scale factors using images acquired of a stage micrometer Secondly you can use automatic scaling and allow AxioVision to calculate the scaling factor for you Here we will discuss manual scaling firs
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