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NIS-Elements Advanced Research User's Guide (Ver. 4.00)

1. a New Press this button to create a new ROI According to which detection tool is selected you can either start drawing a new ROI or click inside the image to let the application detect the ROI automatically ROI picker Select which ROI you want to edit from the pull down menu After Edit Specify which action is taken after a ROI has been edited The Jump Forward option displays another time frame according to set number of frames in the Skip pull down menu Skip Choose how many frames are skipped after an object is edited and the Jump Forward option is selected Skip 1 frame just moves to the nearest next frame E Delete Press this button to delete selected ROI Undo Redo Moves back and forth through the history of changes 102 Image Analysis T Help Displays help file E Finish Press this button to finish definition and close the dialog 6 6 Creating Reports Report Generator enables the user to create customized reports containing images measured data user texts graphics or database entries PDF files can be created directly from NIS Elements AR When you invoke the File gt Report gt New Blank Report command a Report Generator window appears Note Use Adobe Reader to display PDF reports created by NIS Elements Correct performance of other reader software products can not be guaranteed Report Generator untitled report File Edit Page View Help DERRER ajaan Sample number gt
2. Please see the electronic help for more details 9 5 Database If the database module was installed the system can create and control MS Access databases MDB A database can help you to efficiently organize your image archives and manage additional information related to the images The images themselves are stored on hard disk only links to them and the additional data are stored in the database file After you install the Database module the Database menu appears in the main tool bar Please see the electronic help for more details 157 9 6 AutoQuant Blind Deconvolution The imperfection of a microscope optical path causes convolution of the original image signal which makes the resulting image blurry Deconvolution algorithms reduce the impact of convolution on the image Even though Blind means that we do not have to know the convolution parameters accurately some characteristics of the optical path objective shall be known in order for the deconvolution module to work properly Please see the electronic help for more details 9 7 AutoQuant Real Time 2D Deconvolution The imperfection of a microscope optical path causes convolution of the original image signal which makes the resulting image blurry Deconvolution algorithms reduce the impact of convolution on the image The Real Time Deconvolution module algorithm enables high speed processing and therefore is very suitable for use on Live signal from camera
3. The list of optical configurations can be ordered manually using the arrow up and arrow down buttons 42 Setting the Hardware Up e Each configuration can be arbitrary modified within the right side portion of the window 3 5 Objectives If you want to perform measurements on captured images it is wise to calibrate all objectives used to capture images Whenever an image is captured through a calibrated objective the image inherits its calibration 3 5 1 Managing Objectives Run the Calibration gt Objectives command The Objectives window appears Storage _ Calibration Shared 0 002 mm px 2560x1920 Zoom 1x Relay lens 1x Shared 0 001 mm px 2560x1920 Zoom ix Relay lens 1x Shared 0 0002 mm px 2560x1920 Zoom ix Relay lens 1x 0 00007 mm px 2560x1920 Zoom 1x Relay lens 1x 0 00003 mm px 2560x1920 Zoom 1x Relay lens 1x 0 0002 mm px 2560x1920 Zoom 1x Relay lens 1x 0 003 mm px 2560x1920 Zoom ix Relay lens 1x 0 00009 mm px 2560x1920 Zoom 1x Relay lens 1x 0 00007 mm px 2560x1920 Zoom 1x Relay lens 1x 0 0008 mm px 2560x1920 Zoom ix Relay lens 1x 0 0003 mm px 2560x1920 Zoom ix Relay lens 1x LIL Plan EPI 2 5x LIL PLan EPI SLWD 20x LULU PLan EPI ELWD 50x Eco DL PLan EPI SLWD 100x Eco DL PLan EPI 20x CR LIL Plan 1x EPI LIL Plan 40x EPI DL PLan EPI SLWD 50x Eco Plan Apo 4x Plan Apo 10x DIC L HI 3 3 A 0 00009 mm px 2560x1920 Zoom 1x Relay lens
4. 5 1 3 2 Events User events can be pinned to any of the frames of the image in order to highlight a remarkable scene There are number of event types Some of them are inserted automatically while the ND experiment is running others must be defined by user by the 1 button Once there is one or more events within the time line you can manage them via a context menu Right click the event marker a menu appears with the following options Remove All Events Event Selection Range Event Manager Remove Event Edit Event 67 When you open the Event Manager all events within the nd2 file may be edited through the following window The time can be put in and the event type selected from a combo box See Time lapse Acquisition Event Manager 23 Time s Event Typ Descripton e 10 927 Autofocus Autofocus performed e 10 927 Command Executed e 18 687 Command Executed Contrast e 18 687 Experiment Paused Define Events 5 1 3 3 Views There are several views which can display the nd2 files in various ways Some views are available for some dimensions only If a view is available for two or three dimensions of the nd2 file a pull down menu appears in the top image tool bar There you can select the dimension to be displayed Note When you switch the view a new image window opens by default This behavior can be changed within the Edit gt Options command window so that only one view of a image wil
5. The Installation DVD ROM Content The installation DVD ROM contains e NIS Elements software setup file e Drivers and utilities for the HASP Key e Drivers for selected cameras e User s Guide in the PDF file format e Sample image database e Sample ND2 image sequences The Installation Process Caution You have to possess the administrator rights to your computer to be able to install NIS Elements AR successfully 1 When the installation DVD is inserted a selection window appears automatically imaging Software Elements Advanced Solutions for your imaging Word Select the software package to be installed Select the one you have got the license for and which is properly coded in your HASP key The installation wizard welcome window appears Click Next to continue 2 Local Option Destination Folder C Program Files HIS Elements Select whether to install Local Option or not Define the folder where NIS Elements AR should be in stalled We recommend to use the predefined directory If you want to change the directory anyway press the Browse button and select a new one Otherwise click Next Note The Local Option installation provides some advanced features which did not pass the quality assurance procedure yet We recommend to wait until they are released officially 3 Manufacturers Nikon oo SO O O Andor Fhotometrics Hamamatsu Almadging Imaging Source Installation and Startup
6. 3 4 5 6 Application Appearance User rights User Authentication Windows NIS Elements Password protected _ Allow create new user on startup Current user Karolina Macuchova Log As Users Groups Privileges New X Remove LY CopyTo v Duplicate Ie User name Group Privileges Group Michal Zezula lab_1 Common hall y Karolina Macuchova hall Admin Privil 45 Ivo Ulrych lab Default J hall Guest Guest y A guest lab lab Guest Set password _ Enforce user to change his password at start up User can be created removed duplicated or his Group and Privileges copied to another User s Faria Groups and Privileges can be assigned to the selected User s and a user can be enforced to e ae at his next logging into the application 4 WW p Enumerate all Windows users Statistics Export user rights c ProgramDatalLaboratory Imaging Platform Statistics db m Import user rights Make sure you are logged as a user with the Privilege to Modify user rights If not use the Log As button to log in under a different account which has the privilege Set layout visibility Select the Groups tab Here you can see at least one group of items called Default Either select one a group or create a new one by the New button and choose Layout within the list of ite
7. Cameraz Mikon OS L3 G Nikon D5 L3 Nikon D5 UZ LZ USB G Nikon DS5 U2 L2 Ai G Nikon 05 01 G Nikon Desh 2000 G Nikon Deh 200 F G Nikon DS 10M Nikon DOC FS Nikon DS L1 Now select the cameras which will be used with NIS Elements AR 4 Select the modules you want to install and deselect the modules you do not want to install ADI EDF Extended Depth of Focus LIM 20 30 Deconvolution Tools LIM Legacy Deconvolution AQI 30 Blind Deconvolution Metalo Grain Size Metalo Cast Iron Calcium and FRET If your licence contains some additional modules besides the NIS Elements AR core software please select them in this window Note Any module selected will be installed along with NIS Elements AR automatically However you might not be licensed to use it The module will run after you get the corresponding code registered in your HASP key 5 6 Manufacturers Nikon Prior Sutter Marzhauser Lang Ludi 4 51 EXPO Coolled Vincent Associates Physik Instrumente Others Devices Manual Microscope M Mikon Mi E C Mikon Mi T Nikon Ci M Mikon Shutter NI 5H CON Mikon Ti Mikon 3901 801 DIH D FL E mM Mikon TE2000 T Nikon LY Seres C Nikon AFA Remote Focus Assist M Nikon LUSU Lazer amp Shutter Unit Select the devices which will be used with NIS Elements AR Finish the installation by clicking the Install button Note NIS Elements may not be conn
8. Restr The top menu bar containing pull down menus with sets of commands The status bar at the bottom of the screen displaying useful information Image window tool bars which enable to customize the view of each open Docked or floating control windows See the Modifying Layout chapter 2 1 1 Menu Bar Application tool bars where buttons representing various menu commands are gathered for quick access ed image individually All menu commands are sorted according to their purpose to the following menus Main screen File Concerns operations with files such as Open Save Import Send etc Edit Concerns operations with the current image Copy Paste Undo General Options and other commands are available Acquire Concern image acquisition Calibration Concerns image objective calibration and optical configurations Image Concerns operations with images color transformations resizing etc ROI Concerns operations with user defined regions of interest Binary Concerns operations with the binary layer contains thresholding and morphology commands Measure Concerns manual and automatic measurement Reference Concerns operations with reference images To display all reference images use the View gt Thumbnails command Macro Concerns user macros macro creation etc Database Concerns database connections database tables database backup etc requires Database View Gathers commands con
9. turn around X Y Z axis Makes movie of Volume view rotating around selected axis Build up in Z direction Makes movie of appearing Volume view which starts from the bottom up Build up in Z direction reversed Makes movie of appearing Volume view which starts from the top down Time sequence Makes movie of Volume view within the Time loop Combine presets Different presets can also be combined together Simply select items from the list of all available presets 114 Measurement 7 Measurement 7 1 Measurement 7 1 1 Calibration Calibration of the image is of crucial importance to measurement A distance will be assigned to one pixel so that correct and realistic measurements can be performed and objects of different images could be compared to each other A correct calibration has to be made before you begin to measure 7 1 2 Rough Measurement Quick and approximate measurements can be performed utilizing graticules They behave like adjustable floating rulers User can simply align a graticule with the measured object and read the distance value e g the diameter To activate the graticule press the Graticules button Select Graticule Type The type of the graticule ruler is indicated by a picture on the Graticules button placed on the right image tool bar To change the graticule type click on the graticules button with the right mouse button and select the appropriate item from the context menu gt H Recta
10. 0 18 processing P Z Series Lof 2 2 65 00um ZStep 25 00um capturing Remaining diskspace 83GB Detail Info Moving Ti Piezo Drive to 90 00um The window displays the experiment overall progress time elapsed from start of the experiment estimated remaining time remaining free space on the disk where the captured nd2 file is being saved and detailed messages about the experiment work flow such as waiting for next loop stage moving etc You can control the running experiment by the following buttons Next Loop When the time interval of one time loop is long enough the system captures all the included dimensions and waits for the scheduled beginning of the next loop In such case the Next Loop button becomes enabled and you can click it to start the next time loop ahead of the schedule Next Phase After you press this button the experiment jumps to the beginning of the next time Phase after the current loop is finished Start Phase Use this button to start the time phase selected in the combo box Select the phase first and press the Start Phase button 59 Pause Continue The experiment can be paused by the Pause button Let it go on by pressing the same button Refocus If there is a need to focus manually press the Refocus button It pauses the ND experiment and runs Live camera signal Finish This button stops the experiment and saves captured data Abort Use this
11. 1 Display primary secondary etc 2 Docker top bottom left right 3 Column controls can be placed in a docker side by side 4 Tab one column can take multiple controls layered 5 Controls in each tab 2 2 2 3 Modifying Tool Bars _ Large Buttons Main Left Top Toolbar E gt PointingTool Show Toolbar gt ia _ Use Default A AnnotateAndMeasure Default as ModifyBinary e Select the Toolbars item within the list of layouts The right side part of the window changes e Choose one of the tool bars which you would like to modify from the Toolbar pull down menu e If selected de select the Use Default option on the right e Any button of the tool bar may be hidden by de selecting it No buttons can be added to any of the tool bars except the Main Left Tool Bar 17 e The whole tool bar can be hidden by de selecting the Show Toolbar check box e There are two sizes of buttons available Select the Large Buttons option to use the larger one This setting is shared by all tool bars Modifying the Main Left Tool Bar Custom user buttons can be added to the main left tool bar You can define your own buttons which run single macro functions or execute macros Select the Main Left Toolbar from the pull down menu Large Buttons Toolbar Main Left Toolbar Button Properties sane obied binen N 1 Disabled bitmap
12. A separate curve is created for each color channel Run the View gt Visualization Controls gt Histogram command to display the histogram control window Histogram x A Export Ez H fun hos pe we ws wc o oja E Freq eee wt Intensity DIA E E Pos 180 Source Data Source data of the histogram can be viewed if you swap to the Data tab in the bottom left corner of the window The area from where the histogram is calculated can be restricted according to the selected histogram mode The data are obtained from the whole image O The data are obtained from within the probe ca The data are obtained from within the current ROI The data are obtained from the image parts under the binary layer Export hd The source data or the histogram image can be exported to an external file Display the Export pull down menu and select a suitable destination Click the button to perform the export The Export ND histogram Ap button enables to export histogram data of all frames of the current nd2 file The same destination which is selected in the Export pull down menu is applied 78 Image Analysis Note Please see the Exporting Results chapter for further details Appearance The histogram can be zoomed in and out using the zoom buttons on sides of the window There are also other options to adjust the graph appearance e Left slider there is a slider along the Frequency axis Drag it
13. Click the Report button on the main tool bar The following window appears 107 Create Report from File Listing Exporting 2 records Columns Template Filename e Standard Path Records per page we Size Col TA 2 E Filedate i s sE C Dimensions Font Size Coes O Type Custom sampler EA Inthe Columns section you can select fields which will be included in the report If you are exporting images from a database the database table fields will be listed If you are exporting images from a directory image description items will appear e The Template portion of the dialog specifies the layout details If you select the Standard template images will be organized in rows and columns It is possible to set the number of columns and rows If you select the Custom report template a user report template rtt can be opened and used for the report creation If such report does not exist yet you can create it using the Create New button If You Pressed the Create New Button e A wizard appears Select number of columns and rows of the new report template Click Next e Select the paper size page orientation and define margins Click OK e An untitled report template opens containing a grid of images spaced according to the columns rows settings e Edit the top left cell of the image grid You can change the text image boxes position size and m
14. GIF does not support layers or alpha channels Portable Network Graphics Format PNG This is a replacement for the GIF format It is a full featured non LZW compressed format intended for a widespread use without any legal restraints NIS Elements AR does not support the interlaced version of this format Windows Bitmap BMP This is the standard Windows file format This format does not include additional image description in formation such as author sample subject or calibration LIM Format LIM Developed for the needs of laboratory image analysis software Nowadays all its features and more are provided by the JPEG 2000 format ICS IDS image sequence ICS IDS image sequences are generated by some microscopes and consist of two files the ICS file with information about the sequence the IDS file containing the image data The ICS file must be stored in the same directory together with the IDS file Caution ND Images containing multi point XY dimension cannot be saved to a file in the ICS IDS format See also ND2 Acquisition 15 76 Image Analysis 6 Image Analysis 6 1 Image Processing 6 1 1 Processing On Intensity RGB Channels You can decide whether to perform the processing on single RGB channels of the image or on the intensity component of the image only The RGB Intensity setting is global so it is applied to all the other processing commands automatically When processing a multichannel image this o
15. In most cases the following basic procedure is sufficient to connect a device successfully e Install NIS Elements AR and select the appropriate device s during the installation e Connect the device to the PC and switch the device ON e Run NIS Elements AR and run the Devices gt Manage devices command to open the Device Manager Use the Add button to add the device to NIS Elements AR Select the device from the list of installed devices and press the Connect button Select logical devices to be activated Configure device specific settings using the Configure physical Device and the logical Device Paramet ers buttons Close the Device Manager Note MULTIZOOM AZ100M ECLIPSE LV series ECLIPSE MA200 or ECLIPSE L200N 300N microscopes can not be connected from NIS Elements AR while the setup tool of each microscope is active To connect any of these microscopes to NIS Elements AR exit the setup tool and then run the NIS Elements AR 3 3 1 Logical Devices NIS Elements AR handles hardware accessories using the concept of logical devices There are features of different hardware devices which equal and therefore can be controlled equally Such features are called logical devices A typical logical device is Stage XY Different microscopes can be equipped with different XY motorized stages although regarding the user interface they behave equally One physical device a piece of hardware can contai
16. User rights Administrators for installation Operation cannot be guaranteed on all computer models For further information contact your nearest Nikon representatives NIS Elements AR Table of Contents T INStalation and Slant dos 1 AN IS Elements AR Os tala ON lt del titi 1 12 SOM and Ene OPON S A E A bee eee eas A A a ae ow AA 6 ZA Sita and Example e e ade ir eG ol ae 6 ES e y 5 AMERO MOE GI sdagauieltacuetoetniauscubscaastettasues 6 2 APPIcaton APPAIA NCE ute tcs aii Sica Details o ias ldaek sudo 9 2314 NIS BIEMENS AR OCN A A A ee 9 Zalla Meno Bob na RNa 9 2 k2 Applicaton TOON DANS 2er leia ds 11 NUS B e 11 Zo Mage VVINGOW nto iia 11 22 MOVING E alo es 14 Ze A ta ctoae terse atte teen Sees naa ena aso ee atin asa tea ate tea ores anes 14 272 2 kayo Manager bai li a la 15 LS BORNO PANES di decada 20 2 2 4 ANpearanice OPINAS nica 23 O A se sean ee nee sete ss afta Aa 24 ZO Made FIOT rt rn cola 24 23 2 Operations With IMAgES cc od E E ER 26 2 3 3 Adjusting the Organizer Os 27 ZASUSenManad eme aretes edo dido aci a ees 27 3 Seting Ine Hardware UD ib o so despida 31 Sl Working with a Samer la ad 31 3 11 Live Signal trom Camera veritas lentos 32 I2 Capturing Magis kerekein E E teeta america 32 31 39 Canea Ol OM LIVE Mage enesto he ioc eee ie nh a a ac lod 33 3 14 Controlling Cameras by Macro ias eid ci ee Ea aa 33 3A Camera SEUINGS sicec saddens n a a dees seen eens dee ae eee ase 34 30 COMMECICC DEVICE
17. a new report will be created and the exported data inserted to it To Web Browser To HTML Clipboard Some tabular data can be exported as a HTML table It can be displayed in a default Internet browser the Web Browser option or the HTML code can be copied to Windows clipboard ready to be inserted to any HTML text editor To Printer Tabular data can be printed directly This option opens a standard Print dialog window where it is possible to select a printer and print the data 139 Data Export Options Run the Options command and switch to the Data export tab or press the Settings it button within the export pull down menu There are two tabs available Global Settings Options Data export Global Settings Data export Export into Microsoft Excel application 8 Use currently opened file or create a new one Open or create specific file C ES Start at column o and row o Al Export text files into folder J Append current data to previous exported data Export also column headers if not appending exports without statistics Activate target application after export Insert empty lines Export into Microsoft Excel application Select this option if you want to export the data to MS Excel application You can choose if the data will be exported into currently opened file or a new file will be created Or you can set to export the data to a specific f
18. e The filters can be moved within the already defined positions using the Up Down arrow buttons Note When browsing the list of filters details about the currently selected filter are displayed on the right side of the window Illuminator This logical device is used for controlling the specimen illumination remotely There is no standard dialog box for the illuminator control Each device handles this logical device via a user interface specially de signed for it typically containing one button for switching it ON OFF and a slider for regulating intensity Light Path Some microscopes have more than one port where it is possible to attach a light source or a camera This logical device can switch the illumination between these ports Microscope This logical device is used to group standalone logical devices used in certain microscopes To control the logical devices of a microscope from one control window select the Devices gt Microscope Control Pad command Nosepiece This logical device serves for controlling microscope objective changers There can be three nosepiece types attached to a microscope e Manual it can not be controlled via the software e Intelligent the current nosepiece position is read by the application but can not be controlled e Motorized such nosepieces can be fully controlled via the Microscope Control Pad or the Nosepiece control window See Assigning objectives ND Filter A neutr
19. is pressed This button ensures that the motorized stage moves to the coordinates of the currently selected point e Click inside the line you would like to change e Move the Z drive to the new position Click the lt button Note The XY coordinates of one point cannot be adjusted unless you delete it and add a new point Offset All The offset all button can shift the XY coordinates of all points in the same way e Make sure the Move stage to selected point is pressed This button ensures that the motorized stage moves to the coordinates of the currently selected point e Select one point of the list e Move the XY Z stage to a new position define the offset e Press the Offset All button e The coordinates of all points change the same shift which you made with the stage is applied to them 4 2 2 1 Custom Multipoint Definition When you click the Custom button the following window appears There are two tabs where properties of a custom scanning pattern can be defined 52 Capturing Well Plate tab Select the method and set the parameters of the well plate comes 12 E 2 Manual Distance X 9 000 mm Distance Y 9 000 mm Interactive Manual This method will create a pattern which will scan Rows x Columns fields The Distance X and Y para meters specify distances between two fields The scanning will begin at the current position Interactive The interactive method lets
20. n Pry Pr4s Prgo Pr435 4 Measurement Type object field manual Image Type binary Red Red is the mean of red component pixel values It is the usual statistical mean of intensity values of red component Measurement Type manual Image Type color Roughness This feature indicates how much the object is rough 1 means the object roughness is minimal the object is circular Roughness Convex hull perimeter Perimeter Measurement Type object manual Image Type binary 129 RoughnessInf This feature indicates how much the object is rough 1 means the object roughness is minimal the object is circular RoughnessInf 1 Roughness Measurement Type object manual Image Type binary ShapeFactor This parameter is to define whether the object is rough or not Shape factor 4 nm area Convex hull perimeter Measurement Type object manual Image Type binary StartX 130 StartX is the x co ordinate of a pixel on the object boundary When you scan the image from the origin as indicated left gt right the first pixel of the object you hit is the one with StartX and StartY coordinates Measurement Type object Image Type binary Measurement StartY StartY is the y co ordinate of a pixel on the object boundary Measurement Type object Image Type binary StgPosX StgPosX feature is the x co ordinate of the absolute position of the measured field It is available only for
21. 3D model It indicates how much the 3D object is rough EdfRoughness EdfSurface Area Measurement Type object field Image Type binary EDF 123 Elongation Elongation is determined as a ratio of MaxFeret and MinFeret features This feature is useful for shape characteristics Elongation MaxFeret MinFeret Measurement Type object Image Type binary EqDiameter The equivalent diameter is a size feature derived from the area It determines the diameter of a circle with the same area as the measured object Eqdia sqrt 4 Area r Measurement Type object manual Image Type binary ExPurity This feature specifies the amount of white in the measured color if the measured color can be composed of white and a pure spectral color Measurement Type object field manual Image Type color FillArea In case an object does not contain holes then the FillArea is equivalent to the Area If an object contains holes FillArea remains the same while Area is reduced by the area of the holes Measurement Type object Image Type binary FillRatio 124 FillRatio is the ratio of Area and FillArea Measurement FillRatio Area FillArea If an object does not contain holes the FillRatio is equal 1 If an object contains holes FillRatio is less than 1 This feature can distinguish objects with and without holes Measurement Type object Image Type binary IntensityVariation IntensityVariation is deri
22. Applications gt HDR gt Create HDR from ND command 9 15 Metalo Grain Size Analysis After the module installation a new item called Metallography appears in the Applications menu Select the Grain Size command to display the grain size measurement layout Features of the Grain Size Analysis Module e Measurement of single phase structures e g Austenite Aluminium e Measurement of two phase structures e g Ferrite Austenite Ferrite Pearlite e Planimetric Linear Circular and Abrams measurement methods e Customization of the predefined measurement procedures e Support of ASTM and JIS standards ASTM E112 96 ASTM E1382 97 JISG0551 e Easy data export Please see the electronic help for more details 9 16 Metalo Cast Iron Analysis After the module installation a new item called Metallography appears in the Applications menu Select the Cast Iron command to display the cast iron measurement layout 159 Features of the Cast Iron Analysis Module e Graphite classification according to ISO ASTM JIS standards ISO 945 ASTM A247 06 JIS G5502 e Ferrite pearlite ratio measurement e Customization of the predefined measurement procedures e Easy data export Please see the electronic help for more details 9 17 Time Measurement The time measurement tool enables to record average pixel intensities within Regions Of Interest ROIs during a time interval The measurement can be performed on
23. Live camera signal or on a saved nd2 file Before the measurement is started regions of interest shall be defined and turned ON Please see the electronic help for more details 9 18 Tracking Motion characteristics of objects such as living cells can be measured automatically or manually using the Tracking control window Please see the electronic help for more details 9 19 Advanced Tracking requires Local Option If you have this module enabled in HASP the Tracking module gets extended with several advanced features such as 3d tracking etc Please see also the chapter for further details 160 10 Supported Cameras The following cameras are supported by NIS Elements AR Nikon DS U1 L1 Nikon DS U2 L2 Nikon DS U2 L2 Qi Nikon DS U2 L2 Ri Nikon DS U3 L3 Nikon DS U3 L3 Qi Nikon DS U3 L3 Ri1 Nikon DS U3 L3 Vi1 Fi1 Fi2 Nikon DQCFS Andor Ixon Andor Kon Andor Luca Andor Clara Vossk hler VDS 1300 2000 4000 Photometrics Cascade512B Photometrics CoolsnapES Imaging Source DFG 1394 Hamamatsu ORCA ER Supported Cameras 161 162 11 Supported Microscopes Nikon Ci E Microscope Nikon Eclipse 90i 80i Microscope Nikon AZ100 Microscope Nikon Eclipse LV Series Microscope Nikon Eclipse L200 Microscope Nikon Eclipse MA200 Microscope Nikon MM 400 800 Microscopes Nikon Ni E Microscope Nikon Ni U Microscope Nikon TE2000 Microscope Nikon TI Microscope Supported Microsco
24. Opacity dialog window that appears after you run this command e Use Black Level Check this item to ensure that the black slider will be affected by the auto scale functions e Settings opens the Auto Scale Settings window See Auto Scale Settings Keep Auto White Balance Check Box Check this option to switch the Auto White Balance functionality permanently on the live image AWB Press this button to perform the Auto White Balance operation once Auto White Balance Color This button Opens the AWB Color window and enables you to select a color shade which the system shall eliminate make white See AWB Reset AWB Discard all AWB settings with this button 4 Color Oversaturation Switch this button ON and the system highlights all pixels with values reaching maximal values Select colors to highlight the pixels with in the pull down menu These buttons arrange histograms of single channels next to each other horizontally or vertically This button arranges histograms of single channels overlapped Use sliders at the bottom of the graph window to amplify or reduce the green red blue component display in the view lal Save Load LUTs This pull down menu enables user to handle LUTs settings in various ways They can be saved to a lut file and loaded later Or LUTs settings saved along with an image image can be loaded directly from this image by the Reuse LUTs from File command Or you can copy paste the
25. Save Macro As tel commands in the macro editor to save the created macro 8 2 Running a Macro NIS Elements AR provides several ways to run a macro You can run the current macro loaded to NIS Elements AR by choosing the Macro gt Run command or by pressing F4 A hotkey combination Ctrl Alt 1 2 3 9 can be assigned to it in the Macro gt Options window You can run a macro at the beginning of the NIS Elements AR session by assigning the StartUp flag to the macro in the Macro gt Options window You can run a macro saved to a disk by using the Macro gt Run From command A tool bar button can be assigned to run a macro See Modifying Layout Breaking the Macro The macro execution can whenever be stopped by pressing the Ctrl Break key shortcut 8 3 Macro Language Syntax Specifies the NIS Elements AR Macro Language features Variable types The following data types are implemented char text char8 lt 128 127 gt byte lt 0 255 gt int lt 32768 32767 gt 149 word lt 0 65535 gt long lt 2 147 483 648 2 147 483 647 gt dword lt 0 4 294 967 295 gt double lt 1 7E 308 15 digits gt Structures and Unions Structures and unions are not supported Arrays One and two dimensional arrays are supported Local and Global Variables You should declare local variables at the beginning of macro or function only You should declare global va
26. adjacent frames Pro longing the exposure time increases the brightness of the image as well as the image quality the less gain the less noise and decreases frame rate Gain Controls the sensitivity of a camera Increasing the gain increases brightness of the image but decreases quality the more random noise the more streaky noise and color unevenness and increases the frame rate indirectly by enabling shorter exposure times Averaging Averaging is a commonly used technique of decreasing noise in the image In this method 2 4 8 or 16 consecutive frames are averaged together Offset Sets the brightness of the image It is a constant additive positive or negative changing all pixel values of the image With negative offset value the dark image areas become pure black Considering fluorescence microscopy an appropriate offset setting can create continuous black background and thus help to enhance together with gain or illumination enhancement the contrast Contrast Affects dynamics of how the luminosity is rendered There are several modes for different illumination contrast scene situation Gamma The gamma correction maps the intensity of live signal exponentially to the gamma parameter For gamma lt 1 dark portions of the image are enhanced whereas for gamma gt 1 image parts of higher intensities are enhanced AE Compensation In automatic exposure modes the compensation affects how optimum exposure settings Exposur
27. always in pixels Measurement Type object Image Type binary Circularity Circularity equals to 1 only for circles all other shapes are characterized by circularity smaller than 1 lt is a derived shape measure calculated from the area and perimeter This feature is useful for examining shape characteristics Circularity 4 n Area Perimeter Measurement Type object Image Type binary Class NIS Elements AR recognizes 12 classes 1 12 If you have selected class feature function Object Features or Field Features system automatically asks you to specify the class via dialog box In field or scan objects measurements the application asks for a class before measurement is performed on the current image In single object measurement NIS Elements AR asks for a class for every measured object In the case you want to pass by there is a possibility to run the SetClass function Measurement Type object field manual Image Type binary Convexity Indicates convexity of the object edges Convexity Area Convex Hull Area Measurement Type object Image Type binary DensityVariation DensityVariation is derived from density values It is a usual standard deviation of density values Measurement Type object field Image Type color binary EdfSurface EdfSurface is based on 3D model It determines the surface area of an 3D object Measurement Type object field Image Type binary EDF EdfRoughness EdfRoughness is based on
28. black white and Gamma sliders can be reset by a double click Different controls will be available when applying LUTs to monochromatic RGB multi channel or spectral image When LUTs are active the LUTs button in the top left corner of the image window is highlighted red 6 2 2 1 LUTs on RGB Images ns nt R O aw E X G 0 4 0 Bom d ll There are 3 separate windows for each of the RGB channels All channels are controlled at the same time by default To control each channel separately press and hold the Shift key while you drag the sliders or adjust Gamma LUTs Tools Ln Enable Disable LUTs This button applies LUTs to the current image Le Keep Auto Scale Press this button to run the auto scale procedure permanently on the live image When you turn this button OFF the settings remain as if the Auto Scale button was pressed only once Wt Auto Scale This button adjusts the white slider position of all channels automatically with the purpose to enhance the image reasonably If you have selected the Use Black Level option from the Settings pull down menu the black slider will be affected too 81 Reset All Components Discard all LUTs settings and turn LUTs OFF by pressing this button Settings Display the pull down menu and select one of the following commands e Brightfield Opacity You can change opacity of a brightfield channel Define the opacity in the Brightfield
29. button to stop the experiment without saving the captured data Events If a remarkable event occurs during the experiment the user can press a shortcut key and the information will be saved to the resulting nd2 file You can define the key shortcuts after you press the Events button 4 3 Capturing to Ring Buffer The Capturing to RAM technique enables user to record sequences displaying very quick actions lasting tens of milliseconds Make sure that the RAM Capturing buttons are displayed on the Grabber Devices ToolBar see Modifying Layout or use the corresponding commands in the Acquire gt RAM Capture menu Set the Ring Buffer Size The technique uses a ring buffer to store temporary data The ring buffer is a part of virtual memory that is being constantly and repeatedly filled with the live image data Depending on how fast the action to be captured is you should set the ring buffer size in seconds O A Settings e Click on the Settings button e Set the Time buffered before after values These represent the time before and after you click the RAM Capture D button The whole time interval will be included in the sequence e Confirm it by OK 60 Capturing Settings Frames buffered before Frames buffered after Current settings will require buffer with 22 frames The size of used memory will be 27 MB Turn the Buffer ON Click on the Circular buffer ON bu
30. camera ROI function is supported by your camera lts functionality corres ponds to the same button within the Live image window See Camera ROI on Live Image 4 2 5 Combining N Dimensions The dimensions can be combined together creating an N dimensional image as the result e Run the Applications gt ND gt Define Run Experiment command e The following control window appears Be XY Large Image A 4 1 0 e Select one or more dimensions tabs which should be included in the resulting ND2 file Above the tabs there is a preview of how the nd2 file structure will look like e Press the Order of Experiment button and select the item which defines the desired order of loops in the nd2 file e Set the parameters of each dimension acquisition 57 e Select whether to save the nd2 file directly to a file or just to keep it in memory to be saved afterwards To do so select the Save to File check box The path and the file name shall be defined if saving to a file is preferred e Click the Run now button to start the acquisition Note When the experiment is finished a window may appear saying Please wait Finishing ND exper iment writing image data to the harddisk The Cancel button within this window enables you to abort saving of the file Only the data which has already been physically written to the resulting ND2 file will be available this also concerns the Recorded Data Z Intensity Correction T
31. clicking on any object results in only that object being selected All objects can be selected by pressing Ctrl A or by choosing Select All ROIs from the menu over any ROI Alternatively it is also possible to select a set of objects by dragging a rectangle around them with Ctrl key being pressed Moving Objects are moved by simply dragging them cursor indicates that When more objects are selected only the one under cursor is moved by default This can be changed by pressing the Alt key and move all selected objects When object is changing over time any moving results in a new key frame Individual dots representing the key frames can be moved too It is possible to duplicate existing objects by Shift dragging It works on selected objects In case of a key framed object object is duplicated with all its key frames unless Alt is pressed Resizing Resizing a ROI is done by dragging its contour the cursor changes accordingly Note that resizing hit zones extend towards outside of the object as opposed to moving hot zones which extend towards inside of the object Resizing always affects only one object selection is not taken into account The behavior slightly varies depending on the object shape By default resize does not hold aspect of the object width and height can be changed independently By holding Shift key aspect is maintained useful for circles and the center is not moved Rectangle can be also rotated by using Al
32. enables you to reduce the number of channels multiple times You can adjust the number of groups by the and buttons and also change each channel width by moving the white horizontal dividers Gain and Black Level for each group can be adjusted Gamma AAA 1 00 Hold Shift key to control all groups Gray Scale Displaying Mode You can switch to this displaying mode by clicking the E button located in the image tool bar The image is displayed in gray scale but various color gradients can be applied to it 87 LUTs x mws OOOO OOOO A yy N R Mo casae F Z gt ide 4k ioios ne o cma ip 6 3 Thresholding Specifying correct threshold limits is a crucial procedure of the automated image analysis The point is to determine which pixels will and which will not be included in the binary layer and thereby distinguish objects to be analyzed from background To display the thresholding control window run the View gt Analysis Controls gt Thresholding command Thresholding can be performed in the following modes 6 3 1 RGB Mode The RGB thresholding mode is available only if an RGB image is opened There are two ways how to define the threshold limits by choosing reference points within the image or by defining the limit values for each color channel 88 Image Analysis O cre CR To threshold the image use the following tools H7 This button invokes a pull down
33. enhances the image thumbnails automatically Dark images gain details Sorting of Images To order displayed images right click anywhere in the pane a context menu will appear Move to the Sort by sub menu which offers several ordering criteria If the ordering is turned ON already the icon is displayed on the left side of the applied criterion 26 Application Appearance Enable Filter Ascending Sort by Descending Remove Filter and Sort Unsorted Refresh Filename Path Properties Size Filedate Grouping of Images To arrange the view of images efficiently you can use the capability of grouping of images Drag the column name bar to the grouping bar right above the column name bars All files with matching field values of the selected column will be grouped together This can be undone by dragging the column caption back to the others See fig The organizer layout the Calibration column is grouped 2 3 3 Adjusting the Organizer Layout The pane size is adjustable To resize it 1 Place the mouse cursor over the dividing line in the middle 2 The cursor becomes an arrow with two tips 3 Drag it left or right to the new position Also the following buttons appear in the top bar U Resizes the panes to achieve the same size for both of them Resizes the pane to its maximal minimal size one pane is then displayed on the whole screen 2 4 User Management User management is very useful
34. enlarge Pixels are added around the objects If the distance between two objects is shorter than twice the thickness of the border to be added these objects become merge together If a hole is smaller than twice the thickness of the border it disappears from the image Pal Open Open is erosion followed by dilation so the size of the objects is not significantly affected Contours are smoothed small objects are suppressed and gently connected particles are disconnected Pal Close Close is dilation followed by erosion so the size of objects is not significantly affected Contours are smoothed small holes and small depressions are suppressed Very close objects may be connected together B Clean This transformation is also called geodesic opening The image is eroded first so small objects disappear Then the remaining eroded objects are reconstructed to their original size and shape The advantage of this algorithm is that small objects disappear but the rest of the image is not affected 133 Fill Holes Fills the holes inside objects This transformation is handy when objects have a rich inner structure with intensities typical for background After applying the Fill Holes transformation objects become homo geneous Contour This transformation converts objects into their contours Smooth Smooth affects rough edges of the objects makes them smooth a Morpho Separate Objects This transformation detects standalone o
35. finish Please see the File gt Save As command description for more details 6 7 3 Creating QuickTime movie You can easily create a QuickTime movie from an ND document Run the File gt Save As command and select QuickTime Movie File Format in the Save As Type pull down menu The Save As window expands Adjust the mov file settings used compression and press Save to finish Note QuickTime Player must be installed on your PC Warning You cannot create QuickTime movie from an ND document that contains Multi point dimension 6 7 4 Movie Maker requires Local Option Models displayed in 3D Volume View or EDF 3D Surface View requires Extended Depth of Focus can become actors of your movies once the NIS Elements AR movie maker is started The Movie Maker control window appears after you press the oe button located on the image tool bar Volume View or Surface View must be active The user is supposed to create a set of key frames The rest of the movie will be created by interpolation between these key frames This gives the user absolute freedom in how the movie will look like Movie Maker Controls When you start the movie maker the following bar appears at the bottom of the image view tan R 1 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 PRR BR BRHEHRHEHEHEHEHEHEHEHEHEEHEEESE ESE SE SE amp 1 The Clapper button By this button the director s mode can be turned ON OFF See Directors Mode 11
36. following tools l i 1 point threshold tool Q 3 points circle threshold tool g 6 points circle threshold tool Click inside the image to define typical object areas The system will detect similar parts of the image and highlight it by color The threshold limits are indicated in the histogram and can be further modified by dragging the limit lines by mouse Please refer to the Thresholding chapter for further details on thresholding 142 Measurement 7 5 2 Applying Restrictions The number of objects that are included in the results table can be restricted by defining limits Objects which do not fit these limits will be left out of the result table Restrictions EqDiameter um EqDiameter In O Circularity In M MA 4 I J JI Ea 0 49 Bins 20 E 9 08 Right click to the restrictions field to select one or more of the available measurement features e Select the restriction feature you would like to define e Move sliders below the histogram to set the limits The limit values are indicated next to the feature name and can be modified by double clicking the indicated value e Decide whether the defined interval will be excluded or included this is done by setting the Inside Outside option next to the feature name The nearby check box indicates whether the restriction is currently being applied or not If applied the his togram below is color otherwise it is gray Re
37. in order to stretch the view of either lower or upper values of the axis e Drawing style There are two ways the histogram can be drawn e Raw Data tries to draw the source data precisely to the graph e Trend Style interpolates the data so that the histogram lines appear smoother E Auto Scale Vertical zooms the graph of each channel separately to fit the available area When this function is ON the histogram is not proportional i Auto Scale Horizontal zooms the graph so that the marginal zero frequencies if there are some are excluded from display Lin Graph Linear displays the linear scale on the Y axis flog Graph Logarithmic displays logarithmic scale on the Y axis H Show Grid turns ON OFF the grid in the background The graph appearance can be modified Press the Options 4 button a window appears where the following settings can be adjusted Colors Graph background and axes colors can be selected Pen Width Set the width of the histogram s line to 1 2 or 3 pixels Fill Graph Area The area below the histogram line can be filled with the channel color Graph Area Opacity Select the opacity of the Graph Area color in Vertical Horizontal AutoScale Show Grid These options equals the corresponding buttons of the histogram tool bar Interpolation method Select the way of drawing the graph line The Linear smooth and Quick precise options are available AntiAlias S
38. is visible in all T Z dimensions but only in current multipoint select the option Object is always visible and check the Object belong only to Current Multi Point option Measurement e object is visible only in selected time frames in all multipoints select the option Object is visible in range e object is visible only in selected time frames in current multipoint select the option Object is visible in range and check the Object belong only to Current Multi Point option Select one of these rules in the context menu that appears when you click with right mouse button on an annotation object in the ND document Detailed definition of object visibility can be done using commands in the Visibility in Time sub menu 7 1 4 Automated Measurement Automated measurement is the most powerful of image analysis features In combination with user macros NIS Elements AR can become a semi or fully automatic image analysis tool There are several key procedures which a proper automated measurement should involve e Optical system calibration see the Optical Configurations chapter e Image acquisition see the 4 Capturing chapter e Defining threshold creating the binary layer see the Thresholding chapter e Performing the automated measurement e Results presentation Image Layers Involved in Measurement The the binary layer and also the color image can get involved in the automated measurement e Binary layer is typ
39. key frame button to fix the initial appearance Browse the time line modify the object position each time and set key frames by clicking the Add Update key frame button Press the Play Movie button for a preview When all the key frames are properly set press the Create Movie button The movie is created and the AVI file will open in a new image window Save the new file by the standard File gt Save As command Directors Mode In the directors mode it is not needed to press the Add key frame button every time the position of the object is changed The director s mode detects all changes made to the object position and adds a key frame to the time line whenever such change is detected A typical work flow would look like this Turn ON the directors mode by the Clapper button The time line gets highlighted with red color Click to the time line where a certain position of the object shall occur Move the object by mouse or adjust it in some way zoom crop etc at this moment the key frame will be added to the time line automatically Click to the time line where another position shall occur and so on The director s mode shall be used when you know precisely how the movie shall look 112 Image Analysis Special Options A contextual menu appears whenever you right click on any key frame or between two key frames a segment within the time line Set key frame time Sets the selected key frame position
40. lt Less than or equal to gt Greater than gt Greater than or equal Equal l Not equal logical operators The logical operators perform logical operations on expressions The following logical operators are supported amp amp Logical AND Logical OR Logical NOT 154 Working with Macros Expression evaluator Expression evaluator does not support the precedence of operators lt evaluates the expression strictly from right to left so you need to use brackets E g 3 4 2 will be evaluated as 3 6 e 18 To force the native pre cedence of operators you should write 3 4 2 C like functions The system can interpret your own C like functions An entry point to program is the main function in your macro If main is not presented for backward compatibility the body of the macro is also considered as main function The new macros should use main as an entry point The general C like functions also called interpreted functions as opposed to basic system functions from NIS Elements or LUC32_1 DLL have the following syntax Syntax int MyFunction int a LPSTR str double d int retval return retval Return Value The return value can be any basic data types char int word dword int double or pointer Parameters Parameters can be any basic data types char int word dword int double or pointer Example int main char buf 256 my_ function buf WaitText 0 buf
41. may be converted to moving ROIs by clicking the Binary to ROI c button See View gt Analysis Controls gt Tracking 101 ROI Multiple Selection Use the Ctrl key to select unselect more than one ROI When you have selected more ROIs press and hold the Alt key while dragging a single ROI Other selected ROIs will not be moved but remain selected Editing ROI in time You can edit ROIs in time using the Edit ROIs in time command which is accessible from the contextual menu of the Y Turn ROI on off button in the right image toolbar f Move Pik SJ Draw R Detect E Mes m New ly After Edit Select Next Object y Skip 1 frame a Y Delete Undo A Redo 7 Help 59 Finish Use provided tools to edit or create a new ROI Pp Move This tool enables to move the ROI to a different place in the image As you place the cursor over the ROI color of the selected ROI is indicated near the mouse cursor this proves very useful when you have defined many ROls or they are overlapping Simply drag and drop the ROI to another position k Pick Use this tool to select directly in the image which ROI you want to edit 7 Draw Use this tool to draw a freehand curve which defines new region of interest Press the right mouse button to close the curve and finish the definition LS Detect Enables to redetect ROI or to detect a new ROI when the New button is pressed or New item is selected from the pull down menu
42. menu which enables to load save the current threshold settings from to an external file threshold This button erases the threshold settings no binary objects are created i Single point threshold tool Select it and click inside the image to define the threshold The threshold ranges will be adjusted so the selected pixel will fit inside 2 3 point circle tool picks threshold from the radius of 3 pixels 6 point circle tool picks threshold from the radius of 6 pixels 1 Undo reverses the previous threshold operation LA Redo returns the threshold as it had been before the Undo button was used Rethreshold image use this button to update the binary layer according to the current settings The purpose of this button is to obtain the original binary layer in case it has been modified e g by a command from the binary layer a Keep Updating Binary press this button to keep the binary layer up to date all the time You may find this function useful especially when thresholding is applied to the live image 89 A Threshold ND image when an nd2 file is opened this button appears Define the threshold limits on the current frame and use the button to apply it on the whole nd2 file How to use the picker tool to threshold an image 1 Select one of the cross hair i tools 2 Left click on the image over a pixel of the image that should be considered part of the threshold binary layer All pixels with
43. moves the key frame to this position Set as new start of the movie Sets the selected key frame as the first frame of the movie and moves all other key frames proportionally Key frames prior to the selected one will be deleted Set as new end of the movie Sets the selected key frame as a new end of the movie and moves all other key frames proportionally Key frames past the selected one will be deleted Copy Paste key frame Enables to copy and paste key frames Delete key frame Deletes the selected key frame Delete all key frames Deletes all defined key frames Set time segment duration Sets the segment duration Positions of all other key frames will be moved proportionally Hold Linear Spline Select the type of interpolation to be used between two key frames Set Spline for smooth changes motion lightness etc Interpolation Types sie sie gt Hold Linear Spline Ease in out Easing defines the interpolation rate between two key frames No easing means constant rate of interpol ation Use the Ease in option for the first time segment to increase slowly the rate of change On the contrary the Ease out option serves the best at the end of the movie to make the motion slowly stop Of course you can get the benefit of combining them 113 Interpolation Rate Types No easing Ease in Ease out Ease in and out Presets Simple and fast way how to make a movie is to use any of the predefined presets 360
44. object After the click the algorithm presents its guess of the object contour and goes into the interactive phase In this phase it is possible to 99 Redefine the first point by clicking again in the image Cancel auto detect by pressing Esc key Grow or shrink the object area changes the intensity range of pixels making the object by scrolling mouse wheel or pressing PageUp PageDown for finer steps use Ctrl mouse wheel Erode or Dilate morphologically the object by E D or Down Up keys Open or Close morphologically the object by O C keys Morpho separate portions of the object by S or P key if it is not possible to separate the key does nothing 6 5 7 Using ROls for analysis ROIs are used mainly in Time Measurement to measure varying image intensity inside the ROIs Automated Measurement to measure ROIs features or number of binary objects inside each ROI Object Count to restrict binary objects to areas of interest only ROI statistics to measure intensities interactively In all these cases ROI selection is taken into account as well as intersection options in Measurement Options Additionally the Time measurement interprets the ROI usage type Background ROls are used for offset subtraction and Reference ROIs can be used for bleaching correction 6 5 8 ROIs Overview Types of ROI Camera ROI A rectangular ROI which restricts the live camera image See Camera ROI on Live Image Vector ROI A ROI c
45. rated one it expresses the real area Measurement Type object field manual Image Type binary AreaFraction 120 AreaFraction is the ratio of the segmented image area and the MeasuredArea It has a strong stereolo gical interpretation in the case of isotropic uniform random sections it is equal to the volume fraction AreaFraction Area MeasuredArea Measurement Type field Image Type binary Measurement Blue Blue value is the mean of blue component pixel values It is the usual statistical mean of intensity values of the blue component Measurement Type manual Image Type color BoundsLeft BoundsRight BoundsTop BoundsBottom These are distances X or Y coordinates of the left right top and bottom edge of the object bounding rectangle The units of calibration are used if the image is calibrated Otherwise the coordinate value is in pixels Measurement Type object Image Type binary BoundsAbsLeft BoundsAbsRight BoundsAbsTop BoundsAbsBottom These are absolute distances X or Y coordinates of the left right top and bottom edge of the object bounding rectangle within a motorized stage range This feature can be measured only when a motorized stage is connected Measurement Type object Image Type binary 121 Lage range BoundsPxLeft BoundsPxRight BoundsPxTop BoundsPxBottom These are distances X or Y coordinates of the left right top and bottom edge of the object bounding r
46. similar intensity values of the image will also be highlighted An outline will also display around the object s as well as the building solid colored threshold binary layer 3 Continue to click on areas of the image that should be part of the threshold binary layer How to use the histogram to threshold an image 1 Move your mouse cursor over the histogram and hover over the vertical line labeled L lower limit Move the vertical line left or right to set the lower limit of the threshold Use the image and its building threshold to provide feedback on whether the limit is set in the correct place Accepted areas of the histogram for the threshold are colored gray 2 Repeat first step with the vertical line labeled H higher limit 3 You can combine the histogram with picker tools to create the thresholded image Threshold Adjustments Thresholding parameters can be adjusted very precisely in the following way e Threshold limits of each channel can be adjusted by rewriting the values in the top left top right corner of each channel histogram e Threshold range of each channel the colored stripe can be shifted by mouse Place the cursor in the middle of the range a circle appears and drag it left or right Threshold limits of each channel can be adjusted by mouse Place the cursor on the edge of the thresholding range and drag it left or right The two last behaviors can be further modified by pressing additional keys e M
47. source area for LUTs histogram etc lt also affects the AWB auto white balance and the AE Auto Exposure features of the cameras that support it When the probe functionality is not supported the AWB and AE algorithms are computed from the whole image Live Acceleration Would you like to increase the frame rate of the live signal Move the slider to a desired multiplier The system then automatically shortens the exposure time so the frame rate rises and the loss of intensity is compensated by gain software multiplication Target Maximal Intensity Restricts the maximal image intensity after the auto exposure was applied The value represents a per centage of the whole camera dynamic range Maximal Exposure This is a safeguard of the time of the Auto Exposure For quick exposure it is convenient not to set this value too high Overillumination Tolerance Sets how many pixels should be white after the Auto exposure is performed Use lower values 0 01 for very bright shining objects fluorescence For common bright field even 1 may be a good value Optionally you can define the absolute number of white pixels Use Current ROI The currently defined region of interest set by the Define ROI command can be switched ON OFF by checking this item 36 Setting the Hardware Up 3 3 Connected Devices Before you get to work with NIS Elements AR all hardware accessories should be connected properly to the system
48. the Skip combo box Jump forward jumps forward after an object is edited and maintains that object selected In addition to the automation modes there are some hot keys that improve efficiency of editing process e All tools have a hot key indicated with underscore and on the tooltip e Selected object can be changed with Up Down arrows or number keys on the main keyboard for objects with id from 1 to 9 e Current frame can be moved with the Left Right keys by one frame and with PageUp PageDown by number of frames defined in the Skip combo To move between key frames it is possible to use or for pre vious and next respectively 6 5 6 Auto Detect Tool Auto detect tool is available from ROI menu Simple ROI editor Edit ROIs in time editor and some other places It produces a Polygon ROI Using the tool is two step process The first click on an image structure results in preliminary object contour that is drawn on the screen After that user can further tweak the object contour and then finish whole auto detect by confirming with right mouse button User can always cancel auto detect by pressing Esc key The very first click is important because these pixels under the cursor determine intensities from which the algorithm guesses the whole object As a general rule it is better to choose brightest pixels in the bright objects fluorescence and dimmest in the dark object It is also good to click near the center of the
49. the Z drive to the correct TIRF Z position e Click the Set TIRF Pos button 55 Using TIRF e Enable TIRF by selecting the TIRF check box within the Z Series experiment settings e Atleast one value of the Z Pos column of the Multi channel acquisition setup has to be set to TIRF See Multi channel Acquisition Piezo If a Piezo Z drive device is connected the Piezo button appears in the panel Use the pull down menu to select the action which will be performed upon pressing the button MA Keeps Z position and centers Piezo Z This option moves the Piezo Z drive to the home position but compensates the movement by the second Z drive in order to keep the original absolute Z position sum of Z1 and Z2 y Move Piezo Z to Home position Moves the Piezo Z drive to the home position regardless of the absolute Z drive position 4 2 4 Multi channel Acquisition Fluorescence multi channel pictures can be acquired Run the Applications gt ND gt Define Run Experiment command and select the Lambda tab There you can set the multi channel experiment Apo 10x Le Brightfield Brightfield Home no configuration gt New E All L Channels Setup Specify how many channels are to be captured Set a descriptive name of each channel and select the optical configuration that is to be used for its acquisition If no suitable configuration exists you can create a new one by selecting the lt define new gt
50. the current frame 4 Move to Previous Next User event Click these arrows to browse through the frames where user events occur User Event Selection This button selects frames of the ND2 file according to the Event Playing Range settings right click this button and pick the Event Selection Range command from the context menu to view modify them A simple window appears Define the number of frames seconds which will be selected around each event Range Selection Clicking this button selects a user defined range of frames The selection is done by mouse A range can be selected by mouse while holding the Shift key down Single frames can be added removed while holding the Ctrl key down Right click the navigation bar to invoke the context menu A selection can be adjusted deleted or the nd2 file can be cropped Note ND2 files may contain extremely large number of frames so it may be helpful to omit some of the frames from the selection Right click the Range Selection button and a context menu ap pears It enables you to select every 2nd 5th 10th 20th 50th 100th frame The Select All Frames option selects the whole range of frames as if you have selected the first and the last time phase with the Shift key down but preserves the Select Every setting a Disable Selection Press this button to de select all frames of the nd2 file Tips e Right click the selection to invoke a context menu The selection can
51. the object Measurement Type object Image Type color gray 125 Length Length is a derived feature appropriate for elongated or thin structures As based on the rod model it is useful for calculating length of medial axis of thin rods Length Perimeter sqrt Perimeter 16 Area 4 Measurement Type object manual Image Type binary Width LineLength Line length is defined as the length of the object with elongated shape Measurement Type object Image Type color gray Luminance Luminance is defined as a radiant power weighted by a spectral sensitivity that has characteristics of human vision Measurement Type object field manual Image Type color MaxFeret The MaxFeret is the maximal value of the set of Feret s diameters Generally for convex objects Feret s diameter at angle equals the projected length of object at angle a a 0 180 NIS Elements AR calculates Feret s diameter for 0 10 20 30 180 Measurement Type object manual Image Type binary MaxFeret90 The MaxFeret90 is a length projected across the MaxFeret diameter Measurement Type object manual Image Type binary 126 Measurement MaxPeret90 MM axF er et Maxintensity Maxintensity value is derived from the intensity histogram It is the maximal of intensity pixel values Measurement Type manual Image Type color gray MeanBlue MeanBlue value is the mean of blue component pixel values It is the
52. to another area of the specimen to get a better texture e Improve contrast by setting LUTS e Check focus re focus if needed e Turn on the Acquire gt Shading Correction gt Shading Correction command ON 46 Setting the Hardware Up 4 points If you select the 4 points method the system draws four points on the screen subsequently and asks user to move one significant part of the specimen to each position After all four steps are completed the calibration is calculated from the moves of the stage Calibrate Using Objective When you have an uncalibrated image you can use an already calibrated objective to calibrate it Right click the Uncalibrated field on the image status bar and choose one of the objectives from the Calibrate using Objective sub menu Of course the same objective which was used to capture the image shall be selected 3 9 3 Units NIS Elements AR supports the following units e pixels e nanometers e micrometers e millimeters e centimeters e decimeters e meters e kilometers e inches e mils If the image is uncalibrated pixels are the only units available In case of a calibrated image it is possible to select other units which are then used to display all values e g measured length area There are two ways of how to select the desired units e Right click the image status bar where the calibration is displayed Select the units and precision from the context menu e Or click the Curr
53. usual statistical mean of intensity values of blue component Measurement Type object field manual Image Type color binary MeanBrightness MeanBrightness value is the usual statistical mean of brightness values of pixels Measurement Type object field manual Image Type color binary MeanDensity MeanDensity value is the usual statistical mean of density values of pixels Measurement Type object field manual Image Type color binary Meanintensity Meanintensity value is derived from the intensity histogram It is the usual statistical mean of intensity values of pixels Measurement Type object field manual Image Type color binary MeanGreen MeanGreen value is the mean of green component pixel values It is the usual statistical mean of intensity values of green component Measurement Type object field manual Image Type color binary MeanChord MeanChord is the mean value of secants in the 0 45 90 and 135 degrees directions It is a derived feature and is calculated from the Area and mean projection according to the following formula MeanChord 4 Area Pr Pry5 Pro9 Pry35 127 Measurement Type object field Image Type binary MeanRed MeanRed value is the mean of Red component pixel values It is the usual statistical mean of intensity values of red component Measurement Type object field manual Image Type color binary MeanSaturation MeanSaturation is a statistical mean of saturatio
54. when an ND2 file is opened the thresholding measurement and restrictions are applied to a single frame Pressing this button will apply it to all frames of the ND2 document and export the results to a file or clipboard according to the current export settings H Save Load Object Count configuration invokes a pull down menu which enables you to load save the current object count settings from to an external file counting Use Standard EqDia Check this option to enable counting of objects using standard EqDiameter Value of the standard EqDiameter parameter can be edited in the field nearby a Keep updating count refreshes the measured results each time the binary layer changes e g when the threshold is modified 145 146 Working with Macros 8 Working with Macros A macro an executable sequence of commands can make the work very effective NIS Elements AR provides a C like programming language utilizing its internal set of functions The sequence of functions can be created either by recording the performed actions by writing the functions within the macro editor or by modifying the command history the history is recorded automatically during the work The macro can be saved to an external mac file for later use 8 1 Creating Macros Recording a Macro The fastest way to create a macro is to record it e Start the NIS Elements AR macro recorder by selecting the Macro gt Record command e Perform the
55. which opens Remove This button deletes the selected objective 43 Recalibrate Starts the calibration of the selected objective See Objective Calibration Export Enables you to export the complete list of objectives to an XML file The standard Save As window ap pears Import This button enables you to import a complete list of objectives from an external XML file previously created by the Export button Current Unit This button invokes a pull down menu where units for the whole application can be selected Help Displays a help page to the Calibration gt Objectives command Edit the properties of the selected objective in the bottom part of the window Press the Close button to finish this window 3 5 2 Objective Calibration After you press the Recalibrate button in the Objectives window or when creating a new custom objective the following window appears Calibration Method Select method for calibrating objective Plan Apo 4x Manually drawing a line of known length Automatically using stage Select one of the calibration methods e The Manual calibration lets you draw a distance into a picture and assign real length to it see below e Ifa motorized XY stage is available the Auto and 4 points automatic methods appear Press OK to continue Manual Calibration 1 When performing a manual calibration the live image starts automatically and the following window ap
56. you specify the number of fields to be scanned and starts a wizard where you will specify the top left and the bottom right corner of the well plate The distances between wells are calculated automatically Random tab requires Local Option Specify area where certain random set of points will be acquired 53 Custom Multipoint Definition center x 60000 JEjmm wis 30 000 Emm center 0000 fejem Heh 9 000 Elm number fons 3 Rectangle The area will be defined by a rectangle Define X and Y coordinates of left top corner of the area width and height of the area Radius The area will be defined by a circle Define X and Y coordinates of center of the circle Number of Points Define number of points in the area 4 2 3 Z series Acquisition Automatic capturing of images from different focal planes of the specimen can be performed Run the Acquire gt Capture Z Series gt Capture Automatically command There are three different approaches of how to set the experiment E abs Step 5000 pm f 1 600un io steps Feroe 9000 _ om Bottom 0 00 wm Top 90 00 pm EN Y piezo Bottom um Close active Shutter during Z Movement 54 Capturing There are the following ways of defining the Z drive range Range by Top Bottom Positions and Step 1 2 3 4 Press the i button the cube turns blue Run the Live camera signal In case you have two Z drives select the Z dev
57. 1 O O A O Time line By clicking to the time line you can browse the movie frames When a key frame is added a bold line appears in the time line to indicate its position First Frame Play Last Frame buttons Settings by this button you can define the movie length and the frame rate Create Movie The key frame buttons The existing key frames may be browsed by pressing the Previous Next Key Frame buttons The Add Update Key Frame button inserts a key frame to the current position on the time line Presets a predefined rotation can be loaded When you press the button and select the rotation type the existing key frames settings will be overwritten Import Export Key Frames this enables you to save and load the key frame settings A file with the KEY extension will be created during the export How to Create a Movie Run the Movie Maker by the he button Adjust the movie settings by the Settings button a window appears where the movie duration and frame rate can be defined You can use one of the predefined presets or import some previously defined key frames by the Export Im port button Either enable or disable the Director s mode by the clapper button See Directors Mode Go to the beginning of the time line and define the initial Zoom position rotation or component appearance of the object This step is not necessary but very much recommended Press the Add Update
58. 1x amp 0 00006 mm px 2560x1920 Zoom 1x Relay lens 1x 0 00003 mm px 2560x1920 Zoom 1x Relay lens 1x lan Ap 10x DIC M Plan Apo 40x DICMN2 ss MH Pos Pos 5 Pos 6 PS Da Plan Apo 60x OIlDICH 4 Plan Apo 100x Ol DICH dk 400000044 Physical properties Plan Apo 20x DICM Work Dist 1000 um AF Range o Jum 20x Z Step AF o9 um Numerical Aperture 0 75 0 0002 J mmpoxy xXx ZStep Sices 5 Jum Refractive Index A list of used objectives can be created within the Objectives window For each objective the objective name position in the changer storage status and the calibration are displayed A Calculator E icon next to the calibration value indicates that the calibration has been calculated from objective properties If the calculator icon is missing the calibration has been performed manually Use the buttons on the right side to manage the objectives Insert Click the button and select one of the predefined objectives from the list ordered by magnification New Press this button to create a custom objective The new objective will be added to the list Then define the Main properties and the Physical properties of the objective Duplicate If zoom is used the objective calibration must be re calculated accordingly Use this button to make a copy of the objective and define the zoom factor in the window
59. 3 1 1 Live Signal from Camera Live Formats A camera format is a set of attributes of a video signal such as resolution bit depth frame rate etc The live signal is a stream of image data coming from the camera to your computer in real time NIS Elements AR provides two different camera modes Fast and Quality each serving a different purpose The format of both modes can be set within the Camera Settings control window l Live Fast This tool bar button runs the Fast live mode This mode is usually set to low resolution therefore the frame rate is high It is used for searching the specimen or when focusing manually lb Live Quality This tool bar button runs the Quality live mode This mode should be set to high resolution which reduces the camera frame rate It is applied every time the Capture button is pressed 3 1 2 Capturing Images Although it is possible to perform some procedures directly on the live image other image operations require a captured or a frozen image If you try to perform such procedure while the live image is active NIS Elements AR will automatically freeze the image There is a difference between the Capture and Freeze commands 32 Setting the Hardware Up dD Capture When pressed the camera exposure runs till the end of the current frame and the next frame is captured and displayed on the screen it is the first frame with the complete exposure after you have pressed the Captur
60. Contrast Lets say that we very often use the Image gt Contrast command It is useful to add a shortcut button to the tool bar Press the Add button and choose Command from the pull down menu e Anew command CommandQ is added to the list e Now attach the NIS Elements AR command Open the pull down menu on the right side of the Command edit box and click Command List e A list of commands appears Choose _Contrast e Confirm the selection by OK It is possible to create a sequence of commands by repeating this procedure If you are not satisfied with the default icon you can change it by pressing the Change button A window for selecting the icon appears You can select the image from the NIS Elements AR icon set by default or load 18 Application Appearance some other from any file containing icons ico dll You can define the icon for the command in a disabled state too It is handy to define a tool tip a text that appears when the mouse cursor is placed over the icon for your command Simply write the text into the Tooltip box You can change the position of the command in the tool bar using the arrow buttons The Default button discards your changes and inserts the default set of commands to the tool bar 2 2 2 4 Modifying the Menu The Main Menu and some of the Context Menus accessible by right click within the application
61. IMAJe Processing E AAA AA E A A A ATA A 17 6 1 1 Processing On Intensity RGB Channels oococococcoccconcconcconcconcconcconnnannna nn rca 77 6 1 2 NDZ Files o AAA na i a aaa a da E Aa E 17 6 2 histogram and Look UP Tables lt script ii rta a 78 2 USOS vr A NAS AA AAA AS 78 0 ZA O 80 Se WIGS A O a a A dich tcl ero aaa 88 Oo ADIOS faeries raat otc la SAN ae EN rd oa So ing 88 A F A 99 E ae te tee A ee Oe eee a la 91 A IMLCMSILY MOIE se Sic t te a tn hem eae et ne abe Rt ang eed ein eae oad 91 019 42 MEA MOG o rericestcstcs decirte cion tie bin ies diia 91 04D Mar LEIVO eur tula darla rra ivi 91 6 4 1 Controls horizontal tool bar ic A A nee aes 92 6 4 2 Drawing tools vertical tool bar ccc ccc cece cece eee cence ence eee e eee eeae seat eeaeeeneeeaeeaes 93 6 0 ReGgions On Interest ROIS user ita e A ts ca iced Nevins 95 6 5 1 Creating and edling RO Susini ad a ains arica 95 00 72 IYD SO ROL A E E eee 96 0 00 02 Mteracion WIR Se eo aid no dado Ed a MU 97 6 5 4 Simple ROMEO sais conri iii ise 98 6 5 9 Edf ROISTA TIMO stas anto e tao oia a sea cates 98 0040 AU OD ete cuina A A A a ia ci 99 6 59 70 O A tee ae ee aurea 100 6 5 9 ROIS OVINA eat bas eet ee E eee eae ene eos 100 0 0 Cheating REDON secrete forays sa adh ee aad eam ae a Mes eae dc des 103 6 6 1 Working with Report Objects a pa 105 6 6 2 Working with Report Templates ccc cece cece eee eee cena eee eeeaeeeeeeeeeeeaeeeeaes 107 6 6 3 Creatin
62. M378 119NE16 TT Nikon NIS Elements Advanced Research User s Guide Ver 4 00 Thank you very much for choosing Nikon This manual explains installation and use of the NIS Elements Advanced Research For trouble free operation read this manual before using the program No part of this manual may be reproduced and transmitted without Nikon s permission The content of this manual is subject to change without notice Nikon has carefully prepared this manual However we make no expressed or implied warranty of any kind and assume no responsibility for such errors or omissions Be sure to read the instruction manuals for the microscope and PC you plan to use with the NIS Elements Advanced Research Trademarks Microsoft and Windows are either registered trademarks or trademarks of Microsoft Corporation in the United States or other countries Products and brand names are trademarks or registered trademarks of their respective companies The TM and marks are not used to identify registered trademarks and trademarks in this manual To run NIS Elements Advanced Research optimally the following specifications are required Minimum PC Specification CPU Intel Core 2 Duo or higher RAM for 32bit OS 2GB or higher RAM for 64bit OS 4GB or higher Operating system Windows 7 Professional SP1 32bit 64bit English HDD 2GB or more of available hard disk space for installation Display 1280x1024 pixel True Color mode
63. Measurement Type object Image Type binary WaveLen Dominant wavelength This feature is defined as a wavelength of the pure spectral color that is together with white the measured color composed of The color in the purple must be composed of more than one pure spectral colors therefore they have no dominant wavelength Measurement Type object field manual Image Type color Width Width is a derived feature appropriate for elongated or thin structures It is based on the rod model and is calculated according to the following formula Width Area Length 132 Measurement Measurement Type object Image Type binary Width 7 3 Mathematical Morphology Basics The binary image as a result of thresholding often needs to be modified before a measurement is performed Edges of the objects can be smoothed holes in the objects filled etc by using the mathematical morphology commands Note Image Analysis and Mathematical Morphology by J Serra Academic Press London 1982 was used as a reference publication for the following overview The basic processes of mathematical morphology are erosion dilation open close and homotopic transform ations Erosion After performing erosion the objects shrink Marginal pixels of the objects are subtracted If an object or a narrow shape is thinner than the border to be subtracted they disappear from the image Dilation After performing dilation the objects
64. S icsarsen Sic ated o e Sink Voce uate ete el 37 DO LOGICA DEVICES siii o id 37 Jo PS it AS ica 39 3 4 Optical COMMQUIATIONS ct ias 40 20 ODJECIVE peer nen eae E 43 301 Managing OO IES ad ld 43 350 2 OD ECUVS CG allDrallO Mm ss iaiices i oie 44 05S IAS caer deatac emutieei a fetes ooiaed et uae bia e ean em edones O A 47 AO AO Dt het eGo tetas feat o eaten o o ie ee ee 49 Adlone Image Captura N soe dude oda 49 4 2 NDZ ACGUISINION 2 sd is N 49 4 2 1 Timelapse ACQUISITION siii E EAK 51 42 2 Multipoint ACGUISINION sica li 51 ALI NES ACquUISINON eeir e a adele r 54 4 24 Multi channel ACQUISITION ti sio 56 42 5 Combining N DIMENSIONS is coud 57 4 3 Capiuning 10 RNG BUNE 2221 aai rd rE 60 AA Capturing MOVICS erena a ii a a a did 61 Image Fle S eee en N rN E E r E ns 63 Sle CompS PP A 63 oki mage Layers orre E EE cee settasedaed 63 51 2 Image Types m NIS Elements AR uu din 64 nes NO MOS aos sita e ta nl tame es 64 52 WOKING WIth Image PROS a A A A A A ad 12 52 1 Openng oaved IMAGES ae o e bae aa e e nr A 72 5 2 2 Switching Between Loaded Images 0 cece eee e eee e eee e eee e eee eaeeeae eee eaeeeeeeeeeeeeeees 12 OZ O GO SIM AMAS yt er Coat ae 73 9 24 00 WNG Images with VACA A SA 73 5 2 5 Copying Channels by Drag and Drop ooococccocccoccconcconcconcconncnnncnnncnnnrnnncnnnrnnnnnnos 73 0204 Made RONDAS remote RE Mactan santos 14 O lMade Anal SIS ties dilata Donar tadas Mata dais Crd 17 SA
65. Select the columns to display hide from the context menu which appears when you right click one of the column captions e number of selected objects is displayed in the left part of the tool bar One or more objects can be se lected by mouse while holding the Ctrl or the Shift key 144 Measurement Result Total Area 6534 13 pm Area Fraction 16 55 Perimeter Whole Image gt 22 40 Whole Image Threshold 17 05 Whole Image Threshold 5 23 99 Whole Image Threshold 31 39 Whole Image Threshold 3 77 Whole Image Threshold 31 28 Whole Image Threshold 2 35 06 Whole Image Threshold 14 12 94 Whole Image Threshold 15 15 94 Whole Imane_ Threshold 14 73 73 Tool Bar There is a tool bar above the results table which contains the following buttons xX Delete selected objects removes the selected objects The objects can be selected by mouse while holding Ctrl i Invert selection deselects objects selected by mouse and vice versa E Generate binary erases binary objects which do not meet the restrictions settings Show Object Calatog opens the Object Catalog control window with data source automatically set to Object Count Export the measured data can be exported to clipboard or a file using the Export button Please refer to the Exporting Results chapter for further details Ap Export ND data
66. Stage Z The Z Drive device enables movement throughout the Z axis direction TTL Analog Input Output There are six more logical devices available when the NIDAQ controller set is installed TTL Input TTL Output Analog Input Analog Output Calibrated Analog Input Calibrated Analog OutputEach of these logical devices represents a connection to an external device which can receive send signals from to NIS Elements The number of available connections depends on the actual NIDAQ card 3 3 2 Tips Assigning objectives To assign an objective to a position or to change an attached objective assignment click the setup la button in the nosepiece section of the microscope control pad e A window appears which enables you to select one of the available objectives to the corresponding position e Each objective has its specifications displayed in the table These specifications are not editable Motorized Stage Initialization A motorized stage being initialized could strike the objective Before initializing the stage make sure that the objective is away from the stage If a motorized Z drive is available use the Devices gt Objective Clearance command to prevent this Using two independent Z Drive devices Workstations can be equipped with two independent Z drive systems one coarse slow and the other fine fast Typically the first one is used for specimen manipulation in Z axis when changing objectives etc the second one i
67. Status Bar 0 34 pm px RGB 8bit 1232 x 972 pixels 653 464 RGB 107 136 141 e Image calibration e Image bit depth 8bit 12bit 16bit etc e Image size e Pixel coordinates of the mouse cursor along with channel intensities e Binary layer value 0 or 1 e Color mode RGB Monochromatic etc 2 2 Modifying Layout Having a well organized application layout can help you make the work with NIS Elements AR very effective There are the following options on customizing the appearance of NIS Elements AR Custom window placement The live image opened images and all control windows Camera Settings Measurement Histogram LUTs etc can be arranged inside or outside of the application Note To move the Image window out of the main window drag the Image window title bar and drop it when the mouse pointer gets fully outside of the main window Otherwise the Image window would go back into the main window Compact window or multiple windows The control windows as well as tool bars can be floating or docked on sides of the application screen Multiple monitor support The NIS Elements AR window can be stretched to occupy two monitors When you switch from different application NIS Elements AR will be activated on both monitors Customized tool bars Tool bar buttons may be added and removed from tool bars 2 2 1 Layouts A layout in the context of NIS Elements AR is a set of options describing the arrangement of cont
68. TI 7 1 je Me o QUA Wil Mt Date 30 7 2004 gia ri Serie AAAA a Operator DM Result Ss ii 3 defects found Eee marked by arrows gt N a E ui Eae It provides the following tools 103 Draw line Ll Draw rectangle Draw ellipse Draw Arrow Insert text field dl Insert image from file Insert Table all Insert graph IM insert bar code Align aligns selected objects to the position indicated on the button by the line ib Center aligns the selected objects to center HH Distribute distributes the objects uniformly along the specified direction Determines whether to apply the align commands relatively to the whole page or the selected objects only Context Menu Commands Once you insert an object its appearance behavior and position can be changed via the context menu which appears on right click e Ifthere are more than one objects selected the Group command makes them to behave as a single object The Align or Distribute commands enable moving objects within the page or align them with another ob ject s The Resize object commands enables to resize two objects to the same size The Bring to front command changes the order of overlapping objects and brings the current object to front The Send to back command moves the object to the bottom layer The Lock command locks the object disabling any further changes to
69. XY stages and Z drives but also other devices See Connected Devices 9 12 Extended Depth of Focus The EDF module allows you to combine an existing Z stack of images into one focused image by picking the focused regions from each frame and the pieces together Please see the electronic help for more details 158 Additional Modules 9 13 Filters This module is aimed at analysis of filters according to the ISO 16232 standard Please see the electronic help for more details 9 14 HDR requires Local Option The High Dynamic Range module brings the functionality of creating HDR images within NIS Elements AR The following ways to create a HDR image are available Automatic capture Call the Applications gt HDR gt Capture Reflection Free Image command and the HDR image will be captured using computer estimated settings Capture When the Applications gt HDR gt Capture HDR Image command is called you will be asked to adjust the HDR acquisition settings such as number of exposures exposure time range camera settings etc Create from files If a sequence of images which were captured using different exposure times is available the Applications gt HDR gt Create HDR Image command can load the images and merge them into a HDR image Create from an ND file Similarly to the situation when the HDR file is created from a sequence of images it can be as well created from the frames of on nd2 file Just run the
70. a dic dd aaa 157 SZ2740 00 ACQUIS MON a A A A E A DS 157 9 9 ARES AOVANCed Interpretar a e eat Dad 157 OA Calcium and FRE T e iitaids id idea iii 157 AS A e EAA eee een re ne 157 9 6 AutoQuant Blind DECONVOMIION ia iS A O A a 158 9 7 AutoQuant Real Time 2D Deconvolution cece cece cece cence cnet eee e eee eeee ease eaeeeeeeeenaes 158 907 EIM DECOMVOIMMON vaccine sas Aad reas exe ince eee ts eones 158 DBO CAO DMO eraen nse eke eA Saha ae Speen Mie ong ees cote roe ale ena at Mo nee 158 0 10 Deconvolution ICI A A a ro 158 91 Penpreral Devi a a a a oe cue a ei 158 9 12 Extended Depth Of FOCUS se ccciisoranionairc td da aaa 158 OV Ses CRS notte at e en ce 159 A A A A A Pn te ee ee 159 9 15 Metalo Grain Size ANYS a 159 9 10 iMetalo lt Cast ron Analysis insirisci A a A a ai dd ee cok 159 Yi Me Meastre ment coco cnica is e di ee eee 160 SO MACKIE A AA A a o 160 919 Advanced ITACKING AA A A ate E 160 10 Supported Cameras sisters id id aos 161 11 Supported MICKOSCODCS tensas ss false alada ada tn ds tcs Jolie s eel s leas Slane es Rahat 163 Installation and Startup 1 Installation and Startup 1 1 NIS Elements AR Installation Quick Guide e Insert the installation DVD in the DVD ROM drive A window automatically appears e Install the selected NIS Elements AR software version additional modules and device drivers e Plug the provided HASP key into the USB port of your PC e Run NIS Elements
71. age jp2 This command runs NIS Elements AR and opens the starting _image jp2 at the beginning Note e By default only one instance of the application is allowed to run When the application is running and a command similar to our example is called it will be applied to this running instance This does not apply when the q switch is engaged e Some switches do not have parameters 1 2 2 Switches Displays a help screen with the description of switches c Command The application runs the specified NIS Elements AR macro command f Filename The application tries to open the image pointed to by the Filename parameter g Grabber name The image grabber driver of the specified Grabber name will be used and no camera selection window will show at startup As the name of the grabber you can use the name which appears in the startup driver selection dialog or the name of the driver class These are listed in Windows registry HKEY CURRENT USER Software Laboratory Imaging Platform Platform INI CLxGenericProviderGrabber Set the parameter value to last and the most recently used grabber camera will be loaded gn No grabber driver will be loaded The passive version of the software will start h HW Unit Name The specified HW Unit will be used Two HW Units e g two DS U2 cameras can be run by one grab ber driver The available HW Units are listed in the Acquire gt Se
72. al Configurations To display the optical configurations manager window run the Calibration gt Optical Configurations command You can make the following actions from the window e Create duplicate rename delete copy settings and switch between optical configurations e Modify optical configuration properties e Import and export optical configurations to from an XML file Operations with Optical Configurations Once created the configuration appears in the list and can be shared with other users by changing the Private option to Shared see User Management The following operations can be performed on the selected configuration e It can be deleted by pressing the Remove button A confirmation dialog box appears e Its name can be changed by the Rename button A button with the configuration name appears in the tool bar if the Show on toolbar option is selected e The configuration settings may be transferred to another optical configuration by the Copy to button Press the button and select the optical configuration to be overwritten with the current one e A copy of the configuration can be made via the Duplicate button e The configuration can be applied to by the Set As Active button e The settings of all optical configurations can be exported to an external XML file using the Export button e The previously exported optical configurations settings can be loaded from the XML file via the Import button
73. al density filter is a light absorbing filter whose absorption spectrum is moderately flat It is used to reduce the illumination intensity within the optical path The logical device offers two states ON inserted and OFF extracted PFS Perfect Focus System this logical device corresponds to the PFS physical device available with Nikon TE2000 T microscopes 38 Setting the Hardware Up Shutter This logical device can control shutters installed in your system This device is handled via the Devices gt Filters and Shutters control window or straight from the microscope control pad You can select the type and rename the shutter by running a contextual menu command either within the Filters amp Shutters control window Device Manager window or in the main tool bar Note There was a restricted number of shutters which could be connected to the system in previous releases of NIS Elements AR and they were identified by Type DIA EPI Aux1 etc The current version supports identification of shutters by custom Names but the Type attribute can be still found in some windows or used in some macro functions This is to ensure backward compatibility Zoom This logical device is used for controlling the zoom factor Run the Devices gt Zoom Configuration command to adjust the zoom settings Stage XY Stage XY enables the movement of specimen within X and Y axes The system offers user to control stage movements
74. an external library Syntax import DLLname import function_ declaration NIS Elements can call functions from external DLL s You should import the DLL where the functions resides and then make a declaration of the functions You should not import the following system DLL s kernel32 dll user32 dll gdi32 dll com32 dll comdlg32 dll This feature is available only for 32 bit version of NIS Elements Example import luc_13 dll import int RTF_ReplaceVariables LPSTR destfile LPSTR sourfile import int RTF_FindQuestion LPSTR sourfile LPSTR question long length LPSTR defvar import int RTF_ReplaceQuestion LPSTR destfile LPSTR sourfile LPSTR replacement Operators The following operators are supported by the system No operator precedence is supported You should use brackets to define evaluation order other than implemented in NIS Elements AR which is from right to the left arithmetic operators Addition Subtraction Multiplication Division assignment operators The assignment operator assigns the value of the right operand to the left operand Addition 153 bitwise operators The bitwise operators compare each bit of the first operand to the corresponding bit of the second operand The following bitwise operators are supported amp Bitwise AND Bitwise OR One s complement pointer operators amp Address of Indirection relational operators lt Less than
75. analysis systems consist of a computer a camera and a microscope equipped with certain accessories objectives filters shutters illumination rotary changers etc Most of the mentioned microscopic hardware is often motorized and therefore can be controlled via NIS Elements AR In addition it is possible to integrate single settings of all these devices into one compact set called Optical Configuration It is recommended to create several optical configurations containing particular devices settings Then a single click can completely change the current hardware configuration Creating New Optical Configuration 1 Please check that all the devices microscopes cameras etc which you want to associate with the new optical configuration are properly attached to the system and working 2 Choose the Calibration gt New Optical Configuration command The following window appears Adjust the settings of the devices to match the intended state which will be saved to the optical configuration 40 Setting the Hardware Up Name 10x Camera DS Fi1 U2 Camera setting Property Values for Fast Mode ExposureTime 1000 000 ms AnalogGain 6 80x Blue Green Red Active Shutter Default Active Shutter z Default Active Shutter from Light Path None Used devices Microscope Ti Microscope Y A Saas Ti Turreti 2 UV 2A LU4A Shutter 638 nm Ti Tu
76. apping During report creation all the other cells of the grid will be filled automatically according to the settings of the first one e A custom header or automatic page numbering can be added to the report 6 7 Creating Movies Movies is one of the possible ways to present data captured using NIS Elements AR The following function ality is available e A real time movie of a time lapse process can be captured e An ND2 file can be converted to a movie 108 Image Analysis e A3D model or a surface view of an object can be set in motion and make a presentation movie Movies are saved as an AVI which can be played back by any standard video player so you do not need NIS Elements AR to view them 6 7 1 Capturing AVI Movie e Display the AVI Acquisition control window by the Acquire gt AVI Acquisition command e Adjust the advanced settings especially define the file name and the destination folder e Click the Record button The live image appears and recording starts according to the settings e Stop recording by the Stop button or wait until the time defined in the Duration field passes e When stopped the AVI file remains opened in a new image window Settings Press the Advanced button to view the whole control window and set the following options sitio x c Rate MaxFps Duration Infinite Ready 84 7 GB free on drive C C Program Files NIS Elements images Maintain as
77. arious cameras differing from each other in resolution frame rate sensor type etc Despite these differences controlling different cameras is similar The following features are included in the View gt Acquisition Controls gt Camera Settings wndow depending on a particular camera type Common Settings Fast Focus Quality Capture Formats Usually cameras can provide several image resolutions NIS Elements AR enables to create two presets of the resolution settings Fast and Quality The formats then differ in image size and consequently in frame rate number of frames per second fps The higher the resolution is the lower frame rate can be achieved Available resolutions depend on the camera type Note When switching between the formats while observing the live image the size of the image on the screen is maintained the zoom setting is changed instead Only in some special cases the behavior changes and the image size is changed instead of the zoom setting which Is maintained Binning The binning mode provides considerably enhanced camera chip sensitivity by integrating more elements pixels together E g binning x4 integrates the signal from the area of 4x4 chip elements to one pixel of the resulting image Smaller resolutions and faster frame rates are achieved using the binning modes Exposure Mode Exposure mode sets how the Exposure time is calculated Generally there are automatic and manual modes e A
78. ary objects graticules for equally spaced shapes and from binary layers created by tracking 95 Available ROI tools Simple ROI Editor toolbar lt X Pointing C Rectangle A Elipse SJ Polygon C gt Bezier Auto Detect X Auto Detect Al 8 Advanced GUI DrawHoles X Clear 1 Undo A Redo 7 Help ES Finish P Move Pick lt J Draw N Detect d New m New After Edit Select Next Object z Skip 1 frame Delete 1 Undo A Redo 7 Help EJ Finish Turn On Background ROI button Context menu over a ROI 1 Selected ROI Turn RO On button Delete Selected ROIs Duplicate Selected ROIs Lock Selected ROIs Ctrl U Copy Selected ROIs Remove Selected ROIs Touching Image Borders Separate ROI Use as Standard ROI Use as Background ROI Use as Reference ROI Use as Stimulation ROI ASON 2c a fA lt 4 gt gt di Ju DDD 2x16bit 512 x 512 pixels N A ROI is Global ROI changes over Time Frame Interpolation Method Raster ROI Editor Edit ROIs in Time Draw Rectangular ROI Draw Circular ROI Draw Elliptic ROI Draw Polygonal ROI Draw Bezier ROI Auto Detect ROI Copy Binary to ROI Move Binary to ROI Copy ROI to Binary Move ROI to Binary Swap Binary with ROI Simple ROI Editor Ctrl Shift R ROI menu ROI Properties Delete All ROIs Colorize Renumbe
79. assigning a bitmap image and a macro function to the menu command It works the same way as when modifying the Main Left Tool Bar described above 2 2 2 5 Commands These settings enable to run a command or a macro right before and after you switch to the layout Command before Layout changing RunMacrot c MacrostLayoutChangedl mac B 2 2 3 Docking Panes Docking panes are square spaces inside the application window where you can place dock any of the control windows There is one docking pane available at the Right Bottom and Left side of the application screen To Display a Docking Pane e Go to the View gt Docking Panes sub menu and select the pane you would like to display 20 Application Appearance Docking Panes Ctrl 4lt Hum4 Acquisition Controls 1 Right Ctrl Alt 4Wdum6 Analysis Controls gt Bottom Ctrl alt Num 2 Note The Docking Panes sub menu can be also displayed by right clicking into the empty application screen e The docking pane appears either empty or with some window s docked inside e Repeat this procedure to display more docking panes Handling Control Windows Various control windows can be displayed docked within the docking panes or they can be floating See the following picture The Docked Control Window Caption l Histogram x SS To handle the control windows CWs you can Add CW to a docking pane Right click inside a docking pane 3 and sel
80. be adjusted deleted or the nd2 file can be cropped e Detailed info about dimensions will be displayed after you click the leftmost button of the control bar T gt Z gt e Place the cursor over one of the dimensions A tool tip which displays statistics of the dimension appears Playing options Right click the speed bar to display the following context menu and select the playing mode 66 Image Files Back and Forth Forward To Backward The following options can be selected depending on the dimension type Back and Forth Forward Backward Select the direction of playing the image sequence Stop on Events When you check the Stop on Events option and then press the gt Play Sequence button a dialog window appears once the first user event is reached The dialog window displays information about the reached user event Use the Continue button to continue playing to the next event or press the Stop button to stop the playback at the current frame Within the window you can also select the Do not ask again in this session Always stop option the playing will stop automatically on every user event but the window will not appear Repeat Select this option for repetitive infinite playback Fastest Advance This option corresponds to the D Maximum Playing Speed for Every Frame button described above Real Time This option corresponds to the D Real Time Playing Speed button described above
81. bjects that are connected together and isolates them Homotopic Transformations Homotopic transformations preserve topological relations between objects and holes inside them Using a homotopic transformation an object with 5 holes will be transformed to another object with 5 holes Two objects without any holes even if they are very near each other will become again two objects without holes but likely with a different shape and size Opening Closing Erosion and Dilation are not homotopic transformations Typical homotopic transformations in NIS Elements AR are Skeletonize Homotopic Marking and Thick ening 7 3 1 Connectivity Applying the above mentioned transformations has some limitations due to digital images rasterization When speaking about binary image processing a binary image is a set of pixels where values 1 represent objects and values O represent background In the square grid of the image two possible connectivities can be used for processing the 8 connectivity or the 4 connectivity Look at the picture below If the 8 connectivity is used the two pixels represent one object If the 4 connectivity is applied there are two objects in the picture NIS Elements AR works with the 8 connectivity model so all pixels neighboring by the corner belong to one object 7 3 2 Structuring Element Kernel Matrix When applying Erosion Dilation Opening or Closing one of the parameters which determines the transform ati
82. cerning the appearance of the screen displayed control windows zoom etc Devices Contains commands needed to handle all connected devices except cameras Window Concerns opened image windows It enables to select a window to be displayed or select a predefined windows documents layout Applications Gathers commands of additional modules if there are any installed Help Enables access to additional information about the system such as help HASP Info application log etc Organizer In Organizer most of the menus are hidden but the following is added Tools Contains commands for filtering and sorting images 10 Application Appearance 2 1 2 Application Tool Bars There is a default set of tool bars each tool bar containing number of buttons There is also one fully custom izable tool bar the main left tool bar to which any button can be added Every button or whole tool bar can be hidden by user Please see Modifying Layout for further details 2 1 3 Status Bar The status bar at the bottom of the screen displays the following information The Application Status Bar Full Screen AL eu DS Fi1 LocalContrast 15 25 0 0 Achromat 10x 0 XY 100 000 100 000 mm Z 0 0um 1 2 3 4 5 1 Available layouts Note The layout Tabs may be hidden when the Edit gt Options gt Appearance gt Show Layout Tabs option is deselected 2 Type of the current camera 3 The most recently performed command
83. ch control window appears in the same position as it was before it being hidden The controls are sorted to several groups Analysis Acquisition Macro Visualization Acquisition Controls j Analysis Controls Visualization Controls CO Controls Macro Controls P ha Histogram Ctrl Alt H LY Lis Ctrl Alt Shrink Expand dockers Having more docking panes opened a situation where there is not enough room for the control windows can occur In such case the Shrink and Expand commands shall be used e Right click the pane you would like to shrink expand A context menu appears e Select the Expand Shrink command When one of the panes shrinks the neighbouring pane expands to the emptied corner and vice versa 22 Application Appearance 2 2 4 Appearance Options General appearance adjustments can be made in the Options window Run the Edit gt Options command and switch to the Appearance tab Prompts on Image Save dialog The Z value delayed in status bar Enable Z Intensity control Image Window Toolbar _ __ 2 0 Right Toolbar O A C Auto hide bottom toolbar Channel Tabs for RGB and Gray Images _ _ _ ___ Show channel tabs Layouts Show Layout Tabs Show Task Bar mouse wheel scrolls through 7 stack for nd images containing 7 in out for other documents CTRL mouse wheel zoom in out for all documents Keep text size whil
84. change the current image select it from the list or use the Next or the Previous commands represented by Ctrl Tab and Ctrl Shift Tab shortcuts 12 Image Files Mext Ctrl Tab Previous Ctrl Shift Tab Close All Ctrl Shift F4 Close All but Current Shift F4 Close Cascade Tile Horizontally Ctrl Shift H Tile Vertically Ctrl Shift yV 1 C Program Files NIS Eleme ich 1 nd2 Focused 2 Frozen Image windows can be also arranged automatically using the Tile horizontally or Tile vertically commands This will change the size and position of the opened documents and they will be arranged next to each other in the selected direction 5 2 3 Closing Images The currently displayed image can be quickly closed by pressing the cross button in the top right corner of the image window The image can be also closed by invoking the File gt Close command If you want to close all images use the Window gt Close All command If you try to close an image that has been changed NIS Elements AR will display a confirmation dialog box offering to save the changes Use the Window gt Close All but Current command to close all opened documents but keep the current one opened 5 2 4 Saving Images with UAC Because of the security enhancement with the UAC user account control of Windows Vista and Windows 7 it is not possible to save images in those folders that require Windows administrator user ri
85. d Auto Capture Folder The Auto Capture Folder is a control window that can be displayed by calling the View gt Acquisition Controls gt Auto Capture Folder command It displays images within a selected folder To change the folder click the a button in the top left corner of the control window and browse for another folder Any image of this folder can be opened by a double click Any File Manager During installation NIS Elements AR creates file associations to files that are considered its native format for storing images JPEG2000 ND2 The JPEG2000 JP2 and ND2 image files can be then opened in NIS Elements AR just by double clicking their names within any file manager or the desktop Opening Files in Progressive Mode If the image to be opened a single frame in case of nd2 files is too big so that it can not be loaded to RAM in one piece you will be asked whether to open it in a Progressive Mode This means that only a thumbnail of the image will be loaded to RAM When zooming in to view image details the image data of the particular portion of the image will be loaded to RAM progressively piece by piece Many image processing functions and commands are disabled in this mode 5 2 2 Switching Between Loaded Images Commands for managing the opened images are grouped in the Window menu The recently opened files are listed in its bottom part The currently displayed image is indicated by the selected radio button To
86. d tab and choose the Load Default command The application loads the previously saved default layout 2 2 2 Layout Manager Call the View gt Layout gt Layout Manager command The Layout Manager appears 2 2 2 1 Layouts The list of currently available layouts is placed on the left side of the layout manager Each layout may contain information about controls tool bars menu and commands to be performed when switching between layouts 15 The List of Layouts olx el al el dla E4 4 Full Screen Private H Docked Controls Private E 4 Measurement Private nf Controls nf Toolbars if Menu Modifying the Layout Settings Select items within the Global Layout list which you want to be shared by all layouts The check marks on the left of the layout names indicate the layout visibility Select the ones you want to display in the application status bar If an item within some particular layout is not selected it means you do not want to customize it and the global settings if selected within the Global Layout will be used If the item is not selected within the Global Layout either the settings of the most recently active layout will be used Set whether the layout is Private or Shared If set Private it will not be visible for other users See User Management Customize each item according to your needs see below Layout Manager Tools E The New button adds a new layout to the lis
87. d together so you just need to set two of these parameters The remaining parameter is calculated automatically Only the selected time phases will be captured The flag mark indicates the Duration Loops priority For example if the camera exposure time exceeds the defined Interval between frames than the experiment settings are not achievable only the marked column will be pushed to be correct You can set the priority column by clicking on its caption 4 2 2 Multi point Acquisition This window serves for defining XY Z points to be scanned during the multipoint capture experiment This feature is available when a motorized stage XY Z is present in the system Check the Include Z box to display the Z column The list of defined points can be saved and loaded later to an XML file by the Save Load button Run the Acquire gt Capture Multipoint gt Capture Automatically command 51 Points Move Stage to Selected Point aqioal slew Point Name x mm mm 1 0 000 5 200 2 gt 1 200 4 900 Offset All X Y 3 1 800 4 400 m indudez Relative x To Define a Multipoint Array e Move the stage to the first position Press the Add New button A new line containing current coordinates of the stage appears in the list e Move to the next position and repeat the steps until you have all the intended points defined To Change a Single Z Coordinate e Make sure the Move stage to selected point
88. depending on the value of an expression Syntax if expression statement else statement2 The if keyword executes statement if expression is true nonzero if else is present and expression is false zero it executes statement2 After executing statement1 or statement2 control passes to the next statement goto Transfers control of the program execution syntax goto name name statement You cannot use goto to jump inside the block from outside E g following is not allowed goto label If k gt 5 label DilateBinary 3 5 FillHoles You cannot use goto to jump out from the block more then 2 block levels down E g the following is not allowed for i 0 i lt 64 i i 1 for 0 j lt 64 j j 1 if a i gt b i value i goto end crossing 3 right brackets end break Terminates the execution of the nearest enclosing statement Syntax break The break keyword terminates the execution of the smallest enclosing for or while statement in which it appears Control passes to the statement that follows the terminated statement 151 continue Passes control to the next iteration of the statement in which it appears Syntax continue The continue keyword passes control to the next iteration of the for or while statement in which it appears Within a while statement the next iteration starts by reevaluating the expression of the while statement Within a
89. double Exposure function retrieves the current value of Exposure of the specified Mode CameraSet The CameraSet_ functions allows you to adjust camera properties So the CameraSet Exposure int Mode double Exposure function sets a new Exposure value for the specified Mode 3 1 4 1 How to Set Camera Properties Instead of describing each property function it is better to show you a universal way to handle a camera by macro 33 1 2 3 4 3 Display the View gt Acquisition Controls gt Camera Settings control window and find the property you would like to control Make a change of the property from within the control window For example change the exposure time Display the View gt Macro Controls gt Command History window and see the last function which has been called and its parameters e g CameraSet_ExposureTime 1 500 This is the function you will control the exposure time with Experiment with the camera control window in order to determine the right parameter values of the functions to be called within your macro Note See the Function Reference gt Acquire gt Camera gt Properties section of the online help Some of the most common functions are described there 2 Camera Settings A digital camera mounted to a microscope port or a macro scope stand records images ofthe observed scene on a light sensitive sensor and transfers them to a computer NIS Elements AR supports v
90. dow is placed in the specified rectangle in workspace coordinates The coordinates must be in parenthesis without any spaces q A new instance of the application will run 2 Application Appearance 2 1 NIS Elements AR Screen The following items can be found within the NIS Elements AR screen Application Appearance AISA Z O settings _ New 10x 380 3 1 G4 amp La Customize y E color Z series Pollen confocal nd2 Z 20 44 19 00 ym R 089 I NIS Elements AR 3color Z series Pollen confocal nd2 Z 20 44 19 00 um Lole j File Edit Acquire Calibration Image ROI Binary Measure Reference Macro Database View Devices Window Applications Deconvolution Help A y gt pHa Y B Owa Qauxi Qer Ona O auxi O aux w 1 00x G Iz s z 654 y ij NK ls e EJEA O G 817 w Mr olo Thresholding LUTs x P PRTI A rt tortor Kar gt gE DD All FITC M TexRed W DAPI Custom 3e 4mm px 3x12bit 512 x 480 pixels pakar IN a Intensity Profile x mM S a H All Bal t gt pl Al elle BMort OlH Tact Intensity 0 090 Graph Data Measurement Zoomx 1 0 ZoomY 1 0 T 0 120 T 0 130 0 140 mm DEIZ Y Full Screen AEE Measurement du DS Fi1 SetLayout Docked Controls XY 63 000 57 000 mm
91. e It is definable on an nd2 file See ROI in Time Moving ROIs 6 5 8 1 ROI in Time Moving ROIs To define a moving ROI e Open or capture a time lapse nd2 file e Display the first frame of the image and open the vector ROI editor by the ROI gt Simple ROI Editor com mand e With one of the editor tools draw the ROI around an object Close the ROI editor Right click the ROI and select ROI Changes Over Time from the context menu e Play the image sequence and if the object of interest starts to move out of the ROI pause the replay e Now it is possible to drag the ROI to the new position If the ROI is created by the vector editor its size and rotation can be also modified e Play the sequence further and drag the ROI to a new position each time the object in the image moves Note ROI positions between two user defined frames are interpolated If the object of interest was moving at constant speed with a linear trajectory then it would be sufficient to define the ROI positions only in the first and the last frame of the image sequence Tips on Moving ROI ROI Tracking Motion characteristics of a manually defined moving ROI can be measured using the View gt Analysis Controls gt Tracking control window Just click the Add Moving ROls T button to add the current ROIs to the table of objects Automatic ROI Definition Automatic Tracking features may be utilized to define moving ROIs Already tracked objects
92. e button Freeze The Freeze button interrupts the camera exposure and displays the very last complete frame Note If you are using the Fast mode and press Capture NIS Elements will automatically switch to the quality mode to capture the image When the image is captured it is opened on the screen in a new image window 3 1 3 Camera ROI on Live Image You can specify a camera ROI on a live image by selecting the Define ROI command placed in the image tool bar next to the ROI button In a window that appears set the dimensions and the position of the ROI rectangle The live image is then restricted to the defined area whenever the ROI button is pressed The Save ROI command enables to save the current ROI to a file whereas the Load ROI command loads a ROI from a file and overwrites the current ROI A W colt ho Define ROL Save ROL Load ROI 3 1 4 Controlling Cameras by Macro Every camera can be controlled from within a macro by changing its properties There is a CameraGet_ and CameraSet_ macro function available for each camera property e g CameraGet_ExposureTime The number of functions beginning with CameraSet_ and CameraGet_ and their actual names depend on the type of camera s you have currently connected to NIS Elements AR CameraGet_ The CameraGet_ functions retrieve current values of the properties determined by the function name postfix So the CameraGet_ Exposure int Mode
93. e binary objects in several different colors The algorithm ensures that two neighboring objects are never colored by similar colors See Mathematical Morphology Basics 6 3 2 HSI Mode The HSI thresholding mode is available only if an RGB image is opened The thresholding procedure works the same way as in the RGB mode except that the pixel values are displayed in the HSI Hue Saturation Intensity color space and that the Saturation and Intensity channels can be switched OFF Therefore you can threshold over the H HS or HI channels only Switch the channel off by de selecting the check box in the top left corner of the channel histogram 6 3 3 Intensity Mode The thresholding procedure works the same way as in the RGB mode except that it is performed on the in tensity pixel values 6 3 4 MCH Mode The MCH mode is not available when working with a monochromatic single channel image This mode is dedicated to perform threshold on multichannel images but can be applied to RGB images as well e If there are many channels only one channel histogram is displayed Switch between the channels by se lecting their names from the bottom pull down menu e The binary layer s can be displayed in two modes i oo Each channel can create its own binary layer 2 i Y b One layer can be created as an intersection of all channels binaries 6 4 Binary Layer Working with the Binary Layer There are the following b
94. e time and Gain are calculated The Compensation value is expressed in Exposure Values EV Setting the compensation to 1 0 EV makes the image twice brighter e g doubles the Exposure time or Gain AE Lock This option makes the automatic exposure mode to lock the current exposure settings Exposure time and Gain 35 Sharpness Some camera settings provide the sharpness control which affects how sharp edges in the image appear Too much sharpness leads to oversaturated edges Hue Hue shifts image colors across the rainbow Saturation The amount of saturation determines how colors are rendered More saturation produces richer colors Less saturation makes the colors gray White Balance There are usually Red Green and Blue Gain properties that control how colors are rendered It is used to eliminate a color cast from white areas Auto White Balance AWB The AWB button performs an automatic white balancing It adjusts the red green and blue image com ponents in order to get a neutral white color Advanced Settings Separate Channel Settings Having a color camera influence of the settings described above on each color channel can be adjusted In such case three edit boxes with the default values set to 1 0 are displayed below the feature setting These values determine the influence of the feature on each color channel Using Probe The probe Oo enables you to determine a small image area that serves as the data
95. e zooming Allow zoom factors lower than best fit Multiple Windows Lock camera magnification Initial Zoom Best Fit Keep picture window aspect Initial Zoom 100 for Live and Capture The following properties can be adjusted here e Application background e Color scheme selection e Application language e Save dialog buttons Displayed Z dimension value in the status bar Availability of Z Intensity control in the application e Image tool bars visibility and style 23 Bottom toolbar visibility e Channel tabs visibility e Layout tabs visibility e Task bar visibility Initial Zoom of images e etc 2 3 Organizer Apart from the main application layout used for capturing and image analysis NIS Elements AR provides an extra layout called Organizer Organizer makes the work with image files and databases as easy as possible To activate it run the View gt Organizer Layout command or click the button located in the top right corner of the application wndow The screen opens divided into two identical panes To switch from one pane to the other use the View gt Next Pane F6 command To copy files between the panes simply drag the images from one side to the other side The organizer layout E Calibration Uncalibrated 12 items Adrenal Gland jpg C Data Bio 282835 29 7 2005 analcim jpg CiMata Bio 264068 29 7 2005 1253234972 Bs anapait jpg C PatalBio 252130 29 7 2005 1232x972 a
96. ect the control window to be displayed from the context menu If the window is already opened somewhere else in another docking pane or floating it closes and moves to the new destination Close CW Click the cross button 2 in the right top corner of the tab Drag CW Drag any CW by the tab and drop it somewhere If you drop it by the edge of a docking pane it will create another column of this pane If you drop it over the caption of another CW it will be docked in the same pane as a new tab If dropped somewhere else the CW will be floating Note A color frame appears when you place the mouse cursor dragging a CW over the edge of a docking pane or a caption of another CW It indicates that if you drop it its placement will be handled automatically Minimize the docking pane Click the arrows in the top left corner The pane minimizes to a stripe by the edge of the screen It can be restored to its original position by double clicking this stripe or by clicking the arrows again 21 Close the docking pane Click the cross button 4 in the docking pane caption Or you can right click the pane and unselect the Docking View option Dock undock CW To dock and undock a CW double click its tab Display CW Another way to display a CW is to go to the View menu and select the desired control window After that the CW appears on the screen floating or docked Positions of the windows are being saved by the system so that ea
97. ectangle Unlike the BoundsLeft distance this value is always in pixels Measurement Type object Image Type binary BrightVariation It is the usual standard deviation of brightness values Measurement Type object field Image Type color binary CentreX CentreX is the x co ordinate of the center of gravity The units of calibration are used if the image is cal ibrated Otherwise the coordinate value is in pixels Measurement Type object Image Type binary CentreY CentreY is the y co ordinate of the center of gravity The units of calibration are used if the image is cal ibrated Otherwise the coordinate value is in pixels Measurement Type object Image Type binary CentreXabs CentreXabs is the absolute x co ordinate of the center of gravity within a motorized stage range This feature can be measured only when a motorized stage is connected Measurement Type object Image Type binary CentreYabs CentreXabs is the absolute y co ordinate of the center of gravity within a motorized stage range This feature can be measured only when a motorized stage is connected Measurement Type object Image Type binary CentreXpx CentreXpx is the x co ordinate of the center of gravity Unlike the CentreX coordinate this value is always in pixels 122 Measurement Measurement Type object Image Type binary CentreYpx CentreYpx is the y co ordinate of the center of gravity Unlike the CentreY coordinate this value is
98. ected to devices after installation depending on the PC status at installation If so execute the Modify installation command of the Windows Start menu see the next step to perform Repair that corrects the previously installed status NIS Elements is configuring pour new software installation Installing C Program Files MIS Elements v6_gnr_imnagetormat_jpe dll The setup creates a new program group in the Start menu containing the following items NIS Elements AR application shortcut the HASP key information shortcut the Modify Installation shortcut for adding hardware drivers modules etc the Uninstall procedure and the Send Info Tool A shortcut to NIS Elements AR is created on the desktop too These changes affect all user profiles of your local Windows operating system Installation and Startup id NIS Elernents Y HASP Info Y HASP Update EM Modify installation E NIS Settings Utility US NIS Elements AR EM Uninstall Note Clicking the Uninstall command deletes all installed files from disk and removes the NIS Elements AR program group from the Start menu as well as it removes the desktop icon Additional Module Device Installation You may need to install a device or an additional module after the NIS Elements AR main system installation e Goto Start menu gt Programs gt NIS Elements AR program group e Select the Modify Installation command e Follow the installation wizard instructio
99. ects touching edges of ROI or the measurement frame can be treated differently excluded from the measurement or included This behavior can be specified within the Measure gt Options window Statistics and Data Presentation NIS Elements AR calculates basic statistics mean value standard deviation and distribution of all measured features The features to be measured can be selected in the Measure gt Object Features and the Measure gt Field and ROI Features windows Results of all automated measurements are presented and can be exported from the View gt Analysis Controls gt Automated Measurement Results control window 7 2 Measurement Features The following list describes all the features that can be measured within NIS Elements AR Measurements Suitable type of measurement object field manual and the required image layer binary color binary are included in the description See also Measurement AcqTime Calling the Grab Sequence command the AcqTime is set to zero at the beginning Then NIS Elements AR assigns the AcqTime to every newly grabbed image file header which denotes time elapsed from the beginning of the grabbing If the Grab Sequence command is not called than the AcqTime is the time elapsed since the start of NIS Elements AR Measurement Type object field Image Type binary Area Area is a principal size criterion In a non calibrated system it expresses the number of pixels in a calib
100. ed polygon While holding the left mouse button down you are in the free hand mode When you release it each click defines a corner of the polygon The polygon is enclosed and filled by pressing the right mouse button F4 P Draws a polygon It equals the Filled polygon tool but the resulting object is not filled F3 Draws a filled circle Click to determine the center and define the perimeter holding the left mouse button down F8 O Draws a circle Click to determine the center and define the perimeter holding the left mouse button down F7 a Draws a filled circle defined by three points placed on the perimeter Ctrl F8 O Draws a circle defined by three points placed on the perimeter Ctrl F7 E Draws a filled rectangle F10 L Draws a rectangle F9 93 P Draws a filled movable circle ellipse If you grab the ellipse by the center you can move it If you grab it by the border the nearest semi axis is being modified When holding down either the SHIFT or the CTRL key both semi axes change equally forming a circle F12 O Draws an ellipse F1 1 e Drawing by hand F1 N Draws a straight line F2 Y Draws a polyline F5 Us The Auto Detect filled tool Detect hollows using threshold techniques Click to the image to place the probe the detected area is drawn You can adjust the thresholding range by using the mouse wheel or by pressing the keys Ctrl B Threshold tool Clic
101. ed to the image data by pressing the Il button Use the la Show LUTs window button in the image tool bar to display the LUTs window The following actions are available within the LUTs window e Adjusting the Gamma parameter The gamma curve is drawn in gray over each graph You can drag the middle point and move it up or down or enter an exact value to the G field at the top of the graph area e Adjusting the input intensity range The input intensity range can be restricted by moving the black and white triangular sliders to the center All pixels with values outside of the modified input range will be set to maximum minimum values and the remaining pixels will be approximated in order to fill the output full range Note This is the way how images can be equalized For example if the image is very dark and most of the histogram is located to the left while the right part of it is flat you may move the white slider s to the position where the histogram line starts to rise This will incredibly brighten the image and reveal details which were previously hidden in a dark e You can adjust the histograms height non proportionally by moving the slider on the left side of the window up and down 80 Image Analysis Tips e Right click the graph area and de select Draw trend style When ON the LUTs curves will be smoothed to display the data trends rather then represent the actual image data values e The position of the
102. ee the Mathematical Morphology Basics chapter 92 Image Analysis 6 4 2 Drawing tools vertical tool bar The binary image can be modified using various drawing tools Although the way of use of some tools differs there are some general principles Make sure you are in the right drawing mode drawing background E foreground FG Drawing of any object which has not been completed yet can be canceled by pressing Esc The polygon like shapes are drawn by clicks of the left mouse button The right button finishes the shape The automatic drawing tools threshold auto detect have a changeable parameter It can be modified by and keys or by mouse wheel The scene can be magnified by the UP DOWN arrows when mouse wheel serves another purposes You can drag a magnified image by right mouse button A line width can be set in the top tool bar Hints are displayed in the second top tool bar Drawing Tools FG Switches between the foreground and the background editing mode Ctrl SPACE a The Hand tool Serves for moving the image when magnified Ctrl W S Bezier hollow tool The object is defined by placing points on its perimeter The lines connecting those points can vary from straight lines to Bezier curves Use keys to adjust them To finish creation press the right mouse button Ctrl F 11 Bezier fill tool It equals the Bezier hollow tool but the resulting object is filled Ctrl F 12 Draws a fill
103. emplates A report template is a layout defining the appearance of future reports which is ready to be filled with data The data can be inserted by the user on demand or automatically dynamic data To Create a Report Template e Run the File gt Report gt New Blank Report command An empty report appears e In the Report Generator select File gt Change to Template e Edit the report template in the same way as a common report e Insert the dynamic data where it is appropriate measurement results to text boxes or tables the current image to the image field etc e Save the report template rtt via the File gt Save command To Create a Report from Template e If the template is opened inside the Report Generator run the File gt Create Report command Otherwise use the File gt Report gt New Report from Template command inside the main application window to open a template from harddisk e A report opens and the dynamic data is inserted automatically e Save the report print it or export the page s to PDF using the commands from the File menu 6 6 3 Creating Reports from Database Pictures of a database together with the associated table data or common images with the associated Image Info can be exported straight to a report e Switch NIS Elements AR to Organizer by the View gt Organizer Layout command e Select one or more images These images will be inserted to the report
104. ent unit button located in the Calibration gt Objectives command window and select the units 47 48 Capturing 4 Capturing Having NIS Elements AR installed and all hardware accessories set up you can start capturing images The simplest case is capturing of single images one by one There are also other ways of acquiring images and image sequences some of them specialized and needing specific hardware They are described in the sub sequent sections 4 1 Single Image Capture Turn the connected camera and other devices ON and start NIS Elements AR When the application is started display the Camera Settings control window Acquire gt Camera Settings Switch camera to the Live Fast mode and adjust the camera settings to get the live image clear Focus the objective on the scene you would like to capture the image of Switch to the Live Quality mode and adjust its settings especially the Exposure time and Format These settings will be used on every image capture Switch back to the Live Fast mode This is the usual mode for smooth work because less of the hardware resources are engaged Capture the image by running the Acquire gt Capture command D button on the main tool bar A new image is opened and named Captured automatically 4 2 ND2 Acquisition NIS Elements AR being a multi purpose imaging system can be used as a handy tool to study objects live organisms processes etc The
105. eriment Load Use this button to for a configuration XML file to be loaded The file with the current configuration can be created by the Save button Save Click this button to save the current configuration of this dialog to an XML file A standard Save As window appears ND Sequence Options The following options can be applied to time mutipoint and multichannel sequences in ND experiments New This button adds new step into the sequence al Select All Use this button to select all steps in the sequence 50 Capturing a Clear Selection Use this button to clear all steps selection Move Up Move Down These buttons moves the current step up down in the sequence Remove Current This button removes current step from the sequence Remove All This button removes all steps from the sequence 4 2 1 Time lapse Acquisition Detailed studying of long lasting processes is enabled by the time lapse acquisition mode of NIS Elements AR The achievable experiment duration is limited by the hardware abilities of your PC only Invoke the Acquire gt Capture Timelapse gt Capture Automatically command to set the experiment up Time schedule So lti xos F Loops 21 11 The Time schedule table enables you to define consecutive time phases where duration interval between single images and number of images of the phase can be adjusted The Interval Duration and Loops settings are boun
106. for statement the first expression of the for statement is evaluated Then the compiler reevaluates the conditional expression and depending on the result either terminates or iterates the statement body Statements Not Supported do switch case default typedef These statements are not supported Directives Following directives are supported by the system define The define directive assigns a meaningful name to a constant in a program Syntax define identifier token string The directive substitutes token string for all subsequent occurrences of the identifier in the source file Token string can be a value or a string only for 32 bit version of NIS Elements Example define ERROR_SPRINTF 0 define MAINDIR c lmages int main char buf 256 int retval retval sprintf buf s MAINDIR if retval ERROR_SPRINTF Beep else WaitText 0 buf return TRUE include Specifies the name of the file to be included Syntax include filename 152 Working with Macros The include directive includes the contents a file with a specified filename in the source program at the point where the directive appears Example if you do not specify the full path NIS Elements assumes that it is a relative path to a main directory include macrosimy_macro h include c NIS Elements macros my_macro1 h import The import directive is used to incorporate information from
107. g Reports from Database ccccc cece eee eect e nsec nsec eee eee eee eaeeeneeeeees 107 O 7 re auna MOVIES stare ts atte Roxana die de labial plot ia dias 108 641 Capturing AV MOVIE A AA A 109 6 7 2 CONVering NDZ O Ad ee a el a e a 110 6 7 3 Creating QUICKTIME MOVIE o ococccoccccccccncocncncnconnconnconncnnno cnn rnno nono rnnarnnornnananos 111 0 2 MOVE Maker cimentar ins catia Ca anda aa 111 TAME ASUS NE A A A AAA AT AA A A 115 A A e A uta Ot acento ales 115 pe A aca dave aaa a eset ae eaa aeii 115 Fel 2 OU Measurement rd slew bade rada dada 115 Fa MAR ta Me as UTM ED AA AA A AAA AAA 117 1 1 4 Automated Measute Mel hie Pialeneelidaddarenahieemenneunits 119 7 2 Measurement Features tarea en sra o La Da AENA OE a AR veins 120 1 3 Mathematical Morphology Basics cess ai csc snes cc ss a a es nennieien 133 VS COMME CUVILY A se ss ae ed paces as eee ae se eee 134 7 3 2 Structuring Element Kernel Matrix cccccece cence eee eee eeeeeeeeeeeeeeeeeeeenenees 134 MAS EXPONO Results sitio adi ads iii dlls iadi 138 ESO AE A O A A baa e ett een 142 NIS Elements AR ES PMFESNOIGING INe MadS remate ica iaa adds 142 70 2 APPINI SS MICA Si A E gunn ai 143 E A oe 144 AO lalo WIT E el ol AAN ONU O A O 147 e Der a daeb ns estes eet ta te a dia sent ede hee a ewe has 147 S2 RUMAN AMAC O i a A ASAS AS 149 9 32 Macro Language Mia id 149 J AGOINONAll MOQUE S es ts O a ltda estas de earn 157 dla RANES Soria e
108. g structure just the current frame is copied to every position of the target ND2 5 2 6 Image Formats NIS Elements AR supports a number of standard file formats In addition NIS Elements AR uses its own image file format ND2 to fulfil specific application requirements JPEG2000 Format JP2 An advanced format with optional compression rates Image calibration text descriptions and other meta data can be saved together with the image in this format ND2 Format ND2 This is the special format for storing sequences of images acquired during ND experiments It contains various information about the hardware settings and the experiment conditions and settings It also maintains all image layers of course Joint Photo Expert Group Format JFF JPG JTF Standard JPEG files JPEG File Interchange Format Progressive JPEG JPEG Tagged Interchange Format used in many image processing applications 74 Image Files Tagged Image File Format TIFF This format can save the same amount of meta data as JPEG2000 TIFF files are larger than JPEG2000 files but are loaded faster TIFF files have several ways to store image data therefore there are many versions of TIFF NIS Elements AR supports the most common TIFF modalities CompuServe Graphic Interchange Format GIF This is a file format commonly used on the Internet It uses a lossless compression and stores images in 8 bit color scheme GIF supports single color transparency and animation
109. ghts Those folders are C Windows and subsequent folders C Users and subsequent folders excluding C Users login user name C Program Files and subsequent folders excluding C Program Files NIS Elements ARllmages 5 2 5 Copying Channels by Drag and Drop Drag one of the channel tabs and drop it 13 RGE s W Red MM Green Ml Blue e to another image so it will become a multichannel image e inside the NIS Elements AR application window in order to create a new image Note e Even the All and RGB tab can be copied e When extracting a channel from Live image it does not freeze the camera signal ND2 files 1 Drag one of the channel tabs and drop it by the left mouse button It behaves the same way as if performed on a single image 2 Drag one of the channel tabs by the right mouse button and drop it to the application screen A context menu appears that enables you to select whether to create the new image from all frames or just a single frame 3 Two nd2 files that have matching structure can be merged into one easily E g two single channel Z stacks with the same number of Z positions can be merged into a single two component ND2 Z stack by dragging one into the other 4 Drag one of the channel tabs of a single image and drop it to an nd2 file The channel will be copied to every loop of the ND2 file Note This behavior too takes place when copying channels that do not have matchin
110. han three channels only one channel is displayed at one time A pull down menu in the tool bar enables you to select the channel to be displayed P Next to the channel selection pull down menu there is this Auto Scale button By pressing it the system will automatically adjust only the current channel settings 6 2 2 4 LUTs on Spectral Images When observing a spectral image the graph inside the LUTs window does not show the image histogram s but reveals another information about the image Intensity values are on the Y axis and spectra is on the X There are two curves in the graph The top white curve displays the maximal intensity and the color curve displays the mean intensity of each channel Note Spectral image consists of many channels each representing a single wavelength The LUTs behavior depends on the selected displaying mode There are four displaying modes You can select the mode either by a pull down menu in the LUTs tool bar or within the image top tool bar e True Color e Custom Color e Grouped Color 85 e Gray Scale In the bottom part of the LUTs window Gain and Black Level settings can be adjusted either for all channels together or for each group separately depending on the displaying mode Gain brightens the image and the Black Level works just like the left black on RGB mono images Note Each slider gain or black level offers a range of values to be set by moving it but any
111. he Add Current View Cy button on each of the images You can also select the Add New Views Automatically Er button to have all newly opened ND2 files synchron ized The names of all added images appear in the Synchronizer window All documents added to the Synchronizer are automatically being synchronized Set one of the selected images active Any action regarding the view Z position displayed frame etc is performed on the synchronized imagese as well as on the active one Besides synchronization of dimensions you can also synchronize LUTs settings of the images by checking the LUT check box LUTs synchronization works only between LUT compatible images images must have the same number of channels etc If the images are not LUT compatible only the Keep AutoScale and AutoScale actions are synchronized You can select the Enable LUT synchronization by default por button All newly added documents will have the option to synchronize LUTs selected by default To remove an image from synchronizer click the reset button x placed before its name To remove all images from synchronizer press the Remove All Views button 5 1 3 6 ND2 Information Information about current frame of an ND2 document or live image can be displayed using the Show ND In formation command from the image contextual menu It displays information about current frame of the ND2 document or live image All possible information metadata a
112. he Run Z corr button works similar as the Run now button but the acquisition uses defined Z intensity correction See View gt Acquisition Controls gt Z Intensity Correction Save to File An image can be captured and saved manually after the experiment is finished or it can be saved to harddisk on the fly e Check the Save to File box The window reveals an extra section e Insert the Path or use the Browse button to define the destination folder e Specify the Filename Recorded Data The nd2 file contains various Recorded Data which carry e g acquisition times of single frames Some of them can be recorded optionally e Press the Record Data button The following window appears Recorded Data PerFrame Frequency ao mh Recorded Data X Coord Y Coord E Ti Piezo Drive PFS offset PFS status Exposure Time 58 Capturing e Select whether to record the data for each frame Per Frame or with a certain Frequency The data to be recorded shall be check marked e To view the recorded data of an opened ND2 file invoke the File gt Image Properties command and switch to the Recorded Data tab ND Experiment Progress The following window appears while the experiment is running ND Progress Experiment overall progress T A SAA Time elapsed 0 01 20 Time remaining 0 00 50 Experiment Status Time NonEq 14 of 22 T 77 875 Step 5 765 Fos
113. higher value can be entered in the field next of the sliders TD channel A spectral image can contain additional TD transmitted detector channel lts basically a standard monochro matic image LUTs settings of this channel appear on the right edge of the LUTs window True Color Displaying Mode You can switch to this displaying mode by clicking the HA button located in the image tool bar LUTs x ES a bu R Te color LZ gt ide 4 K A 429 459 489 518 548 578 603 638 668 693 728 Brightness am ami i aa Sa Silos 2 7 Black Level Bs l 0 Gamma 1 00 Custom Color Displaying Mode You can switch to this displaying mode by clicking the E button located in the image tool bar You can assign custom colors to sections of the spectrum Sections colors can be added removed by clicking the and buttons placed below the spectral graph To change the section color click the button located in the color stripe A window appears where you can select the color from a palette or define it by Hue or Wavelength settings 86 Image Analysis a Lg iu R custom color LZ gt led Es bb 1K Brightness 2 9 Brightness 3 7 Brightness Black Level RX gt a nn nn O 0 Gamma 0 Hold Shift key to control all groups Grouped Displaying Mode You can switch to this displaying mode by clicking the ME button located in the image tool bar This view
114. i Point nd2 file there is one more option ROI can be defined per Multi Point which is the default It means that ROIs from one point are completely independent of ROIs defined on the other point even if they have the same number This applies also to ROIs defined as Changing over Time in case the image has time axis too On the other hand if the ROI is changed to a Global it will be shared among all points of the Multi Point set Depending on the application purposes ROI objects can have different usage NIS can handle Background ROIs Reference ROIs Stimulation ROIs and Standard ROIs 96 Image Analysis 6 5 3 Interaction with ROIs ROIs can be manipulated anytime directly in the image window Many of the following techniques work in ROI editors too Objects cannot be moved or resized when they are Locked some analysis tools or users may lock them to prevent accidental modification They must be unlocked from the ROls context menu before any manipulation Note that the mouse cursor changes over the ROI to indicate which action will be started after mouse button is pressed it changes when modification keys like Alt and Ctrl are pressed Selection ROI objects can be selected by clicking on an unselected object other previously selected are unselected Clicking on a ROI object with Ctrl key pressed results in altering the selection state More than one object can be selected in this way Double
115. ically used for shape and size measurements area perimeter surface fraction e Color layer intensity or hue measurements are carried out on this layer The area covered with the binary layer is used as the source data Object Field Measurement NIS Elements AR distinguishes two types of automated measurements object and field Object Measurement Groups of neighbouring pixels of the binary layer are called objects Results of the object measurement are usually the object data such as Length Area CentreX Y the X Y position of the central pixel of an object etc Field Measurement Field measurements produce information regarding the whole measurement area measurement frame ROI Such information can be Area Fraction Mean Brightness Density Variation etc Please see the complete list of measurement features in the Measurement Features chapter Restricting the Area of Measurement The area of measurement can be restricted by the measurement frame or by a user defined region of interest ROI Region of Interest ROI ROI is a user defined area of the image Whenever the ROI is displayed by the View gt Layers gt ROI command it is also applied to restrict the area of measurement 119 Measurement Frame The measurement frame is a re sizable rectangular frame which serves for bounding the area accepted for measurement Turn the measurement frame ON via the Measure gt Use Measurement Frame com mand Binary obj
116. ications gt ND gt Define Run Experiment command ND module required Common ND Experiment Options The following options can be applied to all types of ND experiments Name of the experiment phase You can enter a custom name of the experiment Also name of the phase is editable Auto Focus Automatic focusing can be used during the experiment You can select the auto focus method that best meets your needs It can be determined whether the focusing shall be performed at the beginning of the whole experiment or at the beginning of each phase The Define button shows a window where you can define parameters of the selected focusing method Closing Shutter The active shutter can be closed between single image acquisitions Just select the Close active Shutter option in the experiment window Running Macro Commands You can define a command or a macro to be run in various stages of the experiment Select the timing in the Advanced section of the experiment window and enter the command to be run In Out signals requires Local Option requires 4D 6D Acquisition An ND2 experiment can control some accessory devices and can be controlled by another accessory devices at the same time This can be achieved by sending receiving Analog TTL signals through an NIDAQ card If the NIDAQ PCI card is installed an extra tab called Inputs Outputs appears within the ND experiment window See Applications gt ND gt Define Run Exp
117. ice from the pull down menu Set the Z range move the Z drive to the top position and press the Top button move it to the bottom position and press the Bottom button Define the Step size in um or the number of steps to be captured Note In this mode the Home position is assigned to the middle one of all the defined steps automatically e g for 5 steps it is the third step etc The Reset button discards the Top Home and Bottom positions settings Range by Home Position and Range 1 2 3 4 5 Press one of the following buttons the cube turns yellow The range is defined by the home position and the scanning range i w i Symmetric mode Define the Range by inserting a value in um Asymmetric mode The range is specified by two values Below the distance below the home position and Above the distance above the home position Run the Live camera signal In case you have two Z drives select the Z device from the pull down menu Move the Z drive to the position which you would like the Z drive to move around Press the Home button to define the home position Specify the scanning Range Define the Step size in um or the number of steps to be captured Defining TIRF position When the TIRF system is enabled the TIRF option appears in the right portion of the window To set the TIRF position e Select the TIRF check box to enable TIRF e Move
118. idth of the scale bar can be defined Table e Inner and outer borders can be hidden or displayed e Number of rows columns can be modified Graph e The range of displayed values can be limited by defining the min max values e Histogram labels can be edited Aligning Objects You can align two objects to the same horizontal or vertical level Select more objects e g by holding down CTRL and clicking with the left mouse button Right click one of the selected objects and select the Align or Distribute gt Align command from the context menu 105 The objects will be aligned as indicated on the command icon If the Relative to Page option is turned ON the objects will be aligned to the edges or the center of the page Objects of similar size can be distributed uniformly in the horizontal vertical direction Select three or more objects Right click one of the objects and select the Align or Distribute gt Distribute command from the context menu Distances between the objects will be adjusted to equal If the Relative to Page option is turned ON the outer objects will be moved to the edges of the page Sizes of objects can be unified too Select two or more objects Right click the master object to the size of which you want to resize the other objects Select one of the Resize Objects sub menu commands Dynamic Data Results of automatic interactive measurement graphs or the curren
119. ile If this file does not exist it will be created Choose the path and name of the file to be cre ated opened Set the Start at column values column and row indexes These values mark the first cell in the MS Excel application which will hold the exported data Export text files into folder Select this item if you prefer to export the data to a text file Press the button and specify the target folder and the file name Append exported data after previous exports If you check this item the existing data will not be overwritten by the new data Export also column headers if not appending Include the titles of columns in the files which are being created or overwritten If the Append exported data after previous exports is selected no headers will be included in the export 140 Measurement Active target application after export If you check this item the exported file is opened in the default application MS Excel or the default text editor automatically Insert empty lines Check this item to allow insertion of empty lines in the exported data Data export Options You can modify the amount of data included in each export type Data export Global Settings Data export Annotations and Measurements e xport Name Length Area Area Radius Radius SemiAxes SemiAxes Length3D Length3D Counts Counts Taxonomy Comment Statistics The control windows which contain the Export to file functionality are li
120. ing you distinguish objects of interest from background A picture of night sky can make a nice example shining stars may be thresholded and compose the binary layer Then by means of automatic measurement you can e g count them More than one binary layer can be placed over one image ROI layer ROls regions of interest is a strong tool for distinguishing objects from background similar to the binary layer The advantage over the binary layer is that ROIs are vector objects and therefore provide different way of manipulation See Regions of Interest ROIs Color layer The color layer contains the image data captured by a camera It can handle images with the depth of up to 16 bits per color component The dimensions of this layer determines the dimensions of the other layers 63 Note When saving an image only some file formats are capable of saving all the layers The other image formats will save the content of the color layer only See Image Formats 5 1 2 Image Types in NIS Elements AR RGB Images Images acquired by a color camera typically consist of three components that represent red green and blue channel intensities You can display a single color channel using the tabs located in the bottom left corner of the image window Or an arbitrary combination of them can be selected while holding the Ctrl key down RGE 5 W Red M Green Ml Blue Multi channel Images These documents usually arise from fluo
121. istance from the first line The process is completed by right click The following dialog box appears 45 Manual calibration Enter real size of line i 7 Enter the distance between the two lines and select correct units 8 Press OK The objective is calibrated now Automatic Calibration The automatic calibration requires a motorized stage Select one of the following methods Auto The Auto method is fully automatic NIS Elements AR moves the motorized stage acquires two images and calculates the calibration from the shift of the images The auto calibration performs an estimation of the real relay lens zoom factor If the estimation does not match the value specified within the optical path window a warning appears Example of the warning message The Relay Lens is set up in the Camera Light Path setting to 1 30 but the correct value should be 1 00 A on your microscope Please fillin the correct value in Camera Light Path setting Do you want to continue anyway It is recommended to cancel the auto calibration click No check the real zoom factor of your relay lens and check and correct the relay lens settings within the Acquire gt Camera Light Path command window Note The success of this method depends on the texture of the specimen its contrast illumination etc If the combination of these factors is unsuitable the auto calibration may fail If it fails try the following e Move the stage
122. it until it is unlocked The Properties command opens a window where object properties such as color background color border size and color object size font aspect ratio alignment and shape can be adjusted Insert images text fields and other objects to the report 104 Image Analysis 6 6 1 Working with Report Objects When you double click any of the report objects a window appears where the properties can be adjusted Some properties are common to all types of objects and some are special Common Properties The precise position can be determined by defining the XY coordinates of the top left corner of the object Line and fill colors can be selected Line widths can be adjusted within the range of 0 25 to 6 pts Precise width and height of the objects can be adjusted Rotation of some objects can be adjusted Special Object Properties Ellipse Minor and major axes lengths can be modified Arrow The arrow tip shape and size can be modified Text Box e Format of the text can be adjusted like in a standard text editor e The current date and time can be inserted from a pull down menu Image e A new picture can be loaded to the image frame via the Load Picture button e The current image scale is displayed in the properties window It shows the current scale original image dimensions ratio the image must be calibrated to use this feature e A scale bar can be displayed below the image The position and w
123. k into the image to place the probe or more of them to define the initial color level for thresholding Pres keys or use the mouse wheel to change the thresholding range J y Fill an enclosed shape F6 EJ Area of interest All objects outside the selected region will be erased A Text tool Displays dialog box for defining text parameters e Commands tool The last button in the left tool bar displays the currently selected tool You can invoke a pop up menu and select one of the additional commands Commands E The Auto Detect hollow tool Detect hollows using threshold techniques Click to the image to place the probe the hollow is drawn You can adjust the thresholding range by using the mouse button or by pressing the keys T Draws rectangularly crossed lines HK Draws a rose Click to the image to define the center than drag the mouse to set the length of it s arms lt Places a marker to the image Simply click into image selects binary objects de The Connect tool Draws a line s from the place you ve clicked to the nearest object s HH Displays a grid Visible only when using magnification 400 and higher j Smooth see the Mathematical Morphology Basics chapter Contour see the Mathematical Morphology Basics chapter 94 Image Analysis Inverts the binary image Foreground becomes background and vice versa Binary Layer Color and Transparency When in ove
124. k like in different cases 64 Image Files A T Z multi channel image with some loops selected 2111181 MA PE 20 4 gt A T MO Fe AAN Hi DDD Z a A ee DDH All E CAPIL FITS MA Rhodamine Custom Uncalibrated NAA A T Z multi channel image containing 11 channels and the time line hidden a a H lt a po Hg DDD id lt b gt H Gee pph An HDR multi exposure image o o o S o o e tt _ tt _ Playing Controls gt Play Sequence Plays all images of the dimension at selected speed If a selection is applied only the selected images will be included in the playback il Stop Playing Stops playing the sequence at the last displayed frame 4 Previous Position Displays the previous frame of the dimension b gt Next Position Displays the next image of the dimension dw Decrease Increase Playing Speed Changes the playback speed by one step down up D Real Time Playing Speed Sets the playing speed to real time as the image was captured D Maximum Playing Speed for Every Frame Sets the playing speed to maximum while the display of every frame is guaranteed when the speed is set to maximum by the button some frames are usually omitted when playing the sequence depending on your graphic card HH Home Position Displays the frame of the Z dimension that was set as home during the acquisition 65 Event and Selection Controls 1 Insert Default User Event This button inserts an event marker to
125. l be opened at a time L Main View When you open an ND2 file it opens in this view i Maximum Minimum Intensity Projection View These projections analyse all frames of one dimension and pick pixels with the maximum minimum in tensity values These pixels are used in the resulting image Requires Z or T dimension Ll Slices View This view displays orthogonal XY XZ and YZ projections of the image sequence Requires Z or T di mension Volume View This view creates a 3D model of the acquired object Requires Z dimension 50 Tiled View This view displays frames of the selected dimension arranged one next to other Requires Z T or XY dimension 68 Image Files 5 1 3 4 View Examples Main View Max Min Intensity Projection View Volume View 69 Slices View Tiled View 5 1 3 5 Synchronizer Synchronizer enables to compare run and view two or more nd2 files at the same time It automatically synchronizes view of all documents added to the list Run View gt Visualization Controls gt Synchronizer 70 Image Files Synchronizer x EEN al Synchronized Views x C Program Files z newT nd2 A T XY Z t Live AT xr z C Program File WD _00i nd2 AT XY z Ww Open at least two nd2 files you would like to observe Use the Add All Views button to add them to the synchronizer table In case there are more images opened which you do not want to add use t
126. lect Camera window Set the parameter value to last and the most recently used HW Unit will be loaded When using this option you must also use the g switch Installation and Startup i Config Name The application creates a registry entry in Laboratory Imaging Platform Config Name to load from and save its configuration to S Settings File The application will use the specified Settings File to load and save its settings l Language The application will run in the specified language if available Use codes for the names of countries according to the ISO 3166 1 standard m Macro File Application will execute the macro file mac pointed to by the Macro File parameter new If this command line parameter is added to setup executable file the setup checks if any other setup has already been installed If so the setup restarts and runs in independent mode The update mode is dis abled This enables to install two or more builds together on one system passive The application will not connect to any device on startup NIS Elements AR will start with blank Device Manager Camera selection is not affected by this option p Position The application window will be placed according to the parameter left right top bottom the window is placed on the corresponding monitor screen e monitor N the window is placed on the Nth monitor e rectangle x0 y0 x1 y1 the win
127. mera E Nikon G5 US L3 emi cone Multiple Cameras Two cameras can be connected to one system If this is your case and you intend to use both cameras check the Enable Multi Camera option and select the Second driver from the list First driver E Nikon DS U3 L3 Second driver FA Mikon D5 U1 Zeit cone The following set of buttons appears in the top tool bar when multiple cameras are used 31 9 DSFil1 gt a DT 0 DS il 2 Cc 1 2 DE 2 Selecting a Camera Color cameras can be used in a monochromatic mode The actual camera type color mono can be selected by the Acquire gt Select Camera command Note The emulated monochromatic mode is optimized for use with fluorescence specimen where often only a single color signal is being captured The following formula is used to compute intensity of the emulated mono image Ir Wr Ig Wg Ib Wb Where W is channel weight calculated from the channel histogram and I is channel intensity W ensures that channels containing some signal are accentuated while channels without signal are suppressed As a result depending on conditions the brightness of a monochrome image might not change even if the lamp light intensity is changed 3 Adjust the Camera Settings Exposure time camera resolution and other camera specific features are adjustable from the Camera Settings window To invoke it use the Acquire gt Camera Settings command
128. mooths the edges of the graph line Horizontal axis always visible If checked the axis does not leave the graph area while zooming in the graph 79 Overrun Indication If certain amount of pixels with maximum minimum intensity values black white pixels are spotted in the image color dots appears above the graph The color indicates which channel is affected When you place the mouse cursor over the dots a tool tip message appears with details about the percentage of under over exposed pixels The percentage limit setting is loaded from the LUTs settings the color dots do not appear unless the set percentage of black white pixels is exceeded Note If the overrun concerns more than three channels considering multi channel images only one white dot is displayed instead of many color dots Memorizing You can save the current graph to memory and display it later for comparison with another graph MR Displays memorized graph MS Memorizes the current graph Only one graph can be memorized at a time If any graph has already been memorized before it will be overwritten by the current graph MC Clears the memory 6 2 2 LUTs A look up table is a useful tool for image color and brightness modifications You can use LUTs to enhance images for observation purposes so the color modifications will be non destructive to the image data LUTs settings are saved along with the image file If required the LUTs settings can be appli
129. ms All layouts set as shared will be listed on the right side within the Accessible items field Make sure the check boxes next to the layouts which you allow to be used by the group are selected Disable layout modification Select the Privileges tab Select the group of privileges which should have not the permission to modify the selected shared layouts In the Privileges field on the right make sure you de select the Modify Shared Layouts option Apply the new policies Select the Users tab In the list of users select the ones which you would like to apply the policy to users can be selected one by one or by multi selection with the Shift or Ctrl key pressed Then assign the appropriate Group and Privileges by selecting them from a pull down menu on the right side of the window 29 7 Confirm the changes by the Apply button 30 Setting the Hardware Up 3 Setting the Hardware Up 3 1 Working with a Camera This chapter is dedicated to users running the active version of NIS Elements AR equipped with a camera Let s assume the camera works properly is connected to the PC with proper system drivers installed and running if required by the camera Setting Up the Camera 1 Selecting the Driver You will be asked to select the camera driver every time you launch NIS Elements AR You can change the driver later using the Acquire gt Select Driver command Choose the driver that matches your ca
130. n external file via the Save Graticule Mask As and Open Graticule Mask commands A new image containing graticules can be created by the Edit gt Create Full View Snapshot 8bit RGB command 116 Measurement 7 1 3 Manual Measurement Annotations and Measurements x Annotations Yarr 00 gt Length Ce eee er ee rea Angle Radius semiaxes Count amp Taxonomy 000 909 uaj a Length a E a G Espor Length Graph Table Length Distribution O14 043 045 017 019 02 Length area angles taxonomy counts circle radius and ellipse semiaxes can be measured manually over an image The results are being recorded to a simple statistics table which can be exported to a file or clipboard Also the data can be presented as a graph Run the View gt Analysis Controls gt Annotations and Measurements command The manual measurement control window appears Select a tool corresponding to the feature you are going to measure There are several tools for measuring each feature e Measure the objects in the image using mouse e Select where to export results in the Export pull down menu e Export the results using the Export button 117 Example Procedure Measurement of the image of a grain 1 2 3 4 Length 4 01 mm Select the Horizontal parallel lines tool Place the first line on the top edge of the crystal by clicking into the image The positio
131. n of the line can be adjusted while you hold the left mouse button down After you release it the line is positioned Repeat this to place the second line on the bottom edge of the crystal When finished an arrow is drawn between the lines and the result of the measurement is attached A record with the measurement type and the measured value is added to the results table Working with the Measurement and Annotation Objects There are two types of vector objects which can be placed over the image annotation objects and measurement objects A This button of the right image tool bar turns the visibility of annotation layer ON OFF Right clicking the button displays a context menu with the following options Clear All Objects will delete annotation and measurement objects Clear Annotation Objects will delete all annotation objects Clear Measurement Objects will delete all measurement objects Select Annotation Objects will select all objects that are not the measurement ones Select Measurement Objects will select all measurement objects Select All Objects and Deselect will select deselect all annotation and measurement objects Visibility of annotation measurement objects In an ND document that contains time dimension you can set visibility of any annotation object by the following rules 118 object is visible globally means in all frames in all dimensions select the option Object is always visible object
132. n one or more logical devices the list of which appear in the Device Manager after the connection is established Available Logical Devices Analyzer The analyzer is a polarizing filter placed in the optical path between the specimen and the lamp The lo gical device offers two states ON inserted and OFF extracted Aperture This logical device is used for controlling apertures in the light path It is used in complex microscopes rather than as a standalone device Two parameters can be typically set for aperture devices the state ON OFF and aperture size Condenser A condenser is a two lens combination located next to the illumination source in the optical path Its purpose is to collect light and direct it to the specimen being examined The corresponding logical device relates to a changer of different condensers 37 Filter This logical device controls filter changer movements There can be several filter changers connected to NIS Elements AR at a time Each filter changer needs to be set up filter types shall be assigned to pos itions of the changer e Display the filter changer control window Devices gt Filters and Shutters or Devices gt Microscope Control Pad Click the settings button la a window appears e Select one of the available positions which the filter will be assigned to e Click the button a list of available filters appears e Select the filter name from the list and confirm it by OK
133. n values of pixels Measurement Type object field Image Type color binary MeasuredArea MeasuredArea value is the area of the measurement frame or of a mask within the measurement frame if the mask status is on Measurement Type object field Image Type binary MinFeret The MinFeret value is the minimal value of the set of Feret s diameters Generally for convex objects Feret s diameter at angle a equals the projected length of object at angle a a 0 180 NIS Elements AR calculates Feret s diameter for 0 10 20 30 180 Measurement Type object manual Image Type binary MinIntensity MinIntensity value is derived from the intensity histogram It is the minimum of intensity values of pixels Measurement Type manual Image Type color gray NumberObjects NumberObjects value returns the number of objects in the measurement frame Exclusion rules for counting objects are taken into account Measurement Type field Image Type binary Orientation Orientation is the angle at which Feret s diameters have their maximum The diameters are calculated with 5 degrees angle increment Measurement Type object Image Type binary 128 Measurement Perimeter Perimeter is the total boundary measure It includes both the outer and inner boundary if there are holes inside an object The perimeter is calculated from four projections in the directions 0 45 90 and 135 degrees using Crofton s formula Perimeter
134. nglezit jpg C Pata Bio 248817 29 7 2005 1232x972 E This button toggles the display of the directory tree You can switch it off to get additional space to display images e In the nearby pull down menu it is possible to set the file type and only the one will be displayed Or All Images can be displayed e If the Subfolders check box is selected all images from included sub folders are displayed 2 3 1 Image Filter You can define a filter which enables you to display only images which fit the defined conditions One or two conditions may be applied 24 Y Application Appearance Pressing this button invokes the filter setup window Y This button activates the filter Basic Mode This mode enables you to filter files according to one condition Advanced Mode This mode enables you to define two conditions with a relationship between them Either select OR to display files matching at least one condition or select AND to display files which match both conditions Filter Y Search in field Description Search in field Size Field type Text Field type Integer Condition type Values Values Comparison From 800 Occurrence Anywhere To 1200 Exact J Start D All letter strings Any letter string Hint You can type more than one numeric value separated by comma Specified field containing at least one of these values will
135. ngular Grid a Circle O Simple Circle eb Cross gt i Industrial Cross Simple Cross E Vertical Ruler H Horizontal Ruler Define Graticule Properties Right click the Graticules button and select the Graticules properties command from the pull down menu A window appears where display parameters shape color line width density of lines of all graticules can be adjusted Graticules Density The density value the closest distance between two line intersections of the graticule can be set or you can let NIS Elements AR adjust it automatically according to the current zoom factor The units se lection depends on the image calibration calibrated uncalibrated 115 Note Concerning the Cross i graticule The distance defined by Density is divided into smaller sections automatically However for odd density values greater than 10 the markings remain hidden because it is not possible to display them accurately Measure the Image The graticule measurement provides the following options The graticule can be moved by mouse arbitrary The graticule position can be reset by using the Move Graticules to Center command from the context menu A binary layer can be created from the current graticule using the Copy Graticules to Binary command A Graticule Mask can be created from the current binary layer using the Copy Add Binary to Graticule Mask command A Graticule Mask can be stored loaded to from a
136. ns Select the check boxes by the items you would like to add e Finish the installation Sample Database Installation If you chose to install the Sample Database a new subdirectory Databases is created inside the NIS Elements AR installation directory e g C Program files NIS Elements AR Databases The Sample_Database mdb file is copied to there along with database images stored in subdirectories An administrator username pass word to access this database is set to e Username sa e Password sa Software Copy Protection The NIS Elements AR software is delivered with a HASP hardware key The HASP key contains information about the software licence and allows users to run the corresponding software Please connect the USB HASP after the NIS Elements AR installation is finished The utility called HASPinfo is installed to the NIS Elements AR directory It enables the user to view information about the software licence and is accessible via the Help gt HASP Info menu command 1 2 Command Line Options When starting NIS Elements AR from the command line or when editing the desktop shortcut properties you may append some parameters to the main command and thereby modify the startup behavior 1 2 1 Syntax and Example NIS Elements AR executable switch 1 parameter 1 switch 2 parameter 2 etc For example C Program Files NIS Elements AR NIS Elements AR exe f C lmages starting_im
137. ome other features added when in mono mode Gradient Mapping monochromatic images to pseudo color gradients is often used to enhance details in the image which would otherwise not be obvious This button indicates the currently selected gradient When pressed a pull down menu appears where you can select the color scheme you would like to use Try a few of the schemes to see which one suits best E aa Default Rainbow Iron The Manage gradients command in the pull down menu enables to define custom LUTs gradient Use the tools in the Manage gradients window to define custom gradients See View gt Image gt Create Custom LUTs for detailed description of the tools The gradient then appears in the pull down menu ready to be applied 84 Image Analysis 6 2 2 3 LUTs on Multichannel Images LUTs window on two channel image l i 22767 wN 9 9 mjm H G 0 60 20000 30000 0000 50000 ae Hold Shift key to control all channels Most of features mentioned above are also valid for multi channel images There are some other features added when in multi channel mode Each channel is controlled separately by default To control all channels at the same time press and hold the Shift key while you drag the sliders or adjust Gamma When the image contains up to three channels all channels can be visible at one time and you can handle them similarly to the RGB mode When there are more t
138. on of the image S Zoom Out Decreases magnification of the image General Controls q Show Probe button activates the probe The probe affects histograms auto exposure and auto white balance functions a Show Background Probe button activates the background probe Some commands use the BG probe data as reference HH Show Grid button displays the grid for rough measurements ura Show Scale button displays the image scale o Turn ROI On Off button displays the Measurement Region Of Interest EN Show Profile button displays the View gt Analysis Controls gt Intensity Profile control window It allows you to specify a linear section in the image of which the pixel intensities graph will be created iq View LUT Intensity button displays the scale of intensities used inside the image It works on mono chromatic images or a single image channel A Show Annotations button displays the vector layer which typically consists of annotation objects text labels arrows and measurement objects a View Binary button displays the binary layer of the image E View Color button displays the color layer of the image View Overlay button displays the color layer and the binary layer in overlay Tip Right click the icons to invoke a context menu where properties of each tool can be modified Status Bar The status bar at the bottom of the image window displays the following information 13 The Image Window
139. on result is the selection of kernel structuring element matrix type There are the following kernels used in NIS Elements AR 134 Measurement aa st at The bright pixel in the center or near the center of the kernel represents its midpoint 7 3 2 1 Erosion Lets assume the 1 and 0 values represent object 1 and background 0 pixels You can imagine the erosion as the following algorithm Move the midpoint of the kernel to every point of the image Each time look at the neighboring pixels of the kernel and make the following decision e If there are object 1 pixels in all the positions of the kernel set the midpoint to object 1 e If there is at least one background 0 pixel in the kernel set the midpoint to background 0 7 3 2 2 Dilation You can imagine the dilation as the following algorithm Move the midpoint of the kernel to every point of the image Each time look at the neighboring pixels of the kernel and make the following decision e If there is at least one object 1 pixel in any position of the kernel set the midpoint to object 1 e If there are background 0 pixels in all the positions of the kernel set the midpoint to background 0 7 3 2 3 Open and Close Open is performed by eroding the image and then applying a dilation to the eroded image On the contrary Closing is performed as a dilation followed by erosion 7 3 2 4 Repetition issues If the midpoint is not in the center applying e
140. option from the pull down menu in the Optical Configuration column The Comp Color column specifies the color tone in which the channel will be displayed Manual Capture The Acquire gt Capture MultiChannel Image gt Capture Manually command runs the live image and displays a new toolbar with the Capture button After pressing the Capture button one channel is acquired The number of channels depends on the Multichannel Setup After all channels are acquired the Recapture button replaces the Capture button It enables you to capture the multichannel image again All previously obtained multichannel data will be lost To recapture a single channel select it by mouse and press the Re capture button When satisfied press the Finish button 56 Capturing D Capture gt gt This command opens a new image window with the live image and the Capture button After pressing the Capture button one channel is captured The number of channels depends on the settings of the Acquire gt Capture MultiChannel Image gt Multichannel Setup window P Recapture After all channels are acquired the Recapture button replaces the Capture button It enables you to capture the multichannel image again All previously obtained multichannel data will be lost When a single channel is selected it can be recaptured separately Finish Press the Finish button to complete the multichannel capturing Define ROI This button appears when the
141. or the FPS Exposure info in case of live image 4 Name of the current objective 5 Current coordinates of XY Z stage 2 1 4 Image Window Tools affecting the appearance of the current image image are gathered within the image window tool bars There are the following buttons by default 11 B Larve jp2 JOA Tey Yi Hi K a A RGE Red Green W Blue Uncalibrated RGB 16bit 4059 x 3076 pixels mA LUTs T Enable LUTs button applies LUTs to the image See LUTs Ye Keep Auto Scale LUTs button applies the AutoScale command to the image continuously py Auto Scale button performs automatic setting of LUTs K Reset LUTs button discards the LUTs settings dal Show LUTs window button opens the window with LUTs LE Pixel Saturation Indication button turns on off pixel saturation indication without setting on off LUTs Select the highlighting color from the nearby pull down menu Split Components i fn Split Components button turns ON a special view where color channels of the image are displayed separately tiled 12 Application Appearance Zoom Controls E Fit to screen button adjusts zoom to view the whole image within the NIS Elements AR screen Best Fit button adjusts zoom to fit the NIS Elements AR image window in one direction but to fill the screen 11 1 1 Zoom button adjust zoom so that one pixel of the image matches one pixel of monitor CH Zoom In Increases magnificati
142. ove the threshold using the right mouse button the binary layer in the image is not updated continuously but is updated only once after the mouse button is released Such operation saves some computing and therefore is quicker e Hold Shift while moving the threshold limits the threshold adjustment is performed on all channels together RGB images only Hold Ctrl while moving the threshold limits both the low and the high threshold limits move in opposite directions Binary Operations Three basic operations can be performed on the binary layer before it is displayed on screen To turn the operation on click on the up arrow button in order to define the number of its iterations e Clean removes small objects from binary image e Smooth smooths the binary image contours 90 Image Analysis e Fill Holes fills holes within binary objects e Separate separates objects Restrictions e Size define size range using min max value in the edit boxes or using the slider e Circularity define circularity range using min max value in the edit boxes or using the slider Changing Appearance of the Threshold Layer To change the color or transparency of the layer right click the Overlay button in the general controls toobar in the document window A contextual menu appears where you can select one of the predefined transparency levels or run the Colorize Binary Objects command This command displays th
143. pears 44 2 3 4 5 6 Setting the Hardware Up Manual calibration Draw line of known length Select one of the icons to draw a distance to the image If you know the precise calibration value px unit press the Pixel Size button to enter the real size of one pixel Manual calibration Enter real size of 1 pixel 0 001 i Enter the calibration value select units and confirm the calibration by OK If you do not know the pixel size you will have to continue with the calibration on live image Insert a calibration slide to the microscope stage The distance is defined by placing lines Horizontal Vertical Parallel to the image Choose the orient ation of the lines by pressing the appropriate button Note If you are sure the camera angle is 0 90 180 or 270 it is recommended to select either vertical or horizontal lines Otherwise select the parallel lines Click into the image to place the first line Place the second line in the intended position by another click Note You can modify the line position while holding the mouse button After you release the button no further changes can be made When Parallel lines were selected draw the first line by clicking twice inside the image The line can be moved and adjusted arbitrary by mouse When Satisfied finish the first line by right click The second line can be placed by another click to the image this time only to adjust the d
144. pect ratio of to 1 Live Choose the format of the camera The Live Fast or Capture Quality mode settings will be used while capturing the AVI movie Rate The number of frames per second of the movie can be defined Possible values range from 1 to 50 fps There is also the Max Fps option When selected the system captures every frame sent from a camera If the frame rate is beyond hardware capabilities of your system some frames will be omitted 109 Duration Several predefined lengths of the movie may be selected 60 min 10 min 1 min 10 s or the Infinite option is available When selected the movie will be captured until the Stop button is pressed or the hard drive is filled to capacity File the path and the name Define the path to a directory and the file name of the AVI file If you capture more then one movie without changing the file name setting the other files will be numbered automatically File Codec It is common to encode compress movies in order to save some disk capacity This can be done using various types of codecs Press the Open button on the right and a window appears Select one of the codecs available in the system or use the Default option Remember that the codec you choose will be required for the AVI playback as well when playing the video on other computers Note Video codec is a software tool which can code and decode video files We recommend to use one of the following codec
145. pes 163 164 NIS Elements AR User Manual Laboratory Imaging s r o Za Drahou 171 17 CZ 102 00 Praha 10 Published 09 201 1 No part of this publication may be reproduced or transmitted except with the written permission of Laboratory Imaging S r O Information within this publication is subject to change without notice Changes technical inaccuracies and typographical errors will be corrected in subsequent editions
146. ption is disabled the operation is applied to channels automatically Note In case the command does not open a window where it would be possible to make the choice the setting from the last performed processing command the global setting is applied 6 1 2 ND2 Files Processing Most of the image processing and binary image processing commands can be applied to one frame all frames or selected dimension of the nd2 file When you use such command with an ND2 file the following options are added to the command dialog window Sharpen Slightly on RGB Image Apply To Current Frame All Frames 2 Selection Time ese Current Frame The operation will be performed on the current frame of the nd2 file All Frames The operation will be performed on all frames of the current nd2 file Selection The operation will be performed on all frames of the selected dimension If you selected some of the frames loops of the nd2 file by mouse the Selection option appears in the pull down menu Opt for it to process only these selected green highlighted frames loops Example 6 1 When you process an image containing T and Z dimensions selecting the Z Series dimension performs the operation on all Z frames of the current T loop 7 6 2 Histogram and Look Up Tables 6 2 1 Histogram A histogram displays frequencies of pixels of a certain intensity value The intensity values range from O to 255 for 8bit images
147. r Hide ROI Labels Shift Alt Q Show Tracks of Selected ROIs Other ROI functions 6 5 2 Types of ROI Individual ROIs ROI objects are 2D objects They can have different shape rectangular elliptical polygonal or bezier One shape can be created with one or more tools For instance polygon ROI can be defined using both Draw Polygonal RO and Autodetect ROI In case of an ND file which has time axis e g time lapse individual ROI objects can be defined as Global or as Changing over time Global objects do not change any of their characteristics over time Objects that are defined as changing over time can change their position and shape in time however objects cannot change their shape e g from ellipse to polygon This is feature is useful for track moving or warping objects To track moving objects use the Edit ROIs in Time editor available as a Local Option or just change any global ROI into changing over time from the objects context menu and move it or change its shape see in teractive edit on two or more frames Such frames are called key frames 2 shows trajectories with little boxes representing the key frames All frames in between key frames are interpolated from these two key frames Frames before and after first and last frame respectively are taken from that first or last frame Therefore it is not necessary to reposition or redraw the ROI on every frame In case of Mult
148. re displayed by default You can edit which information is displayed in the Label Properties dialog window Right click the ND2 in formation field and select the ND Info Properties command Use options and tools in this window to customize the display of the ND information you can change text properties type of displayed information accuracy of the displayed number up to three decimal places format of the information duration of display and other 71 5 2 Working with Image Files 5 2 1 Opening Saved Images NIS Elements AR offers several ways to open an image file using either The File gt Open command To invoke the Open dialog box where you can select the file to be opened run the File gt Open command This command is also called when you click the Open button on the main tool bar Organizer An image can be opened by double clicking its filename within Organizer Run the View gt Organizer Layout command F10 to switch to the Organizer See the Organizer Recent Files List You can quickly access the last opened images using the File gt Recent Files menu Open next previous first last Commands These commands enable you to continuously open the subsequent images from a particular directory or a database table The File gt Open Save Next gt Open Previous File gt Open Save Next gt Open Next File gt Open Save Next gt Open First File gt Open Save Next gt Open Last commands may be use
149. reated by the simple ROI editor It can be freely resized and rotated See ROI gt Simple ROI Editor Raster ROI A ROI created by the raster ROI editor similar to the binary layer editor see Binary Layer The raster ROIs behave the same as the vector ROls but can not be resized or rotated See ROI gt Raster ROI Editor Usage of ROIs The ROls can be also distinguished according to their purpose Right click the ROI and select its purpose from the context menu Standard ROI A standard ROI restricting the area of measurement used by the automatic measurement functions Background ROI The ROI specifying the background characteristics This ROI is used as the data source by the Image gt Background gt Subtract Background Using Background ROI command 100 Image Analysis Reference ROI A special ROI used by the View gt Analysis Controls gt Time measurement control window to reduce the influence of photo bleaching on the resulting data Stimulation ROI Confocal microscopes use this type of ROI to aim stimulation lasers ROls in time When performing time lapse experiments the observed objects may be moving If a ROI has been created over such object you can make it move along with the object Right click the ROI and select one of these options ROI is global Position of a global ROI throughout different frames of the ND2 file is static ROI changes over time Position of a moving ROI can differ frame by fram
150. rement results can be transferred out of NIS Elements AR so they could be used further There is a standard Export pull down menu in some of the control windows intensity profile histogram measurement etc which enables the export 138 Measurement settings A Clipboard Web Browser HTML Clipboard Printer EN Report All Measurement Types The set of commands of the menu differs according to the type of data tables or graphs which can be exported Click the arrow button to display the pull down menu When you select the destination the pull down menu hides and the icon of the selected export type appears on the Export button The export action is performed after the button is pressed once more Export Destinations It is possible to export tabular data or a graph To MS Excel Tabular data can be exported to MS Excel A new XLS sheet opens and the table is copied to it automat ically To File Tabular data can be exported to an MS Excel sheet xls or a text file tab delimited txt To Clipboard Data tables and graph images can be exported copied to MS Windows clipboard Then the data or the image can be inserted into any appropriate application text editor soreadsheet processor graphics ed itor typically by the Paste command To Report Tabular data and graph images can be exported to NIS Elements AR report If a report is already opened for editing the data graph will be appended to it Otherwise
151. rescence microscopy Instead of 3 color components RGB mul tichannel images can be composed of arbitrary number of user definable color channels Note If there is a image that contains more than 8 components the tabs in the bottom left corner of the image are replaced by the wavelength dimension similarly to how other dimension loops of nd2 files are displayed ND2 multi dimensional files datasets An nd2 file is usually a sequence of images One ND2 file can contain multiple images organized according to what type of acquisition they came from There are the following acquisition types also called dimensions available Time lapse Multi point Z series Multi channel Large image The dimensions can be combined together 5 1 3 nd2 files 5 1 3 1 Control Bar When an ND2 file is opened its structure is pictured at the bottom of the image window There is a time line with all captured images indicated by gray markers The blue highlighted marker indicates the currently observed image Below the time line loops of each dimension are indicated by rectangles In case the multi channel dimension is included and the number of channels does not exceed 10 the channels are indicated by the color tabs at the very bottom of the image window Browse the nd2 file by clicking inside the time line You can also display a single loop by selecting the corres ponding blue rectangle There are some examples how the nd2 file control bar can loo
152. return TRUE int my_function1 char buf strcpy buf This function has a pointer to char array as a parameter return TRUE 155 156 Additional Modules 9 Additional Modules 9 1 3D Analysis If you have this module enabled in HASP some extra commands and features for analysis of multiple binary layers over Z become available 9 2 4D 6D Acquisition The 4D 6D Acquisition module enables users to combine several acquisition types into one ND experiment A multi dimensional data set comes out from such an experiment 9 3 API Advanced Interpreter This module dramatically extends the macro programing capabilities It adds the following functionality to the main application User interaction functions Contains functions which enable interaction with user like type value text combo boxes list boxes and many other Windows standard controls with easy and intuitive code handling Support of importing functions from external DLLs Functions from external DLLs can be imported at the start of the macro and used during the macro exe cution Please see the electronic help for more details 9 4 Calcium and FRET Installing the Calcium and FRET module adds several tools to the system e A control window suited for capturing FRET images e Automated FRET calibration algorithm e The Create FRET Image window for passive creation of FRET images from files e Calcium ion concentration measurement ability
153. riables only at the beginning of macro You can run two nested macros declaring the same global variables but they must be of the same type You can declare global variables by prefixing the declaration global Eg global int Number_Rows global char buffer 200 The global keyword in front of the variable definition assigns the variable to the global scope Such variable is then accessible from all function scopes within the macro interpreter Statements Supported for The for statement lets you repeat a statement a specified number of times Syntax for init expr cond expr loop expr statement First the initialization init expr is evaluated Then while the conditional expression cond expr evaluates to anon zero value Statement is executed and the loop expression loop expr is evaluated When cond expr becomes 0 control passes to the statement the following the for loop while 150 The while statement lets you repeat a statement until a specified expression becomes false Syntax while expression statement First the expression is evaluated If expression is initially false the body of the while statement is never executed and control passes from the while statement to the next statement in the program If expression is true nonzero the body of the statement is executed and the process is repeated Working with Macros if else Conditionally executes a statement or group of statements
154. rlay mode e The Insert key switches between predefined overlay colors e Ctrl Up Down increases decreases the binary layer transparency Erasing Single objects Single binary objects can be erased in the following way e Run the View gt Analysis Controls gt Binary Toolbar e Select the Delete Object tool from the toolbar e Click inside the objects to be erased Multiple Binary Layers An arbitrary number of binary layers can be created within one image Click this Create New Binary Layer l button in the image tool bar to add a new binary layer The binary layer that you are currently editing can be selected in the nearby pull down menu Binary layers can be managed from the View gt Analysis Controls gt Binary Layers control window 6 5 Regions of Interest ROIs In laboratory imaging experiments users are often interested in just a part of the image To define such an interesting part of the image we use one or more Regions of Interest ROIs These regions are used later in analysis and measurements Note Do not confuse image ROIs with Camera ROI which is used to reduce the active area to just a part of the camera sensor CCD or CMOS Such reduction usually results in higher frame rate See Camera ROI on Live Image The Camera ROI Enable button Fad Frozen 6 5 1 Creating and editing ROIs ROIs can be created and edited using commands gathered in the ROI menu ROIs can be also created from bin
155. rol windows tool bars and menu items Blue tabs representing active layouts appear in the application status bar The following layouts are placed there by default e Full Screen e Docked Controls e Measurement Other layouts can be added and managed via the Layout Manager To hide show the layout tabs within the status bar go for View gt Layout gt Layout Manager and de select the Show Layout Tabs option 14 Application Appearance To Create a New Layout Full Screen Docked Contots AUST ea Ds e Modify the current layout so that it suits your concept of work An asterisk appears next to the layout name to indicate it has been modified Right click the layout tab and select the Save Current Layout As or Save As Default comman4d If you do not need to create a new layout but would like to save the changes made just right click the current asterisk marked tab and select the Save command from the menu e Write the new layout name and confirm it by OK e A new tab appears and the layout is saved to the list of layouts To Reload Previous Layout Settings You may want to undo the changes made to the layout Mostly it can be done by the Reload command Or by selecting the Load Default command e Right click the asterisk marked recently modified but not saved tab and choose the Reload command The application restores the last saved state of the layout e Right click the asterisk marke
156. rosions or dilations by odd number of iterations causes image to shift by 1 pixel Normally the total image shift would be determined by the number of Iterations in pixels NIS Elements AR eliminates this shift it changes the position of the midpoint 1 pixel down rightwards within the kernel for even operations For opening and closing it is possible to eliminate this shift totally However if you run the erode or dilate processes again and again using the kernel with even dimensions and odd number of iterations then the shift becomes significant 7 3 2 5 Examples Please see the following examples of some of the binary functions applied to an image In the following se quence of images the functions were applied subsequently 135 a Threshal CloseHolesBinary 2 4 Morphol arateObjects 4 1 Qs e e See another sequence of functions 136 Measurement CloseB 1 4 gt q Sc A T a E Y q St gt a OpenBinamy 1 4 Skeleton Pruning 3 inary 3 te 7 Ax gt x no E x n pa roe ao o i k E Bi The following examples were created by calling each of the functions separately Original Image OpenBi CloseB ag rary 1 4 Od q 4 do 137 ll L F Convex Huh Contourf Quench 5 Dista nce Function og 4 Or C ZonesOfinf uerice HomotopicMarking 7 4 Exporting Results Some image analysis data and measu
157. roup enumerates shared items optical configurations objectives etc which will be visible for its members NIS Elements administrators can create any number of groups and select the items to be visible Privileges There are basic groups of privileges which allows users or restrict them to perform certain actions This concerns for example modifying macros reports selecting cameras etc There are four levels of privileges by default but further can be created by an administrator e Admin e Common e Guest e Default NIS Elements AR authentication If you have chosen NIS Elements AR Password protected user authentication method a login dialog window appears at every start up of NIS Elements AR Enter a user name and a password for existing user account If the option Allow to Create a New User at Start up is enabled you can create a new user account without needing an administrator This account is assigned to the Default group of users and Default group of privileges automatically Creating a shared layout Please read the following example on user management Step by step we describe the creation of a shared layout which can not be modified by members of the Common and Guest groups 1 Run the View gt Layout gt Layout Manager command Set at least one of the defined layouts as Shared see Modifying Layout 2 In the left column of the window select User Rights The following window appears 28
158. rret2 1 ANALY LU4A Shutter 405 nm Ti Emission Wheel 1 EM400 7 LU4A Shutter Argon Ti Excitation Wheel 1 EX330 380 LU4A Shutter 543 nm le geese te Ti Shutter 1 EPI Opened Y LU4A Shutter SH5 Ti Shutter 2 DIA Opened Illuminator DIA LU4A Shutter AOTF Closed 7 tluminator EPI LU4A Shutter 638 nm Closed Y LightPath Show on toolbar Analyzer Y Condenser C PFS Offset Zoom SILLA BRA dH anne y Objective 3 Plan Apo 20x DIC M Camera amp Devices Controls 3 Type the name of the new optical configuration to the Name field Use a short descriptive name the name is used on the button in the main tool bar when you select the Show on toolbar option 4 In the left column select which device settings to associate with the Optical configuration Camera setting A list of the current camera properties appears on the right It is being updated dynamically Channel setup These settings determine how channels of newly captured images will be named and what color will be assigned to them The available properties depends on the current camera setup color mono triggered acquisition engaged or not dual view engaged or not etc In the mono camera mode you can either assign the name the emission length and color to the channel Manually or leave this task to NIS Elements AR the Automatically option In such case information of
159. s Cinepack Provides maximum compatibility and small files Intel Indeo Provides the best quality compression rate Microsoft YUV A non compression maximum quality codec Size The size is determined by the resolution of the connected camera but can be adjusted by the following options Original 1 2 Original 1 4 Original Sizes counted from the applied camera resolution NTSC TV PAL TV Standard TV resolutions Free Size an arbitrary Width and Height in pixels may be defined No matter what size is selected the video always displays the whole scene If the defined size has different aspect ratio than the live image the empty space is filled with black Maintain aspect ratio of If the Free Size option is selected the aspect ratio may be defined One of the width and height dimen sions are then counted automatically 6 7 2 Converting ND2 to AVI You can easily create an AVI movie from an nd2 file Run the File gt Save As command and instead of saving the file as an ND2 simply select the AVI format in the Save As Type pull down menu The Save As window expands as follows 110 Image Analysis Use Default Video Compression Mo compression Output Timing Parameters Experiment Loop e Use Time Loop Use Original Aca Time A l 5 a Use Z stack Loop C Specify interval 100 iyin 00 _ Use Z stack and Time Loop Adjust the AVI file settings and press Save to
160. s Real time deconvolution can be applied to the Live image continuously or just at capture time It can be also used for processing saved images Please see the electronic help for more details 9 8 LIM Deconvolution requires Local Option The imperfection of a microscope optical path causes convolution of the original image signal which makes the resulting image blurry Deconvolution algorithms reduce the impact of convolution on the image Standard 2D 3D Blind 2D 3D and Fast Wiener methods are available Please see the electronic help for more details 9 9 Local Option Local Option is a pseudo additional module When Local Option is installed see Step 2 NIS Elements AR will provide some advanced features which did not pass the quality assurance procedure yet We recommend to wait until they are released officially 9 10 Deconvolution legacy The imperfection of a microscope optical path causes convolution of the original image signal which makes the resulting image blurry Deconvolution algorithms reduce the impact of convolution on the image The al gorithm used is constrained iterative maximum likelihood estimation algorithm the actual name of the al gorithm is Lucy Richardson Please see the electronic help for more details 9 11 Peripheral Device lf a peripheral device is connected to NIS Elements AR some extra commands and features become available This concerns typically motorized
161. s used for auto focusing The following text explains how NIS Elements handle the two Z drives 39 Absolute Z The current positions of both Z drives Z1 Z2 and the absolute Z position sum of Z1 and Z2 are displayed in the main status bar If there is not enough space to display all these values only the absolute Z value is displayed Z1 and Z2 still appear in a tool tip The absolute Z value appears in the Z Series Setup Active Z The concept of Active Z enables user to select the preferred Z drive e The active Z drive is used when performing auto focus e The Devices gt Enable Mouse Joystick Z in Live command applies to the active Z You can select the Active Z device in the Devices gt Mouse Joystick and Auto Focus Z menu Regular Z drive Piezo Z drive If you have a regular Z drive and a Piezo Z drive installed some functionality is added e The Move Piezo Z to button is added to the Z Series Setup window when Piezo Z is selected e The Devices gt Keeps Z position and centers Piezo Z and the Devices gt Move Piezo Z to Home Po sition commands appear in the Devices menu The button and the Devices gt Keeps Z position and centers Piezo Z command provide corresponding functionality They move the Piezo Z device to its home position and compensate this shift by moving the second Z drive in order to maintain the absolute Z position 3 4 Optical Configurations Typically laboratory computer image
162. satisfy the criteria You can define also an interval with lower and upper bound 1 Search in field select the field where NIS Elements AR should search for a given expression 2 If the selected field is of a numerical type e g Size Calibration File date etc you can specify whether you want to find the exact value or a value in a given range This is selected by the Condition type radio button 3 If the field type is Text the Occurrence setting determines the way of evaluating the expression Anywhere If the given sequence of characters is found anywhere in the field the system will evaluate it as a match For example you have entered set to the Values field The filter will select records with the following field values set reset settings preset etc Exact If the given sequence of characters exactly equals to the content of the field it is evaluated as a match Fields containing the set value only will match Start If the entered string is found at the beginning of a field it passes the condition For example tk tk fields containing set settings setup will match All letter strings It is possible to search for more expressions These should be entered separated by commas If you want to enter an expression with a space insert it quoted If this option is selected only records where all of those expressions appear any
163. series of actions you would like to record Finish the recording by the same command again its name changes to Stop Recording e It is recommended to check the macro in the macro editor before saving Run the Macro gt Edit command to display it e Save the macro to a file via the Macro gt Save As command Creating a Macro from History You can create a macro using the list of recently performed commands 1 Run the Macro gt History command to display the Command History control window Command History A Execute Create Remove 1 Reset selection macro redundant history DefineThresholdintensity 0 166 0 DefineThresholdProcessing 2 3 0 DefineThresholdRBestrictionSize 0 0 0000 DefineThresholdBRestrictiontircularity 0 Threshold EnableLUTs 1 LUTs Eeep utoScale 1 ScanObjects ImageSave s C Program Files NIS Eleme 1 Fa 3 4 5 6 7 8 3 _DefineThreshold aeu U fx Number of commands Free Memory 3 2 GB 2 Push the Create Macro button 3 Select what portion of the command history will be used in the macro 147 e a Create macro from selected commands or whole command history PA Selection This option will use only commands which you have previously selected by mouse within the Command History window Group selection holding Shift or Ctrl is available Whole History All commands listed in the Command History window
164. settings within the documents opened in NIS Elements AR the copy paste Modify Image Through LUTs Press this Il button to apply the LUTs settings to the image data the original image will be overwritten Until you press this button no changes are made to the image data Auto Scale Settings Press the arrow next to the Reset LUTs button and a pull down menu appears Invoke the Settings command 82 Image Analysis Auto Scale Settings io a oe JC cone Shared with Histogram under over exposed settings The Low and High fields determine how many of all pixels of the picture are left outside the sliders when Auto Scale is applied 0 10 AWB White color in some images may suffer from having a color tint The AWB Auto White Balance mode adjusts the image to get pure white instead of this tint Similarly to LUTs auto scale the AWB function can be used once or permanently on the live image If you know the tint your white color has you can select this color by the color picker that appears after pressing the button AWE Color E EE Choose the color by mouse You can also enter the RGB values to the fields below A preview of the chosen color appears in the rightmost rectangle 83 6 2 2 2 LUTs on Monochromatic Images AA AA 0 G 1 32 255 All features mentioned above are also valid for monochromatic images except that the AWB function is not available There are s
165. sted in the top pull down menu Select the control window of which the export shall be modified Export Name A control window such as View gt Analysis Controls gt Annotations and Measurements stores information about the measurement in multiple tables according to the measurement type area length etc In this column you can select the table you would like to modify Sheet name In this column you can define an arbitrary name which will be used to name a sheet or a txt file to which the data will be exported 141 Options for This section of the window enables you to select the items to be included excluded from the selected table during export 7 5 Object Count The Object Count tool is designed for automated object detection and counting It enables users to threshold the image automatically measure the binary objects and export the measured data to a file in a straightforward way Object count can be performed even on Live image Run the View gt Analysis Controls gt Object Count command to display the control window The recommended workflow through the dialog is to move from left to right starting with thresholding then using restrictions and finally reviewing the result and export out of NIS Elements AR to a text file or an MS Excel spreadsheet 7 5 1 Thresholding the Image The threshold limits shall be defined by picking object typical colors from the image Select one of the
166. strictions Circularity rea Circularity Note Infinitude can be defined by entering oo or inf Reset The Reset button on the right side discards restriction settings of all features 143 Bins This setting determines the number of columns in the histogram Restrictions from the Image A reference object can be picked from the image and used to set the limit values e Select the restriction feature you would like to set by mouse Lets assume you have selected Circularity e Right click the thresholded object which is going to be used as source A context menu appears e Select whether to INCLUDE or EXCLUDE the selected object and all others with higher lower circularity e The restriction will be applied and indicated by colors within the image Restrict Circularity INCLUDE selected objects and all with LOWER Circularity INCLUDE selected objects and all with HIGHER Circularity EXCLUDE selected objects and all with LOWER Circularity EXCLUDE selected objects and all with HIGHER Circularity 7 5 3 Results The following results appear in the results table e Total Area is the area of the whole image in square pixels or square calibration units e Area Fraction is the ratio between Total Area and the area of the thresholded objects e Number of objects number of objects after applying restrictions e Table of results measurement results for each object are displayed in this table
167. systems equipped with a stage Measurement Type object field Image Type binary 5tgPosX 5tgPosY StgPosY StgPosY feature is the y co ordinate of the absolute position of the measured field It is available only for systems equipped with a stage Measurement Type object field Image Type binary SurfVolumeRatio SurfVolumeRatio is a feature with a strong stereological interpretation if you measure on fields that are sampled systematically and independently of the content of the sections then the feature is an unbiased estimator of the surface area of objects inner structure per volume of the whole sample SurfVolumeRatio 4 n Perimeter MeasuredArea Measurement Type field Image Type binary 131 Time Time assigns the time to a field object after a measurement has been performed See the SetRefer enceTime function help Measurement Type object field Image Type color binary VolumeEqCylinder This parameter is based on the rod model Length is interpreted as height and Width as the base diameter of a cylinder Bases are considered to be spherical VolumeEqCylinder z d I d 4 x d 6 where l max MaxFeret Length d min MinFeret Width Measurement Type object Image Type binary L VolumeEqSphere Supposing a profile was created as an intersection of a ball and a section that contains the center of the ball VolumeEqSphere is the volume of the ball VolumeEqSphere 1 Eqdia 6
168. t image can be inserted to reports Insert an object which can contain dynamic data text picture table or graph Right click the object and select Insert Dynamic Data Insert Dynamic Picture from the context menu A window appears Select one of the available sources and click Next Finish the source definition and click OK The data appear on the report page List of Available Dynamic Data Sources Data inserted by user available for image text table the system will ask you to type a text or browse for an image to insert during the report creation When creating a dynamic object of this type a query text which will be used to prompt you for the data can be defined System data available for text table enables you to insert some general data such as date the name of the user account currently logged in the page number or the page count Macro available for text table enables you to insert expressions values or results of a macro Measurement available for text table graph enables you to insert results of automatic interactive measurement Database available for image text table this data source is displayed only when creating a database report template see below It enables you to insert a link to any of the database records The real purpose of the dynamic data turns up when creating reports using report templates 106 Image Analysis 6 6 2 Working with Report T
169. t key while dragging Exact values can be entered in ROI Properties dialog which is accessible from the ROI context menu 97 ROI Properties e Current Object Selected Objects CO CO on keyframe 3 of 4 Delete Keyframe cnt cen 09 om 5 Rotation 15 0 Colorize All Objects 6 5 4 Simple ROI Editor When creating more than one ROI the Simple ROI editor is the tool of choice With this tool it is possible to make all possible shapes with different methods All interaction techniques work here as well Simple ROI Editor toolbar Pointing C Rectangle 7 Elipse J Polygon Bezier LS Auto Detect XQ Auto Detect All B Advanced GUI Draw Holes X lt Clear 1 Undo A Redo 7 Help ES Finish Note that many actions have a hot key P for Pointing tool R for Rectangle E for ellipse L for Polygon B for Bezier A for Auto detection H for drawing holes and others to speed up the process of interaction 6 5 5 Edit ROls in Time requires Tracking requires Local Option The Edit ROIs in time editor simplifies editing ROIs that are changing over time ROIs created and edited in this tool are silently converted into changing over time in case they were global All operations can be rolled back by Undo Redo 98 Image Analysis Edit ROls in Time toolbar Y Delete Undo A Redo 7 Help 2 Finish P Move Pick Draw IQ Detect a New Cc New 3 Af
170. t of layouts X The Remove button deletes the selected layout The first two layouts cannot be deleted Othe Activate button makes the selected layout active E The Load Default button loads original settings of the selected pre defined layout Full Screen Docked Controls Measurement so that it looks just like after the program installation 0 a 0 O The Apply to All button ensures that the executed changes will be applied to all layouts EF The Import button enables to load a previously saved set of layouts from an XML file When you try to import a layout with already existing name you will be prompted to choose whether the imported layout replaces the existing colliding layout or is not imported or is imported and renamed inl The settings of layouts can be saved to an external XML file Use this Export button In the windows that appears define the destination file name and check which layouts will be included in the exported xml file 16 Application Appearance 2 2 2 2 Controls The layout of docking panes and the control wndows arrangement has to be modified directly in the application The right part of the window provides only the overview of the structure El Primary Display Right Docker E Column 1 H Tab 1 E Tab 2 E A Annotations and Measurements Bee A Preview a Bottom Docker E Column 1 i Secondary Display Tree View Levels There are 5 levels in the tree view
171. ter Edit Select Next Object j 1 frame al SN Del New b Ss e The editor has two major tools Draw and Detect Both produce polygon but in the first case it is drawn manually around a structure on the image whereas in the later it is automatically detected after one click see Auto detect tool When Draw or Detect is selected it is possible to start drawing or detecting immediately as indicated by cursor symbol In case of editing an existing object it will be hidden after this first click is made so the new one can be drawn unobstructed Esc key is always available to cancel and revert to the previous state Other tools are for Picking Moving and Deleting objects In this editor there is always exactly one object selected Its name and color is reported in the first combo box In the image here reported there is object 1 red currently selected To toggle between creation of new objects and editing of existing ones there is a New button To further help with tracking the objects in time there are two automation modes which can be selected in the After Edit combo box Do Nothing maintains the current frame and selected object User must advance manually by hotkeys or ND navigation control as well as select objects by picking them or by means of hotkeys Select Next Objects selects next object after one is edited and maintains the current frame After all objects has been edited it jumps forward by the frames specified in
172. the light path emission wavelength will be used to determine the channel name and color Microscope setting If there is more than one shutter available and you would like to associate a shutter with the optical configuration select which one is the Active Shutter from the pull down menu Select which parts 41 of the microscope shall be included in the configuration by checking them in the Used devices dialog box Objective An objective mounted to a motorized nosepiece can be included in the configuration Select the objective from the pull down menu Objectives which are currently assigned to any position of the nosepiece are listed See Objectives Note The objective must be assigned to a nosepiece position via the microscope control pad or the nosepiece control window beforehand 5 If some of the device settings still need to be adjusted click the Camera 8 Devices Controls button and select the appropriate control window from the pull down menu Adjust the settings within the control window the optical configuration will be updated automatically 6 Click Finish to save the new optical configuration and to close the window 7 You can create more optical configurations by repeating the procedure The optical configurations are saved to registry immediately A backup of optical configurations can be made by running the Calibration gt Optical Configurations command and clicking the Export button Managing Optic
173. tton to enable recording of Live image to RAM e The ring buffer functionality gets activated Capture the Image Sequence e Press the RAM Capture button e A new image window containing the captured image sequence opens Note When the Camera is switched to Live mode the circular buffer ring button indicates the buffer activity If the RAM Capture button is pressed afterwards the captured image sequence will contain frames from the whole time interval before and after the button was pressed If you press the button while in the Frozen mode or without the time buffer turned ON only the Time buffered after portion of the sequence will be captured 4 4 Capturing movies AVI or MOV acquisition is an easy way of recording actions in real time It produces avi or mov video files which can be played by any standard video player so you do not need NIS Elements AR to view the resulting video Please see Creating Movies 61 62 Image Files 9 Image Files 5 1 Composition 5 1 1 Image Layers The following image layers can be saved within a NIS Elements AR image r a E ay A a a a E ee or rn aa T k i 21 ILS a 4 So Sos an Annotation layer In this layer vector objects are stored The results of manual and automatic measurements text labels and other annotations can be included Binary layer s The binary layer is usually the result of thresholding By threshold
174. universal ND2 N dimensional file format is what makes this possible One ND2 file can contain multiple images organized according to what type of acquisition they came from There are the following acquisition types Note Each acquisition type gives a name to the corresponding dimension of the resulting ND2 file For example we can speak about two dimensional TZ ND2 file which means the file contains the Time lapse dimension and the Z series dimension Time lapse T A sequence of images can be captured over a period of time and create a time lapse image Multi point XY Images from different areas of the slide can be acquired Z series Z Several techniques utilize Z stacks of images Such Z stack can be for example converted to a 3D model of the specimen Multi channel 2 Fluorescence signals can be captured selectively using excitation an emission filters each of them cre ating a different color channel Large image M requires 4D 6D Acquisition 49 When the area of interest exceeds the camera field of view you can capture a large image composed of multiple image frames and let it be stitched by an automatic algorithm An nd2 file can be created captured either containing one dimension or combining several of them e Single dimensional documents can be created automatically using motorized accessories or manually using commands from the Acquire menu e To create a multi dimensional image use the Appl
175. uto Exposure Calculates Exposure time and Gain in order to achieve optimal brightness of the scene Image quality is the priority therefore longer exposure times are being preferred In most cases the auto exposure can be run once by pressing the Auto Exposure button or continuously by selecting the nearby check box 34 Setting the Hardware Up Note Depending on particular light conditions the result of Auto Exposure may not be optimal for image acquisition Warning You can use one optical configuration when working with live image and switch to another optical configuration just for the image acquisition very often this is performed automatically i e by a macro command If the on acquisition optical configuration has the Auto Exposure camera mode turned ON it may produce over under saturated images Since the Auto Expos ure algorithm estimates the optimal exposure time by analyzing last several frames it is not able to make the estimation correctly if the change of optical configurations would cause Sig nificant change in image brightness To prevent this we recommend to use Manual Exposure mode in the optical configuration used for capturing e Manual Exposure User selects both Exposure time and Gain manually Exposure When in manual exposure mode this option controls the exposure time which is expressed inversely 1 x second It is the time of charge accumulation in a camera chip between two
176. uttons at the bottom of the right image tool bar 91 This button displays the binary layer only of the image It can be edited by hand using the binary editor Binary gt Binary Editor The binary layer can be displayed together with the color layer using an overlay mode Several binary layers can be created for one color image Manage them using the View gt Analysis Controls gt Binary Layers control window is This button switches to view the color image only The binary layer as a result of thresholding can be modified by hand using the binary layer editor It is a built in application providing various drawing tools and morphology commands Go for the Binary gt Binary Editor command or press Tab New buttons appear on the tool bars 6 4 1 Controls horizontal tool bar E Reverses the changes made to the binary image U X Clears the binary image fills the entire image with the background color R o Loads the image previously saved by the Save button L Y Temporarily saves the current binary image It can be loaded anytime before the binary editor is closed by the Load button S Y Displays this help page E Stores the changes and quits the editor ESC or TAB The other buttons of the horizontal tool bar are simplified versions of mathematical morphology functions 8 Dilate j g Erode j Pal Close Pal Open Separate Objects j Clean B8 Fill Holes Please s
177. ved from an intensity histogram It is an usual standard deviation of intensity values This feature describes the inner structure of an object or a field Measurement Type object field Image Type color Green Green value is the mean of green component pixel values It is the usual statistical mean of intensity values of green component Measurement Type manual Image Type color HueTypical HueTypical is the hue value with maximum frequency in a hue value histogram This feature describes the most frequent hue color in an object or field Measurement Type object field Image Type color binary HueVariation HueVariation is the usual standard deviation of hue values This feature describes hue color distribution of inner structure of an object or field Measurement Type object field Image Type color binary SumBrightness SumBrightness is defined as the sum of brightness in every pixel of the object Measurement Type object manual Image Type color gray SumDensity SumDensity is the sum of individual optical densities O D of each pixel in the area being measured This feature describes for instance the amount of a substance in biological sections Optical density is evaluated according to the following formula O D log PixellntensityValue 0 5 62 5 Measurement Type object field manual Image Type color binary Sumintensity Sum Intensity is defined as the sum of intensity in every pixel of
178. when a single workstation is shared by number of users Some user accounts may have administrator rights to NIS Elements while other s privileges are quite restricted A per person system may be established or user accounts may be shared The following principles are utilized within NIS Elements AR MS Windows accounts support NIS Elements AR can assign privileges to MS Windows accounts Whoever is logged in the operating system and runs NIS Elements obtains a default set of privileges System administrator can than further restrict or extend the user s rights NIS Elements AR password protection It may be not comfortable enough to log the current user off from MS Windows when a user change is required in NIS Elements For such case a completely independent list of user accounts which is not connected with MS Windows users can be created Then just restart of NIS Elements is needed to switch the user 27 Private vs Shared storage Every user created item setting important in the work flow can be protected from unwanted change in two levels First the creator of the item optical configuration layout objective etc can save it as private Unless its status is changed to shared no other user will even see this item within the lists throughout the application The second level of protection concerning all shared items is provided by assigning users to Groups and Privileges groups Groups Every user is a member of a group The g
179. where will match 25 e Any letter string This option is for entering multiple expressions as above but this time every field with an occurrence of at least one from the given expressions is matched 2 3 2 Operations with Images e To open an image double click its thumbnail NIS Elements AR will close Organizer and display it in the main window e To select multiple images either click on the first and the last image holding the Shift key continuous group selection or click individual images with the Ctrl key down e You can copy one or more images from one folder to another by drag and drop e To delete selected images press the Delete key All those operations and some other can be invoked also from the context menu which appears each time you right click on the image thumbnail Open 3 Copy Cut Select All Invert Selection Delete Thumbnail Displaying Options You can adjust the way images are shown in the organizer Thumbnail View Press this button and select the size of displayed image thumbnails Selecting the Details with preview option will display images below each other with all available information aside go Rotating Images There is a possibility to rotate images from within the organizer It affects not only the image thumbnails but the image data too Press the corresponding buttons 4 Autocontrast Press this button to turn the Apply autocontrast to thumbnails option ON lt
180. will be used for the new macro 4 After you selected one of the options the macro editor appears containing the new macro source code Use the editor to fine tune it Note The Remove Redundant command is automatically performed before the macro is opened in the editor Writing Editing a Macro If you posses programmers skills a macro can be written by hand in the built in macro editor Run the Macro gt Edit command to display it SBH a pou amp ROlo x qag WaitText 2 Detection of poppy seeds Pre definition of threshold DefineThreshold DefineThreshold int RLow int GLow int BLow int RHigh ZEB A SmoothBinary BinaryToReference The macro editor provides e Undo Redo functionality e Command insertion from the list of available commands 148 Working with Macros Interactive command names list Press Ctrl Space to display a simplified list of all commands while typing the macro Syntax hints parameter types and names appear as you type a command name Bookmarks can be placed to the code so you can easily roll to the important parts of the macro Breakpoints can be placed to the code A breakpoint forces the macro to stop the execution at a certain point so you can check the state of variables and therefore inspect the macro functionality thoroughly Syntax highlighting Help on commands with detailed description Use the Save Macro lal and the
181. window may be modified Items of the context menus can be hidden by de selecting them similarly to the tool bars The main menu can be modified as follows Modifying the Main Menu H Edit H Calibration Image ROL Binary aMeasure H Reference H Macr o View H Window H Applications e Select the Main Menu in the topmost pull down menu e Any item may be added to the main menu a Separator a Menu Command a sub menu Menu Popup and even a new menu Main Menu Popup by the Add button 19 e Select the existing menu item under which you would like to place the new item e Click the Add button and select the item to be added from the pull down menu e Edit the Item Properties Note The Default button discards all changes and loads the main menu original configuration The Re move button deletes the selected item The arrow buttons move the selected item up down The Use Default check box when selected applies the default settings to the menu Item Properties Text This is the text which appears in the pull down menu You may add amp before any letter such letter will be considered a keyboard shortcut when browsing the menu Hot key One or more hot key shortcuts may be assigned to the command Just press the Add button and press the key combination Press Remove to remove the selected hot key Enabled Disabled bitmap Command These fields serve for

NIS-Elements Advanced Research User's Guide (Ver. 4.00)

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