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1. 25 EA bensen asao niai aeai ir Arts aa 27 3 1 Store Captured Mages oiiiosi sscciinciccionisi nococanacacinnsis 27 3 2 Capture a single iMmagZe sssssssssssssscsrrorseserooronsrrerr 27 3 3 Capture a time lapse sequence cccoconoococcccccccnnnonicccnns 27 3 4 Creating a pattern of images to be captured in a AAA A A A II 27 3 4 1 Select the wells to be CAPtur dA ssrsrrrerrrrrrrrrrrnn 28 3 4 2 Select positions to be CAPtur A srrrrrrrrrrrrrrrrran 28 3 4 3 Create identical capture patterns in all wells 28 3 4 4 Create random capture patterns 29 3 4 5 Create patterns using the current position 29 3 4 6 Clearing the capture Patt rM ssssssersrrrrrrrrrrrrrrrraa 29 3 4 7 Settle time after stage MOVEMeENL ssoererrrrrrerrrrnnna 29 3 4 8 Capture the PO ai 29 3 5 Capture timelapses at several locations in parallel 29 3 3 1 Storing Captured pattern timelapses in one group 30 3 3 2 Storing parallel timelapses in separate groups 30 4 View captured IMAGES iii na ind 31 AL View an MAp nacidos 31 AZ VIEW a TIMO 31 4 3 View one position of several in a timelapse 31 4 3 1 Capture pattern timelapse stored in single group 31 4 3 2 Capture pattern timelapse stored in individual EA A PN AR SRA SEA ack 32 4 4 Move flip or zoom the cell image ssccsssseoeees 32 4 5 Holographic image display ccccccccsssssssssss
2. J a al El E F igure 105 The several plot display colors that can be selected a a gt as Ju Eng Se en E na Area 178 1 pm _ Volume 2 22E3 pm Thickness max 21 9 pm Thickness avg 12 4 pm Frame 25 in Lapsus s avg Dele Dele Dele Dete Mase Perimeter length 48 9 um MI At Eccentricity 0 30 3 Irregularity 0 07 11678 a a Show labels Show grid lines T Only current frame Figure 106 Identifying the cell represented by a certain data point 7 3 1 Identify data points as cells Hover with the mouse cursor over a data point in the scatter plot The corresponding cell will then be identified in one of the cell images that have been added to the plot Figure 106 Data for that cell will be displayed simultaneously When hovering over a certain cell in an added image frame the data point representing that cell will be identified in the scatter plot and data for that cell will be displayed Figure 107 Area 379 0 um Volume 3 65E3 pm Thickness max 14 5 pm Thickness avg 9 63 ym Perimeter length 73 4 ym Eccentricity 0 47 Irregularity 0 12 ss avg um Frame 25 in Lapsus Figure 107 Identifying the data point representing a certain cell X axis feature Thickness avg um NM Yaris testure ara Figure 108 The X and Y axis labels 7 3 2 Change the scatter plot axis units The axis labels of the scatt
3. Vessel growth area e Vessel media volume Snow cell count per mi Figure 134 Text boxes for cell culture vessel area and volume The images are then analyzed and the results are presented in a Cell Count Report Figure 135 Cell Count Report Area um distribution Nbr of cells in vessel 1 86e 005 24 42 Nbr of cells per ml 3 71e 004 36 30 Confluency 23 7 A 6 147 0 154E3 292E3 43183 5 7083 7 0983 84863 986E3 1 13E4 Report date 2012 08 31 11 42 Volume um distribution ture at 2010 12 01 13 23 20 2012 07 30 16 14 55 140 th area 25 cm Pe I a volume 5 ml mages 6 112 98 34 70 56 42 8 14 0 image edge 115 0 0 1 653 33183 4 96E3 6 6183 8 263 9 92E3 11664 1 32E4 ged area 4 424 mm es 386 Figure 135 The Cell Count Report The report contains data on e Number of cells in the vessel e Number of cells per mi e Confluence e Report date e Capture time points e Vessel growth area e Vessel media volume e Total number of image frames used for the analysis e The total area of the images e The total number of cells in the images e The number of cells that are placed on the image edge and which therefore are not included in the morphological analysis although they are included in the cell count Of the edge cells 50 are included in the the cell number while 50 are not included If the confidence interval is too large larger than 10 15 more images sh
4. Otsu Otsu in blocks Auto Auto Auto Adaptive mean Auto Adaptive gaussian Double Otsu Double adaptive mean St Auto o gt Adjustments Auto S Background threshold Miscellaneous Auto Double adaptive gaussian Auto Minimum Error NM Y Use pre smoothing Method Adjustment Y Join nearby markers Object definition Min object size Figure 65 The Adjustments tab showing the different methods to calculate threshold settings The different threshold settings calculation methods Figure 65 will result in slighty different cell identifications lt is advisable to try out which method that works best for every type of cell sample e Manual allows the user to set the global threshold level using the slider e Minimum error sets the global threshold level using the minimum error threshold method histogram based Otsu sets the global threshold level using the Otsu method Otsu in blocks the image is split into blocks which are thresholded separately using Otsu method This is a form of adaptive threshold Adaptive mean sets an adaptive thresholding using a mean filter Adaptive gaussian sets an thresholding using a gaussian filter adaptive Double otsu double thresholding is a method where both a wide and a narrow threshold mask is used The narrow image is morphologically reconstructed under the wide image The final image is used as threshold mask The res
5. New C Existing Y New C Existing New C Existing New C Existing 48 333 21 567 15 333 23 233 48 167 55 900 21 000 61 400 Cancel Figure 44 The Configure Destination window Chapter 4 View captured images In order to view and adjust captured images select the View Images tab Figure 45 MCF 10A time laps File View Databzsgeussl Sel A oe een Soran Figure 45 The View Images tab 4 1 View an image Start by selecting a Project and a Group in the Image Frame List side window Figure 46 which is found to the right of the Main Viewing window An image frame presentation list for the selected group will appear Both holographic images and phase contrast images are presented in the list as well as information pertaining to the images Highlighting an image will make it appear in the Main Viewing window ces Project 2012 06 07 11 48 MCF 10A time lapse E Group 2012 06 07 11 48 MCF 104 Medica rod New Delete Rename New Delete Rename 5 Count 37 Confl 9 6 oo Count 21 Figure 46 The Image Frame List side window lologram x20 2012 06 07 11 48 09 Take 3 2 62 2 62 A i LI a ie UTV a F iale fr Hologram x20 rom oem MM oe 4 2 View a timelapse Start by selecting a Project and a Group in the Image Frame List side window Figure 46 which is found to the r
6. User Manual for HoloStudio M4 2 6 2 with HoloMonitor M4 GPNAS E HoloStudio 2 6 2 Software Manual 2015 Phase Holographic Imaging AB Contact us Phase Holographic Imaging Scheelev gen 22 SE 223 63 Lund Sweden 46 0 46 386080 info phiab se www phiab se Introduction to the HoloMonitor M4 The HoloMonitor M4 is a cell analyzer for adherent cells If can count cells and analyze adherent cell morphology and confluence The HoloMonitor M4 can also be used for longterm timelapse captures which do not harm the cells Cells can be tracked through the timelapse capture both for movement and for morphology The HoloMonitor M4 is incubator proof allowing for experiments to be performed in a cell culture incubator Figure 1 The HoloMonitor M4 with a flat sample Stage The HoloMonitor M4 uses digital holography This technique is based on measurements of how the cells shift the phase of light that passes through the cells This kind of live cell imaging does not require any kind of labeling or staining The cells can be analyzed while growing undisturbed in their Usual cell culture vessel The HoloMonitor M4 works with most of the common cell culture vessels such as 6 well plates petri dishes and IBIDI slides The HoloMonitor is equipped with a flat sample stage Figure 1 which can be replaced with a manual XY stage Figure 2 or a motorized XYZ stage Figure 3 When the M4 is equipped with a
7. found to the bottom right of the window in order to apply the cell identification adjustments on all the captured images Alternatively click Apply All Quickly preview the rest of the checked 15 20 21 22 23 images to make sure they are all well segmented Note that for cell counting the cell area identification does not need to be exact but the cell markers blue dots need to be correctly placed For confluence measurements the threshold setting needs to be correct Proceed to the Cell Count tab Acquire the cell numbers for one vessel Highlight or check the image frames you want to include in the plot Click the Add buttons which are found in the lower right corner of the window to add the data from the highlighted or checked image frames to the cell count images list The cell count results are given as Nbr of cells in vessel and the confluence results are given as in the Cell Count Report in the main window Type the vessel growth area in the text box below the Cell Count Report window and choose the unit from the scroll bar to obtain a correct cell count area or a Vessel media volume for a corect cell count volume Export and save the Cell Count Report by clicking the Save Report button Quick guide Proliferation studies When the Startup Make sure that the instrument has been placed in an incubator for at least three hours while switched on If the instrument is
8. Capture bes Project Oooo C EX Group New Delete Rename IS Y New Delete Rename Timelapse Total time s y Interval s y Min interval 1 0 Number of timepoints 2 Capture pattern 5 Capture 2 images Figure 35 Activated Timelapse function 3 3 Capture a time lapse sequence To enable slow events to be recorded and studied a movie can be created from images captured at intervals i e a timelapse movie In order to capture images for a timelapse study check the Timelapse box Figure 35 in the Capture window Enter the total time for the timelapse and select the desired time unit seconds minutes or hours Enter the interval between the capture time points and select the desired time unit seconds minutes or hours The minimum interval between captures is given to the right of the interval time unit box The number of timepoints will be given when total time and interval are entered Click the Capture button 3 4 Creating a pattern of images to be captured in a sequence When it is necessary to capture several images in a sequence e g for cell counting purposes or for parallel timelapses it is convenient to create a capture pattern When this capture pattern is applied the instrument automatically captures the set number of images in the set pattern Check the Capture Pattern box Figure 36 in the Capture side window Capture FJ Project Ooo Y FT
9. Figure 43 the set capture pattern will be captured at as many timepoints as are set for the timelapse Capture Leal Project 2015 04 28 13 17 Ta Group 2015 04 28 13 18 New Delete Rename Testar New Delete Rename test B1 1 Timelapse Total time Min interval 2 1 Interval Number of timepoints 121 Capture pattern Settle time after stage movement S p election O Select wells Select positions Clear pattern Setup storage J J _ Identical pattern in each well Generate ifo Jrandom positions in each well Capture 2662 images AAA AAA a Stage position Vessels Figure 43 A combined timelapse and capture pattern capturing sequence 3 5 1 Storing Captured pattern timelapses in one rou By default all captures will be stored in the same group This becomes impractical if the intention is to capture a time lapse at each location 3 5 2 Storing parallel timelapses in separate groups When using Capture pattern the option to create separate groups for each location is available This is done by clicking on Setup storage Figure 36 which will show a configuration window Figure 44 In the Configure Destinations window deafault is set to adding all captured frames into a single group When checking Multiple destination groups the images will 30 be sorted into one group for each capture position Figure 44 The group
10. instructions of the Collect Wizard Diagnostics Quick guide Time lapse capture and movie export 12 13 Startup Make sure that the instrument has been placed in an incubator for at least three hours while switched on If the instrument is to be operated at room temperature start the HoloMonitor 30 minutes prior to use Start the computer Start HoloStudio M4 Image capture Go to the Live Capture tab Choose create a project and a group Put the cell sample on a spacer plate or vessel holder of the correct type Make sure the image is focused Activate the Timelapse function and enter the total time and the interval between the time points Press Capture Use the View Images tab to ensure that the captured imges correspond to your settings When image capture is complete proceed to the View Images tab Run through your images by clicking the Autoscroll button If needed recalculate the images Export individual images or a movie Go to the Export Images tab Highlight or check the image frames you want to include in the movie or image export Click the Add buttons in the lower right corner of the program to add the data from the highlighted or checked image frames Adjust the coloring and viewing angles of the added images Preview the movie Export Images or movies 13 Quick guide Parallel timelapse captures Make sure that the instrument has been placed
11. such as how to capture an image and how to analyze the images The last chapter contains a trouble shooting guide HoloStudio M4 2 6 outline Goo va File View Database About Figure 4 The main tabs of HoloStudio 2 6 Main tabs overview HoloStudio M4 Tracking is divided into seven functional parts that are represented in seven different tabs Figures 4 and 5 1 Live Capture which concerns the live viewing and capturing of digital hologram images 2 View Images which concerns viewing captured images 3 Identify Cells which concerns the segmentation of the image resulting in the identification and outlining of the cells 4 Track Cells which concerns the tracking of individual cells through a series of captured frames 5 Analyze Data which concerns the analysis of cells in the captured hologram images as well as display and export of the results 6 Cell Count which concerns the counting of adherent cells in their cell culture vessels 7 Export Images which concerns the visualization and export of images and movies The Main Viewing window The Main Viewing window Figure 5 shows the actual live cell image when the Live Capture tab is open and when the other tabs are open it shows the currently selected stored image The Side windows Basic functions are found in side windows to the left and right of the Main Viewing window Figure 5 If the side windows are collapsed they can be exp
12. 6 3 1 Select the cell to be adjusted Activate Select in the Select Mode side window Figure 79 Click on the cell to be adjusted A new set of functions will then be available in the Change Tracking side window Figure 85 The cell that will be adjusted is noted in the Change Tracking side window 6 3 2 Switch the tracking from one cell to another After selecting the cell to be changed click the Modify Location button in the Change Tracking side window Then click the cell that actually should be followed instead of the selected cell Now the colored border is transterred to the new cell to be followed Figure 86 The software will recalculate the tracking automatically from the present image frame and forward through the time lapse gt Click to change Cell 10 to this cell instead Figure 86 Transfer the tracking from one cell to another The left image shows the selected cells and the right image shows how the tracking is transferred 6 3 3 Discontinue a cell tracking Sometimes a tracking needs to be discontinued e g when a cell leaves the image area After selecting the cell to be changed click the Unset button in the Change Tracking side window Figure The tracking will be discontinued from the present frame A new button Set location will appear in the Change Tracking window Figure 87 Change tracking Make changes for Cell 2 Set location Unset Undo for this frame Undo for all frames
13. 90 frames Figure 140 The Image Frame list 53 9 1 Add and remove image frames Below the Image Frame list Figure 140 there are buttons to add image frames to the Main Window The side wndows become active only when one or several images have been added By clicking the Add Highlighted button Figure 140 which is found below the Image Frame list the data from a single image frame or from several frames can be added when the frames are highlighted in the Image Frame list The shift key can be used to highlight several consecutive images and the ctrl key to highlight non consecutive images Alternatively the box to the left of each image can be checked and then the Add Checked button is clicked All image frames can be added by clicking the Add All button Clicking the Remove buttons Figure 141 will remove either only the highlighted or all of the added images X Remove X Remove all Figure 141 The Remove buttons The Main Viewing Window contains a view of the currently active added image as well as thumb nails of all the added images Figure 142 By clicking a thumb nail a new image will be displayed in the Main Viewing window Group 10A Me Frame no 9 roup MCF 10A Mex 012 06 07 11 48 12 20 An ma A Figure 142 The Main Viewing window 9 2 Edit the images 9 2 1 Zoom move or flip the image The Perspective side window Figure 143 shows h
14. A i es e Identify cells a Track cells q q Analyze data 3 Cell count fort images o Figure 130 The Cell Count tab 8 1 Count cells Choose the Cell Count tab Figure 130 A text will appear which says that five image frames or more must be added in order to analyze images for cell count confluence cell area and volume Figure 131 Create a cell count report by adding at least 5 frames from the frame list to the right Figure 131 The Cell Count instruction text Highlight or check the images to be analyzed in the Image Frame List to the right Click the appropriate Add button Figure 132 The Add buttons are found below the Image Frame List The added image frames will be shown in the Source Frames window below the Cell Count Report Figure 133 ye Count 50 Cont 18 ag i 359 frames E Hologram W Phase cont E Only checked Add highlighted Add checked Add all Figure 132 The Add buttons Nbr Group Project Commen ES dagl scanlab Take 1 m dagl scanlab Take 2 dagl scanlab Take 3 dagl scanlab Take 4 dagl scanlab Take 5 dagl scanlab Take 6 Anant LA da sonia Taka 7 1 3 Figure 133 The Source Frames window o NN Oo WwW amp uy MM 51 Fill in the correct cell culture vessel growth area and the volume of the cell culture vessel medium content in the text boxes below the Cell Count Report Figure 134
15. Coloring side window Additional functions are found as buttons and in a menu which is found at the arrow tip Figure 147 By left clicking the R button Figure 147 the coloring in the image is rescaled to better utilize the optimal dynamic range of the image Note that this button needs to be operated every time the image coloring is off e To add a new color to the colorset left click the plus button Figure 147 and select a new 55 color A colored triangle representing the new color will appear beneath the histogram Figure 146 Alternatively right click the x axis at the position where a new color is wanted and select Add Color from the menu that appears e Change the color by using the right mouse button to click on a colored triangle beneath the histogram and select a new color alternatively left click the arrow button and select Change Color Figures 146 and 147 e To change the color span left click and move the desired colored triangle beneath the histogram using the cursor Figure 146 e To save a new colorset with the current color settings left click the arrow button and choose Save As Figure 147 e To save the current color settings to a previously saved colorset left click the arrow button and choose Save Figure 147 Note that this will overwrite the settings previously saved to this colorset FE Add color Change color F Save colorset Save colorset As Delete colorset Figure 14
16. Dox Regions can be deleted by highlighting the region in the Regions window and then clicking Delete which is found to the upper right in the regions tab Figure 116 Scatter plot Histogram Area um 221 H 37 6 57 2 76 9 96 5 116 1 135 8 Average optical thickness nm Figure 113 An X axis region green and a Y axis region blue Scatter plot Histogram Area um 37 6 57 2 76 9 96 5 116 1 135 8 Average optical thickness nm Figure 114 A linear region blue and an arbitrary region green Type No of cells covered Part of population 2 a X Thickness avg um 7 BB Region Feature region Y Area um 1 0 46 X Thickness avg um ZB Region2 fFeature region X iS ns 179 82 49 T GB Analysis 1 Region 1 Feature region se Figure 115 The Regions tab X Delete XM Delete all Figure 116 The Delete buttons 48 7 4 Display results in histograms The results can also be displayed as histograms where one cell parameter is offset against the cell number Figures 117 and 118 Analysis 1 Histogram D MRI oo A 120 1004 ie a da te A A a Figure 117 The Histogram tab Scatter plot Histogram 1207 0 47 9 915 1350 1786 2221 2657 309 2 3528 3963 439 9 4834 5270 5705 6141 6576 7012 7448 7883 8319 Area um Figure 118 A Histogram When Only Current Frame Figure 119 is checked the data
17. Measure button is clicked again the measuring function is deactivated 7 2 Analyze results in plot 7 2 1 Start a new analysis Choose the Analyze Data tab Figure 96 Od ne Ec File View Database Analysis About f ene fg Live Capture Identify cells E Track cells Analyze data B cet Bt L Export images Figure 96 The analyze data tab A window will appear where a new analysis or a stored analysis can be selected Figure 97 New analysis Start a new data analysis ES Open Load a previous analysis Figure 97 The Select Analysis buttons After clicking the New Analysis button a new message will appear Figure 98 Generate the analysis by adding source frames from the frame list to the right Figure 98 The Add message Select a Project and a Group in the Image Frame list window Figure 99 which is found to the right of the Main Viewing window Figure 5 deal Project 2012 06 07 11 48 MCF 10A time lapse New Delete Rename E Group New Delete Rename 2012 06 07 11 48 MCF 10A Medica r d Hologram x20 A 2012 06 07 11 48 09 Take 3 2 62 2 62 El 6 pz gt Holagram x20 Figure 99 The Image Frame list To add data highlight image s and transfer to the plot by using the Add Selected button Figure 100 which is found below the Image Frame list window Several images can be added simultaneously if they are all highlighted
18. The shift key can be used to highlight several consecutive images and the ctrl key to highlight non consecutive images Add selected Add checked Add all Default color Figure 100 The Add buttons below the Image Frame list The images that are included in the analysis are shown in the Source Frames tab Figure 101 which is found below the Main Viewing window oo Filter Regions Export Number Group Project Commen t E 5 MCF 10A Medica r d MCF 10A time lapse Take 3 2 62 2 62 6 MCF 10A Medica r d MCF 10A time lapse Take 4 2 62 2 62 7 MCF 10A Medica r d MCF 10A time lapse Take 5 2 62 2 62 8 MCF 10A Medica r d MCF 10A time lapse Take 6 2 62 2 62 ELINA times ls beg s n X Remove X Remove all E E a INNE a MICE_INA Mandira r d MA E Figure 101 The Source Frames tab which is found below the Scatter plot tab If several images are added to the plot the data from all images will be displayed in the same plot unless the Only Current Frame Figure 102 is checked Then only the data from the current frame are displayed The Only Current Frame function is found below the Scatter plot E Only current frame Figure 102 The Only Current Frames function 7 2 2 Remove data from plot To remove data belonging to a single frame from a plot highlight that frame in the Source Frames list Figure 101 Then use the remove button that is found above and
19. analyzed M lder et al 2008 Figure 159 The holographic setup Laser light Phase shifted laser light Figure 160 Light waves passing the cell will be delayed causing a phase shift of the light waves Phase shift Every morphology calculation performed in the software is based on the phase delay i e the phase 62 shift of the light caused by the sample When the light passes through the cell it will travel slower Once through the cell it will regain its former speed This delay will cause the phase of the light waves to be offset or shifted compared to the reference light The phase shift is the basic raw data in digital holography Phase shift P The optical cell thickness can be obtained by combining information on the phase shift and wave length of the light with the refractive index of the cells 1 38 and the refractive index of the surrounding medium 1 34 Wavelength 1A Refractive index n Optical thickness T P x A Ncen Nmedium For the cells we use an average refractive index based on publications worldwide Dunn and Richards Kortum 1996 Farina and Verkman 1996 Rappaz et al 2005 Small variations in the refractive index do not cause any significant changes in the calculations The refractive index of the surrounding medium should not deviate too much 0 08 from the refractive index of the cells If should also not be identical with the refractive index of the cells The opt
20. and viewing angles of the added images Preview the movie 14 16 Export Images or movies Quick guide Cell counting and confluence measurements 18 When the Startup Make sure that the placed in an incubator for at least three hours instrument has been while switched on If the instrument is to be operated at room temperature start the HoloMonitor 30 minutes prior to use Start the computer Start HoloStudio Image capture Go to the Live Capture tab Choose create a project and a group Put the cell sample on a spacer plate or vessel holder of the correct type Select the correct vessel type in the vessel list in the stage position side window Ascertain that the live image looks well Press Capture Use the View Images tab to ensure that the captured images correspond to your settings Move the sample Press Capture Move the sample and capture again Continue capturing images until at least five images have been captured image capture is complete proceed to the View Images tab Make sure that the captured images look good Proceed to the Identify Cells tab Identify cells Adjust the cell identification in the first image frame using the Adjustments and Manual Changes tabs Make sure all the images you want to include in the cell count are checked in the Image Frame list on the right side Click the Apply Checked button which is
21. as cell division the Ignore box can be checked and the warning is then ignored Figure 83 In the example Figure 84 the segmentation needs adjusting and the user would then click Identify to go to the cell identification tab Source frames Tracked cels MON Possible errors Y Show icons in image Y List ignored warnings l Message Time index o Date Tracked cell E Ignore Show A Volume increase 169 4 2010 09 02 12 23 01 E Cell 1 L E ignore Show A Volume drop 47 division 5 2010 09 02 12 28 01 Mc E Ignore Show A Volume increase 86 9 2010 09 02 12 48 01 Mc a Figure 83 The Warnings tab 41 There may be a problem with the cell identification or tracking of the cell Previous frame This frame Next frame Volume increase 22 Figure 84 The warning in the segmentation for the previous current and next image frame 6 3 Adjusting the cell tracking Sometimes the software will track the wrong cell e g when cells are moving very close to each other and then separate again This needs to be adjusted manually Note that the adjustments will be active from the frame of adjustment and forwards through the timelapse Select mode Add cells Select a Change tracking Make changes for Cell 2 Modify location Unset Undo for this frame Unde for all frames Delete Figure 85 Changing the cell tracking
22. colorset will be applied to the currently viewed frame and saved e To save several images with the current colorset check the box of the desired images in the Image Frame List side window Figure 46 left click on the arrow button in the Histogram side window Figure 55 Choose Apply To followed by left clicking Checked Frames The current colorset will be applied to the checked image frames and saved Note that changes regarding color will not in any way affect the raw data and that the original gray scale image always can be retrieved 4 6 Move flip or zoom the cell image To zoom the cell image click the image in the Main Viewing window and then use the scroll button on the mouse To move the cell image to a desired location in the Main Viewing window click hold and drag the image using the left mouse button To flip and move a holographic 3 D image click hold and drag the image using the right mouse button on the image 4 7 Recalculate a holographic image If a holographic image is not correctly focused as in this example Figure 56 the software focus can be recalculated after capture in the View Images tab by changing the software calculation settings Figure 56 An unfocused holographic image Software focus I Automatic Manual 11444 Update More Figure 57 The Calculation Settings side window set to manual 34 4 7 2 Recalculate the focus manually After image capture
23. i NEE N E EE EA E ET hh aah alte te At 25 A a PA 38 Undodoralk Eramos nai 42 Undo torihls Frame suscita dis li laca ds del da 42 Unser Duto as 42 Use Plain Image Mass iii id S6 AA E 54f VES as 24 A A A Hee eee Sie SA 9 NeW Imaces tada deis 44 NIE WER PH NS aida 25 54 A EEEE ne E 39 64 WAC asc asec ta O EE 32 XA Si ata Si 52 LE rs tea ateen fbi cece oestenemuseetarateddaarecdd 49 X axis label MSO Mii taa 49 Datos ea 100 PER a R N S MORRA INNER GR r 47 A O A E RR ROR 47 A A A r ARR UREA EE 54 Zooni the CELL TING riel 32 ZOOM Le IS IM di 24
24. most images are well focused Some cell samples are more demanding and need to be adjusted manually Check Manual in the Software Focus side window Figure 57 The text box next to the Manual button shows the software focus distance in mm The distance can be changed either by entering a value manually or by using the slide bar beneath the text box To determine which focal distance that will result in a well focused image is offen a matter of trial and error To find a starting value choose an image that was captured at the same time and that is well focused and note the focal distance of that image Highlight the image that needs to be recalculated Select Manual in the Software Focus side window and enter the focal distance of the well focused image in the text box Click Update If the image focus needs to be improved enter a new focal distance and click Update Continue until the image is well focused Use the arrow button to apply the update to either the current frame or to all checked frames Software focus Automatic Manual Update y More I Figure 58 The Calculation Settings side window set to automatic 4 7 1 Recalculate the focus automatically To replace the manual changes with the original computer focus Select Automatic in the Software Focus side window Figure 58 and then click the Update button If the result is a well focused image use the arrow button to apply the up
25. motorized stage samples are directed to move using the computer Figure 2 The manual XY stage Figure 3 The HoloMonitor with a motorized XYZ Stage Introduction to HoloStudio HoloStudio is a specially designed software for capture and analysis of digital holographic images with the HoloMonitor M4 HoloStudio enables the user to Capture images both as single captures and as time lapse captures The procedure is simple the user captures images of the sample using the HoloMonitor M4 and the image is analyzed in the software HoloStudio The imaging procedure does not affect the cells in any measurable way To simply count cells takes approximately one minute Confluence measurements are performed simultaneously To analyze the cells for area thickness or other parameters either the built in analysis functions can be used or the data can be exported as xml files for further work in Excel or similar programs Cell movement and morphology can be traced over time through a timelapse sequence for both individual cells and for a cell population The raw data will remain intact as all changes made by the user only concern how the results are displayed The changes do not affect the raw data Applications Some applications have been described in application notes such as cell motility cell death cell cycle and toxicology The application notes are available on our webpage http www phiab se publicat
26. range of the image This button needs to be operated every time the image coloring is off Add color Change color A new color can be added to the colorset by oler clicking the plus button Figure 31 and select a new color A colored triangle representing the new color will appear beneath the histogram Figure 30 Alternatively right click on the X axis and select Add Color Save colorset As Delete colorset Apply to k Figure 31 Coloring side window functions Change the color by using the right mouse button to click on a colored triangle beneath the histogram and select a new color alternatively left click the arrow button and select Change Color Figures 30 and 31 To change the color span left click and move the desired colored triangle beneath the histogram using the cursor Figure 30 To save a new colorset with the current color settings left click the arrow button and choose Save As Figure 31 To save the current color settings to a previously saved colorset left click the arrow button and choose Save Figure 31 Note that this will overwrite the settings previously saved to this colorset To delete a colorset select it in the Colorset list by left clicking it Then left click the arrow button and select Delete Colorset Figure 31 Colonng Colorset 8 89 15 67 20 96 R E MOA Figure 30 The Coloring side window 26 Chapter 3 Capture images Note that only holograph
27. stage Then put the cell sample on the stage An image will appear in the Main Viewing window If the image is correctly focused the slide bar in the Software Focus side window will be in the green area Figure 17 The thicker the plate the further away from the objective the sample will be Essentially the image is focused if the correct distance plate is used If the image is out of focus try a different distance plate or a different combination of plates until the software focus is in the middle of the green 2 3 Focus a live holographic image using an M4 with a manual XY stage The manual XY stage is delivered with holders for standard cell culture vessels If the correct holder is selected and then the vessel is placed on the stage the cells will be in focus 2 4 Focus a live holographic image using an M4 with a motorized XYZ stage The motorized XYZ stage is delivered with holders for standard cell culture vessels 23 2 4 1 Focus the image semi automatically Place the sample on the stage Wait for the image to stabilize Make sure that Automatic is activated in the Software Focus side window Figure 17 Adjust the hardware focus using the Microscope Settings side window Figure 23 which is found to the right of the Main Viewing window Figure 5 To the left in the Microscope Settings side window there is an objective representation and a black bar that represents the sample stage Figure 23 The hardware f
28. the pixel with the highest measured thickness in the cell For healthy normal cells this usually corresoonds to nucleus and nucleoli Peak pos Y pixel Position Y in image for the pixel with the highest measured thickness in the cell For healthy normal cells this usually corresoonds to nucleus and nucleoli Perimeter length um The length of the perimeter around the cell The calculations are based on a threshold setting that distinguishes background from cell Perimeter length pixel The length of the perimeter around the cell The calculations are based on a threshold setting that distinguishes background from cell Phaseshift The shift of the light caused by the cells when the light passes through Phase shift is the original parameter that is measured with digital holography The different ways to look at the Phaseshift may reveal different effects of cell treatments Phaseshiff avg The average measured phaseshift within the cell area Phaseshiff max The maximum measured phaseshift within the cell area Phaseshiff min The minimum measured phaseshift within the cell area Phaseshift std dev Standard deviation of the measured phaseshift within the cell area Phaseshiff sum The sum of the measured phaseshift within the cell area Roughness is calculated by subtracting a mathematically smoothed image from the actual image in each pixel and gives an indication of the smoothness roughness of the cell The ca
29. to the right of the Source Frames list Figures 101 and 103 X Remove X Remove all al Figure 103 The Remove from Plot buttons 7 3 Display results in scatter plot The cell morphology data are first represented as a scatter plot with the segmented cell images displayed beside the plot Figure Fel Det gick inte att hitta referenskdllan The data point for each cell is represented as a colored box If several frames are represented in the plot the data points from all images will be displayed in the plot simultaneously while the cell image window shows the frame that is highlighted in the Source Frames tab The data points will be displayed in the plot with the color shown in the color box in the Source Frames tab Figure 105 which is found below the Scatter plot tab To change the display color left click the color box for the added frame and select a different color Histogram 908 4 734 5 560 6 E 3 Pi gt lt 386 8 212 9 39 0 0 88 2 26 3 63 5 01 6 39 Thickness avg um Frame 10 in Lapsus lt 9 Identify Save Center X axis feature Thickness avg um ES Y Auto scale Y Show grid lines 5 Only current frame f Define region Save plot Y axis feature Area m Je 3902 Y Show labels Figure 104 Results displayed in a scatter plot shown with the corresponding image frame 5 e Pa IO eo y ME E
30. up of the entire database seessoooossssssseosseso 57 11 Troublesh otil2s s sssssss sssososstvss vssass ssdesdsdenssess rsnsesssssnskessed 59 11 1 Live IMACiNO sss ssss ovnss soss s ssvossossosassnes ssnsnonssssssssssses 59 11 1 1 The Live Capture tab is iNACtlv ssserrerssrrerrrrnn 59 11 1 2 It is impossible to focus the live image 59 11 1 3 The cells are very bright and blurry showing no MEF SUP UCLUT OS a RANKAS 59 11 1 4 The cell image is completely Whit 59 Tel 5 The Cell image is Dll aa 59 11 1 6 The live image focus was OK but it slowly turned bad and now it can not be set AZAN sserrrrrrrrrrrrrrrrrrrr rn so 60 TZ CAUCA 60 11 2 1 The cell image in the Main Viewing window is WIEN 60 11 2 2 The capture button is INAC IVE oonnnnnnnnnnnnnnnnnnncos 60 11 2 3 In a series of captured images not all images were AN 60 11 35 View Mat CS estoica iia censos 60 11 3 1 No image frames are visible in the Image Frame LSE WNUOW aiia a isae 60 11 3 2 The cell image in the Main Viewing window is WII Cd AA TT ANA 60 11 4 Cell IdentifiC tiOm s sssssssssossscsssssssovssuvoskovessvosssonssonsnss 60 11 4 1 No image frames are visible in the Image Frame FAST US N 60 11 4 2 The cell image in the Main Viewing window is WINE AAA SA RSA RAA odio 60 11 4 3 The automatic cell identification looks strange 60 11 4 4 Some cells are incorrectly segmented as two or MO
31. view other morphological parameters select a different feature in the cell features list which is found below the diagram Export tracking data Select the Tracking tab and click export to create an xml file containing all the tracking data The cell tracking image containing the tracks can be saved by using the snapshot button in the Tracking tab The spatial tracking diagrams can be exported by using the Export Plot button in the Plot Movement tab Quick guide Data backup 9 Backup of individual projects groups or frames Click Database in the top menu Go to Export When the browser window opens create a new folder at the selected destination Select the projects images or groups that you want to export back up Click Export Backup of the entire database folder Click Database in the top menu Click Settings Determine the Root Directory for the database folder HstudioimageDB This is a road map to find the folder Go to HstudioimageDB 10 Copy the entire HstudioimageDB folder 19 Manual PART TWO A user guide Chapter 1 Start up and Close down 1 1 Start up at room temperature e Switch on the laser and the instrument It needs 15 minutes of pre warming before the laser is stable e Start the computer e Open HoloStudio If you want to work with HoloStudio without using the instrument e Start the computer e Open HoloStudio When HoloStudio is not connected to an instrument the L
32. 0 13 18 Count 36 Confl 13 Take 66 57 256 41 15 70 Hologram x20 E SAIN AT AC OA390 1410 Hologram Phase contrast Only checked 70 frames Auto Scroll Figure 48 The Auto Scroll button It is possible to select Check frames with comment as an alternative Figure 47 That allows you to search for a comment such as the particular location XY coordinate that was saved with the image Figure 49 This will check every X frame in the group starting from the selected frame Enter a value for X PO Ok Cancel Figure 49 The Check Frames window 4 3 2 Capture pattern timelapse stored in individual groups For each position in the multi position timelapse images were stored in a separate group Go to the group of interest Highlight the first frame in the Image Frame List side window Below the Image Frame list there is an Auto Scroll button Figure 48 Click that button to show the image frames as a movie 4 4 Move flip or zoom the cell image To zoom the cell image click the image in the Main Viewing window and then use the scroll button on the mouse To move the cell image to a desired location in the Main Viewing window click hold and drag the image using the left mouse button To flip and move a holographic 3 D image click hold and drag the image using the right mouse button on the image 4 5 Holographic image display All holographic images will basically appear
33. 7 Additional functions found in the Coloring side window 9 3 Create an AVI movie When the set of images look good click the Use For All buttons in both the Perspective side window and the Coloring side window Figures 143 and 146 Preview the movie by clicking the preview button Play Figure 148 The number of slides per second are not shown as in the finished movie but rather at a set speed Preview gt Play HH Pause Figure 148 The Preview side window If the preview looks good click the Export Movie button in the Export window Figure 149 Export 3 frames added Export images Export movie Figure 149 The Export window Clicking the Export Movie button will open an export window Figure 150 Select the destination folder by browsing By moving the slide bar it is possible to set the number of frames per second that will be shown in the AVI movie Settings Destination fie roms Ms as as Tae a 5 0 frames per second Y Use compression Figure 150 The Export Movie side window 9 4 Export images When the image has been set up nicely and looks good click the Export Images button to open an export window Figure 151 Select the destination folder by browsing Check the box to add frame comments to the file name The Image format can be selected in the drop menu When the box for Use Plain Image Images is checked no coloring or 3 D will be displayed in the exported
34. 7 3 4 Create hide and delete plot reZiONsS 47 7 4 Display results in histOgGraMs ccccssccccsesccesssees 48 7 4 1 Change the histogram axis UNISs sssrrrrrereerrennnr 49 7 4 2 Change the histogram X axis intervals manually 49 7 4 3 Change the number OPfDINS ooooooonnnnccccccoconnnnnnnnnos 49 7 5 Export plots and cell data sssssssssccsssccessscceesees 49 EDAD OLPC CU AGILE asics ta 49 7 5 2 Save plots and histograms 50 AN AAA PPP son nns sens ess E E 51 8 1 COUNETCIS s2see0sveses rers nssessssesosbasoskereneneresserensssnesstedesernds 51 8 2 Adjust the histogram proportions ccccccsseeeees 52 8 3 Remove data from plot sscccccccccrssssssssssssceesscees 52 8 4 Export TeSUltS ssssssvnssocs sssvsssssssossevssnsrn ssssssnsssessbssnss sese 52 9 Export images and MOVIES csssscsssccccccccsessceesccsessceescees 53 9 1 Add and remove image frames ccccccsscsssscesssseees 53 OZ Nit CT AR a sne bedrar dee 53 9 2 1 Zoom move or flip the iMAZC ssmmrrrrrrrrrrrrrrrrrrrrr rna 54 9 2 2 Adjust the image disPlaY sssssssssrrrsrrerrrrrrrrrrr nn 54 9 2 3 Holographic image COLlOriM8 ooonnnnnnnnnnncnnionicinnno 39 9 3 Create an AVI I VIC 25ss oso sussebensorediossos ssssssspsndensdebosenke 55 DA Export Iman ui in e aaa oaei 56 10 B CK UD id 57 10 1 Back up of individual projects groups or frames 57 10 2 Back
35. Delete Figure 87 Set Location 6 3 4 Continue a discontinued cell tracking When a cell has been unset it will still be present in the Tracked Cells list but noted as not present In order to resume the tracking in a different frame select the cell in the Tracked Cells list and click the Set location button Figure 87 Then click the cell in the frame where the tracking should be resumed 42 6 3 5 Undo manual changes If the manual changes need to be undone start by selecting a cell Then click either the Undo for this Frame or the Undo for all Frames button Figure 85 When clicking Undo for this Frame the manual change in the current frame will be Undone and the tracking will be recalculated according to the change in settings When clicking Undo for all Frames the manual changes in all frames will be removed and the tracking will be recalculated according to the change in settings 6 4 Tracking cell morphology Once the cell movement has been tracked the cell morphology can also be followed over time by using the Plot Features tab Figure 88 Cell 5 DON EE 5 2200000 p ELLELLELS Jm H wn a E e index 11 2010 09 02 13 03 01 55 mir 29 PY e Cell feature Area um v Y Show value balloons Y Show guide line Y Auto scale 5 583 5 Save plot Figure 88 The Cell Morphology Tracking tab 6 4 1 Display cell morphology parameters for individual cells By c
36. E EEE TA E A E A SEEE enced eke E E TTET 36 manuale han tae 37 Mantal PART ONE c sd 11 Manual PART TW Oia at 20 Microscope SEMI tii aE 23 Mi orao N eeri enen eE bd pean tens ester 63 NUS e donna 63 Mini SER ta tt de o 36 Minimum OE cis 37 Modify Location DUNA a 42 Mota lt al 63 A a e o eco xiueetah lease steed 63 move he colima kati sinne tet N a 32 ns AAA A 54 move the live UMA CS e605 aid 24 A A A RA 32 54 O O LO 55 DO A a tee 27 A NNE RER ERS Wee asd in cated 2 OMO TES Wicca 21 Only Current Frame e 45 48 Optical path lenoth ave UD iia 63 Optical path length max UM ooooonnnnncnnnnnocononocononnnnnnnnnnnnnnnnnncnnos 63 Mo A caer tte seh eet E ease alata ade 38 A eneste steder 37 Ostia DIOCKS acetil d 37 Parallel time lapse Cap iii 14 Peak pos sie asset eae aie A 63 Peak DOS Y unida li 63 PREM ss a dia 39 Perine terden OU dais 63 PO tia 25 Phases EIE oaeee T Far ASIA SE LELE E 62 Phases A o a a ts enten EC CECT PN per 63 PhRaseshlll AV 0 naa asia 63 Phases MA ess lesa ee 64 Phases hii mi sn a el a le 64 Phases litt std do A 64 Phases hi SUN ennen eke eves sh eececra eMac ende 64 NS eeacaeh aca adaceensasceupemeasaaelaseesdans 47 plotarea manuals aio cds 47 plus DUC ON os aren RAN ih era RKA SER cates 55 POU CSW AMI a dde 20 A A inde died Re otalt red 37 AA ARR RS NI EN IO 55 o N 27 32 ReEDUO Nadar ld iii 26 33 55 AAA A et toe eee we 39 Recalculate a holographic IMage ooooonnnnnnnnccocococononnnnnconnnncnn
37. Group New Oooo _ Timelapse Capture pattern Settle time after stage movement S Selection New Delete Rename Delete Rename Clear pattern J Capture 0 images Figure 36 Activated Capture Pattern function J Setup storage a Figure 37 Capture Pattern window showing selected wells 3 4 1 Select the wells to be captured Click the Selection button Figure 36 This will make the Capture Pattern window appear displaying a representation of the currently selected type of cell culture vessel Figure 37 If necessary change the current type of vessel in the Stage Position window Figure 38 Even if the selected type of vessel contains only one well start by activating the Select Wells button Figure 39 and then click the wells In the example below Figure 40 the wells A2 and A3 will be captured but not Al and B1 3 as they are not selected 28 Stage position Vessels Nunc Multiwell6 hal Ibidi Microslide Nunc Multiwell6 Petri dishes 40mm X 55 500 mm Y 34 500 mm Remember Clear Figure 38 Change vessel type in the Stage position window Delete Rename _ Timelapse Capture pattern Settle time after stage movement S p Selection O Select wells Select positions Clear pattern Setup storage J _ Identical pattern in each well Generate 0 random positions in each well C
38. IIS 60 11 4 5 Two cells are segmented AS OM oooonnnnincccccnncnnccoso 60 11 5 ANALYZE Cal AAA TI 61 11 5 1 No image frames are visible in the Image Frame TAS WINA OW TEE EDISON 61 11 5 2 The cell image in the Main Viewing window is WUC ia bina yam A ANNAN SINE ERE NERE 61 11 5 3 The dots in the dot plot disappeared 61 ILG Cell Track mG ainiin aeaaeai a i 6l 11 6 1 The tracks are very irregular oooooocnnccinininninnncos 61 11 7 Export images and MOVIECG cssssscccccssssssssssees 61 11 7 1 No image frames are visible in the Image Frame O et ee a Rae ROOS 61 11 7 2 The cell image in the Main Viewing window is A stom E E ven Soames hones E 61 Addendum A Morphological parametere cccccsscssssseees 62 Holographic microscopy ccccccccssssssssssssccsssccssscoesscoees 62 Holographic teChnOlOQ W ssssssssrrrrrrrrrrrrrrrerrrerrrrrrrrnnnn 62 Phase SN fl skies six A 62 Threshold sel MoS 2 65 tase ddess tesa tendons denne bes bris 62 TN OLE eiaa andes A loser 62 Cell Morphology Parameters ssccccccccrssssssscrsssccesscees 63 1 a 4 Lu APR NN es Nerd A O sees seserdrnsNsnsk des rede 64 The Software Manual In the first part of the manual there are quick guides to the most common procedures such as cell counting timelapse captures and cell tracking In the second part of the manual there are detailed guides of how to perform different procedures using HoloStudio
39. Move the slide bar until the white rectangle is in the middle of the green area Thereafter the hardware focus has to be adjusted In the Microscope settings side window there is an image to the left with an objective representation and a black bar that represents the sample stage Figure 23 The focus distance can be adjusted in large steps by left clicking holding and moving the black bar with the mouse cursor This allows the stage to move up and down The distance between the objective and the sample is shown in the text box Software focus Figure 24 The Software Focus side window The double gray wheel with red knobs in the Microscope settings side window is used for intermediate outer wheel and small inner wheel focus adjustment steps The wheels are moved by left clicking holding and moving the red knobs As the computer updates the image with small intervals it is recommended to await the results of one change before making further focus changes Keep adjusting the hardware focal distance until the cells are in focus Calibration Calibrate background Cell refractive index 1 38 Medium refractive index Figure 25 The Calibration side window 2 5 Improve calibrate the holographic image If the quality of the focused live image is not adequate it can be calibrated in two different ways 1 Recalibrate only the image background Click the Calibrate Background button in the Calibration side windo
40. OO o dei E 39 63 E pOh e e 49 EXPO rss nen endre ie eee are 43 Export TINGS S faser aan pert cae sa tea ea ANA NEN STARS ER 9 56 Export Movie td ltda S6 A A EL SER dee ate ooh 25 flip and move a holographic 3 D IMage6 oooooooonooononaaoaconanannncnnns 32 ip and movethe DIM e aid 54 flip and move the live 3 DIMA ii brass run sn 25 TOC AGI ANC Gatien o eii 34 POCUS ACT A a EE E CEEE PAPERA NAS 23 focus dislance assin ti 23 focused mantal y sore feck A heed ATR 23 Generale DUO dd dai 29 A te cc cee ea ae a et elec ie eee et 25 32 CHOU ie sce OE alte es Sc ech sees Ma sal ae dee ees AA Digco2 TOW OC SIAR nesie leo co OS 37 Nard Wate foCUS tiara di ba 231 A A A A 32 histogram data display COIOL sssssesssssserssrsrrrrrrrrrrrrrrrrrrrr rr rr rr 48 AAA II ANSER AN RRRERRIRNT 48 A A E Po nee REAR 23 holoeraphic Image Col siii din 3 Holographic techno d y soosse aaae 62 Hall convex er a 63 Identical Patterns in Each Well ccccoonnnnnnnnonononacononoconnnos 28 Identity A A A RR 9 dental ta ia beds 36 113 8 75 424 EL A A RER NEN 33 Mare MAC a 53 Image Frame lst WII Wii a aai 45 Image Frame List Wind Wo 38 image frame presentation liSt ooononnnnnnnnnncccnncconcnononononnnnnononos 31 a A A IR 39 63 Toin Nearby Ma irc as 37 A SS TEA 23 A 32 54 A A II ee 23 PAS AI Te COM A A 47 Ive Capture aD Mii lala io dais ener eins 22 Mamta bS dd iatrossnrensnuecolescn tnebensensbeenorkiaariadeiledes 9 AE TT
41. ab Acquire the cell numbers for one vessel Highlight or check the image frames you want to include in the plot Click the Add buttons which are found in the lower right corner of the window to add the data from the highlighted or checked image frames to the cell count images list The cell count results are given as Nbr of cells in vessel and the confluence results are given as in the Cell Count Report in the main window Type the vessel growth area in the text box below the Cell Count Report window and choose the unit from the scroll bar to obtain a correct cell count area or a Vessel media volume for a correct cell count volume Export and save the Cell Count Report by clicking the Save Report button Acquire cell numbers for several vessels at several time points Repeat points 6 11 and 12 22 for each cell culture vial and for each measuring time point To compare data from different time points note the cell number and standard deviation values found in the Cell count report in an Excel sheet Quick guide Morphology analysis When the Startup Make sure that the placed in an incubator for at least three hours instrument has been while switched on If the instrument is to be operated at room temperature start the HoloMonitor 30 minutes prior to use Start the computer Start HoloStudio Image capture Go to the Live Capture tab Choose create a project and a group Put the cel
42. anded by clicking the black arrow tip found in every side window header Additional functions or parameters are found in the More menus in some of the side windows These functions are usually not needed for the user but rather for the service engineers Information concerning the different side windows can be found by clicking the Information buttons which are placed in the side window headers Figure 6 Main tabs E Hstudio AS TI FM ive Live Capture 2o Identify cells A Track cells la Analyze data Cell count aa Export images Capture Preset O Holographic x20 N 225 4 um Project New Delete Rename 2012 10 12 10 08 Deierborg2 N 2 Viewer options se Fo Y Group New Delete Rename ge morania ope Uncut Rotate 2 Laser pattern V Show ruler Timelapse 9 Hologram Y Show color bar a E Light effect 9 Phase Background MN Amplitude Colorsetlyulcano NM 8 89 15 67 20 96 0 00 um v a Software focus 9 Automatic Manual w More w Camera properties E O DA Figure 5 Overview of a main tab Figure 6 Information button 10 Manual PART ONE Quick guides to commonly used procedures Quick guides index Start up and Calibrate the instrument occccccccncncnnnnnnnnnnnnnnnnnnnno 12 Time lapse capture and EXPO A A ee 13 Parallel time lapse cap lUTES 3 ate slates os eel ee i
43. apture 21 images Figure 39 Activated Selection function 3 4 2 Select positions to be captured Activate the Select Positions button Figure 39 to select the capture points Select the capture points by clicking in the well representations The selected points will be shown as red dots Figure 40 3 4 3 Create identical capture patterns in all wells To create identical capture patterns in each of several wells check the box for Identical Patterns in Each Well Figure 39 Now every added capture point will appear in all selected wells Figure 40 Selected wells each displaying a different capture pattern 3 4 4 Create random capture patterns To generate random capture patterns first fill in the number of capture points in the text box and then click the Generate button Figure 39 Identical Patterns in Each well and the generation of random capture points can be combined to generate random patterns that are identical in the selected wells 3 4 5 Create patterns using the current position Use the Remember button in the Stage Position side window Figure 41 to add a capture point located at the current stage position The current stage position in X Y as well as Focus is remembered in the Capture pattern The remembered locations are shown as red dots in the image and if clicked the stage returns to that position Stage position Vessels Microslide m X 16 469 mm Y 16 167 mm Remembe
44. ar second moment Texture entropy Entropy is a statistical measure of randomness that can be used to characterize the texture of the cell image Texture homogenity Measures how many different gray levels the image holds Texture maxprobability High values occur if one combination of pixels dominates the pixel pairs in the window Thickness avg is the average thickness of the cell um The calculations are based on the phase shift the wavelength of the light and the refractive index of the cell Thickness max is the maximum optical thickness of the cell um The calculations are based on the phase shift the wavelength of the light and the refractive index of the cell Volume is the optical cell volume um 3 It is based on Area and Thickness References Dunn and Richards Kortum 1996 Three Dimensional Computation of Light Scattering From Cells IEEE J Selected Topics Quant Elec Vol 2 898 905 Farina and Verkman 1996 Cell Volume and Plasma Membrane Osmotic Water Permeability in Epithelial Cell Layers Measured by Interferometry Biophysical J Vol 71 3511 3522 Haralick et al 1973 Textural Features for Image Classification IEEE Transactions on Systems Man and Cybernetics Vol SMC 3 610 621 M lder et al 2008 Non invasive label free cell counting and quantitative analysis of adherent cells using digital holography J of Microscopy 232 240 247 Rappaz et al 2005 Measurement of the integral r
45. ard drive Figures 157 and 158 Note that the root directory is different for different computers Copy the entire HstudioimageDB folder In order to access the data in the folder HoloStudio must be directed to use the copied data base ds gt Dator OS C PHI Figure 157 The pathway to the PHI folder ey Bin 2013 12 09 10 28 Filmapp de Defaults 2013 07 10 11 34 Filmapp de Doc 2013 07 10 12 09 Filmapp de Drivers 2013 07 10 11 34 Filmapp 2013 07 10 12 10 Filmapp 2013 07 10 12 19 Filmapp Tf 2013 07 17 10 39 Filmapp A PHILibs_2 6a02 2013 11 18 11 01 Filmapp ey PHILibs_2 6a04 2013 12 09 10 34 Filmapp de Settings 2014 01 29 11 42 Filmapp m VesselContainers 2013 07 10 12 09 Filmapp Figure 158 The PHI folder that contains the HstudioimageDB database Chapter 11 Troubleshooting 11 1 Live Imaging 11 1 1 The Live Capture tab is inactive Why The software cannot find the HoloMonitor Fix Check that the USB and power cables are properly connected Try restarting HoloStudio Why The drivers are corrupt Fix Start the Windows Device Manager and check for any drivers with exclamation marks Try disconnecting the USB cable from the instrument and connect it again and wait a couple of minutes If the problem persists try to run the installation program again The driver files are located under C PHI Drivers as subfolders If a driver has an exclamation mark shown in device manager it may be possible to re i
46. as OK but it slowly turned bad and now it can not be set again Why There is probably a drop of water hanging from the top lid of the cell culture vessel As the drop grows larger it will increasingly disturb the image focus Fix Gently tilt the cell culture vessel and make the drop Slide to the side of the vessel 11 2 Capture 11 2 1 The cell image in the Main Viewing window is white Why The image dynamics are not optimal Fix Click the R button in the Histogram side window Save by clicking the arrow button and then Apply To and then Frame 11 2 2 The capture button is inactive Why The capture button is inactive when no project and group has been selected The program then does not know where to store the captured images Fix Select or create a Project and a Group 11 2 3 In a series of captured images not all images were good Why There might have been a scratch or a smudge on the vessel where the bad images were captured Fix Capture extra images and then discard the bad ones Why Floating cells disturbed the automatic software focus Fix Follow the instructions in section 4 5 to recalculate the software focus 11 3 View Images 11 3 1 No image frames are visible in the Image Frame List window Why No Project and Group has been selected Fix Select a Project and a Group Why If the Only Checked box is checked only checked images will be displayed If no image frames in the current Group h
47. as been checked no frames will 60 be visible Fix Uncheck the Only Checked box 11 3 2 The cell image in the Main Viewing window is white Why The image dynamics are not optimal Fix Click the R button in the Histogram side window 11 4 Cell Identification 11 4 1 No image frames are visible in the Image Frame List window Why No Project and Group has been selected Fix Select a Project and a Group Why If the Only Checked box is checked only checked images will be displayed If no image frames in the current Group has been checked no frames will be visible Fix Uncheck the Only Checked box 11 4 2 The cell image in the Main Viewing window is white Why The image dynamics is not optimal Fix Click the R button in the Histogram side window 11 4 3 The automatic cell identification looks strange Why The chosen threshold setting method might not suit the cells Fix Try a different threshold setting method Why If the cells grow at a very high density the threshold setting methods are not able to identify individual cells If cells are growing very closely they cannot be separated out Fix Adjust the threshold setting using the Adjustment slide bar Make sure that only the background and not any cells are defined as background Then use the confluency or the cell dry mass parameter to determine the amount of cells instead of the cell number Why There is background noise in the image that causes
48. changes in settings and display has caused the dots to be outside the range of the plot values Fix Set the plot area to Automatic and click the Reset View button 11 6 Cell Tracking 11 6 1 The tracks are very irregular Why The cell segmentation is not properly done Fix Go to the Identify Cells tab and ascertain that the segmentation is properly performed 11 7 Export images and movies 11 7 1 No image frames are visible in the Image Frame List window Why No Project and Group has been selected Fix Select a Project and a Group Why If the Only Checked box is checked only checked images will be displayed If no image frames in the current Group has been checked no frames will be visible Fix Uncheck the Only Checked box 61 11 7 2 The cell image in the Main Viewing window is white Why The image dynamics is not optimal Fix Click the R button in the Histogram side window Addendum A Morphological parameters Holographic microscopy Holographic technology HoloMonitor creates label free cell images by dividing red laser light into a reference and an object beam As the object beam passes through the sample a phase delay is imprinted on the light beam By subsequently merging the sample and the reference beam an interference pattern is created The interference pattern is the hologram It is recorded by a sensor and then used to numerically reconstruct a so called phase image which is displayed and
49. d folder In order to access the data in the folder the data must be imported into the software again using Database Import Destination C Users kersti Desktop Ny mapp 2 Browse Export Select what to export gt E MY MCF 10A time lapse 2012 06 07 4 WD Deierborg2 2012 10 12 El Y Birgits Fest 2012 10 12 Je K penhamn vatten 2013 04 03 E NRK52e 2013 04 19 EJ MY K 2013 04 22 H test 2013 08 23 j t El IR h9 iwr treatment day6 2013 09 03 gt gt gt jel rameens and giannis cells 2013 09 03 GMM Angelika 2013 09 16 GW du 2013 09 30 GRY ethan 2013 10 02 GRD basel 2013 11 06 le 0 projects 0 groups 0 frames selected Select all Select none Close Figure 155 Select projects groups or images for export 10 2 Back up of the entire database First locate the database Click Database in the top menu Figure 152 Then click Settings Determine the Root Directory for the database folder HstudioimageDB Figure 156 My Image Database Root directory C PHI HstudiolmageDB Number of projects 15 Number of groups 29 Number of frames 2089 Storage size 9 7 GB 18 7 GB free Change location Clear Figure 156 Database settings information containing the root directory of HstudioimageDB Find HstudioimageDB on the computer h
50. date to either the current frame or to all checked frames Figure 59 Example of an incorrectly calibrated Figure 61 An example of a recalibrated image image 4 7 3 Recalibrate a holographic image If a captured or imported image is not correctly centered due to aberrant calibration of the instrument the image will look strange Figure 59 lt can be re calibrated Clicking the Recalibrate button Figure 60 in the Software Focus side window will result in a re centered image with an adjusted focal distance Figure 61 4 7 4 Using a background hologram The images are noise improved by using a background hologram to subtract noise from the image If the background hologram is not correctly set it might instead disturb the image calculations There is an option not to use the background hologram Figure 60 software focus I Automatic Manual 1 444 Update Y Relative center X 0 264 Relative center Y Max size focal length um Min size focal length um Recalibrate Use background hologram Figure 60 The More list with the Recalibration button and the Use Background Hologram function in the Calculation settings side window 35 Chapter 5 Cell identification The base for all image analysis is the cell identification The software has already made a segmentation suggestion which might be very good but it might also need adjustments The cell number and confluence of each image is immediately giv
51. dow click hold and drag the image using the left mouse button To flip and move the live 3 D image click hold and drag the image using the right mouse button 2 8 Change the holographic image display To change how the live holographic image is displayed open the Viewer Options side window Figure 27 on the left hand side of the Main Viewing window Figure 5 Viewer options 0 FFT 3D Uncut ET Rotate Y Show ruler Show color bar E Light effect Background IN Laser pattern Hologram 0 Phase Amplitude Figure 27 The Viewer Options window By clicking the Center button Figure 28 the image will be centered in the Main Viewing window E Figure 28 The Center button By pressing the Snapshot button an image of the current view will be saved Figure 29 o Figure 29 The Snapshot button 25 Different image display options are shown Figure 27 They can be activated by checking the boxes Some boxes can be checked in parallel thus making it possible to combine functions e Checking FFI Figure 27 displays the Fast Fourier Transform which represents the frequency domains e Checking Uncut displays the image as it is first reconstructed e Checking Laser Pattern displays the original interference pattern resulting from the merging of the object and reference laser beams e Checking Hologram displays the reconstructed image which is based on the laser pattern The h
52. e Why The instrument was not switched on for 30 minutes before use at room temperature or put in an incubator at least three hours before use If the instrument is warming up the focus will change all the time and the focus settings will need to be adjusted continuously Fix Allow the instrument the warming up period Why Something causes the instrument to vibrate Fix Check that the cables connected to the M4 are slack and not touching anything Ensure that no vibrations come from sources like centrifuges or hard working air vents 11 1 3 The cells are very bright and blurry showing no inner structures Why The image dynamics are not optimally set Fix Press the R button by the histogram in the Coloring side window 11 1 4 The cell image is completely white Why The image dynamics are not optimally set Fix Press the R button by the histogram in the Coloring side window 11 1 5 The cell image is black Why The laser unit is not switched on or connected properly Fix Start by checking that the laser unit is connected properly both to power and to the instrument If the laser is connected properly and the image still is black please contact instrument service Why The images are either zoomed in or out to much Fix Use the mouse scroll to scroll back Why The image dynamics are not optimally set Fix Press the R button by the histogram Histogram side window in the 11 1 6 The live image focus w
53. e captured images look good Proceed to the Identify Cells tab Identify cells Adjust the cell identification in the first image frame using the Adjustments and Manual Changes tabs Make sure all your images are checked in the Image Frame list on the right side Click the Apply checked button to the bottom right of the program in order to apply the cell identification adjustments on all the captured images Alternatively click Apply All Quickly preview the rest of the images to make sure they are all well segmented Adjust the segmentation as needed Proceed to the Track Cells tab Track cells through the timelapse Click the button for New Analysis Highlight or check the image frames you want 18 22 23 24 2i 26 27 28 29 to include in the tracking Click the Add buttons in the lower right corner of the program to add the data from the highlighted or checked image frames Activate the Add Cells function in the Select Mode side window Add cells to be tracked by clicking them Follow the tracking by using the Timeline which is found below the Cell tracking window Spatial tracking Select the Plot Movement tab which is found behind the Tracking tab The directions of the cell movements are given in a diagram Morphological tracking Select the plot Features tab which is found behind the Tracking and Plot Movements tabs The Area over time is given as a default To
54. e objective The software focus describes how well the computer is able to calculate an image based on the light information that reaches the sensor Put the cell sample on the stage An image will appear in the Main Viewing window lf the image is correctly focused the green bar in the Software Focus side window will be in the green area Figure 17 Software focus Automatic J Manual 1299 Figure 17 The Software Focus side window Figure 20 A holographic phase image that is out of focus 7 FramelD 2924 Cell culture surface objective ag Figure 18 The suitable distance between the cell Figure 21 A holographic phase image th culture surface and the tip of the 20x objective of focus gt gt s atis totally out 22 2 1 1 Automatic versus manual focusing Automatic focusing mostly results in well focused images Some cell samples are more demanding and need to be focused manually Check Manual in the Software Focus side window Figure 22 Figure 22 The Software Focus side window The text box next to the Manual button shows the current software focus distance in mm That distance can be set to a different value either by entering a value manually or by using the colored slide bar beneath the text box Move the slide bar until the image looks good 2 2 Focus a live holographic image using an M4 with a standard sample stage Choose the correct spacer plate and put if on the
55. e procedure Fix 2 If the cell culture medium is replaced with cold PBS or cold cell culture medium immediately after taking the multi well plates and petri dishes from the incubator there will not be condensation Replace the PBS with medium again after the analysis Why The laser intensity is not optimal for the chosen cell sample Fix Make sure that the laser beam intensity is 59 adequate When auto exposure is on camera properties window in the left side window the exposure time should be less than 10 ms If the exposure time is long start by checking that the laser unit is connected properly both to power and to the instrument If the laser is connected properly and the exposure time still is very long please contact instrument service Why The objective and the laser window need cleaning Fix Clean with an ethanol soaked Q Tip and dry after with a dry Q tip htto www phiab se su cleaning movie ort contact use the Why If the cells are very thin or very sparsely distributed in the vessel the auto focus has difficulties to find focus Fix Set the software focus manually instead of automatically Activate the manual focus mode by clicking the Manual button in the Software Focus side window in the Live Capture tab Thereafter drag the slider to the green area in the focus bar and then adjust the hardware focus Thin cells have a very narrow focal distance and focusing has to be performed with great car
56. efine region Figure 111 The Define Region button 47 123 1 i a n Define new region Arbitrary region X axis region a Save as Image Y axis region a 7 ty Linear region ENT mu Figure 112 A list ld types In both cases a list of region types appears Figure 112 Select a region type by clicking it Then click in the plot where the region setting should begin For the linear region and the X and Y axis regions the second click in the plot determines where the region ends For the arbitrary region every click in the plot will add to the region Finish the region by clicking the starting point again The starting point will expand to indicate that the region is closed If clicking the region does not make it stick try clicking a little slower e X axis region where X axis values are the region cut off values Figure 113 e Y axis region where Y axis values are the region cut off values Figure 113 e Linear region where clicking in the diagram window results in straight lines that cut off the region from origin Figure 114 e Arbitrary region where repeated clicking in the diagram window results in an irregular figure that outlines the region To finish the region close it off by clicking in the starting point Figure 114 The regions are summarized in the Regions tab Figure 115 which is found below the Scatter plot tab Regions can be hidden by unchecking the Display
57. efractive index and dynamic cell morphometry of living cells with digital holographic microscopy Optics Express Vol 13 9361 9372 65 A RSA UNS E E NN ae 32 54 A arial OAN 25 A e reee cisacd 20 Adaptive gauss aian eane a 37 ACADEMICO dl lacs o ea Sal as 37 Add All os ses tee ase RENNIE SST escape ESSER SANN ERNA 53 AO DUM T atada dass dd dota cata 40 51 E A A A SNS BIN a Eden NEN 40 Add hoeke enron alii ar s 53 Add Highitohted tdi 53 Ad Or REMOVE orros E NN 37 A E 45 Adusien SIGS Da io 37 AJUSTES WIG O tocada 361 AMP A AA 25 Analyze Datatab oc ii N 44 APP adria 38 Apply ENE rec Ler 38 AP o lo ls 38 Arba TS GION ie 47 ATO eno 39 AEF ORE a a RENEE 63 Area UM 2 AAA II SARA ak hated ida 63 Auto apply CO St 39 Auto apply GANG CS rt dba 38 PNAC OTN AG O SRA one ts tices AA E 23 A IAE 47 Dac k A A A ON 57 Background CO issn rr 55 A 008 0 10121 19 3 arena a a RNA AAA NANNA a 49 Boxed Dread actions 63 Boxed breadth and Midland 63 Boxed Center POS A a 63 B xed Cener poSI ii 63 BORG UTEN to e ae EN 63 Capture DU a 27 CAPS PALM a lo ci tas RA OS 28 Capre patera as nats diia sssesscnsscis 30 Capture Pattern window ClOSeC sssssssssessesesresserrrrrsrsrn rr nr aa 29 capture patterns random ssssssssrerrserrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrr rr rr rr rna 29 CAD IRS WAI Were lores 27 cell core adjust the SIZS OTIS seissen Ne 37 AD O E R 9 CellC ount REPO ennei E tee teh caee 51 Cel C oint AD 07 rs a sd i
58. en beside the image in the Image Frame List window Figure 63 Note that the software will suggest a cell identification at the time of capture This automatic identification needs to be evaluated for each image A images IIA leony eens We Track cens Lx Analyze data Figure 62 The Identify Cells tab 5 1 Identify cells Choose the Identify Cells tab Figure 62 5 1 1 Select an image The Image Frame List side window Figure 63 is found to the right of the Main Viewing window Figure 5 Select the project and group where the images are saved Figure 63 Highlight the image of interest to make it appear in the Main Viewing Window ce Project 2012 06 07 11 48 Bem Delete Baname MCF 10A time lapse a Group New Delete Rename 2012 06 07 11 48 MCF 10A Medica rod 100 frames Ed Hologram x20 A 2012 06 07 11 48 05 ka Count 37 Confl 9 Take 3 2 62 2 62 Ed a Hologram x20 2012 06 07 11 48 10 Count 21 na a Figure 63 The Frame List side window 36 5 1 2 Automatic threshold settings The software will automatically make threshold settings according to the default segmentation method Figure 64 Figure 64 Identified cells There are several other methods to calculate the threshold settings in the Methods list in the Adjustments window Figure 65 which is found below the Main Viewing window Manua Auto Minimum Error
59. er plot can be changed by using the listed X and Y axis parameters found below the scatter plot Figure 108 For a full list of axis label parameters go to Addendum A 7 3 3 Change the scatter plot axis display manually The Plot Area settings Figure 109 are found below the Scatter plot The area of the scatter plot i e the values of the X and Y axis is set automatically to fit in all values from all added frames To set the plot area manually deactivate Auto scale by unchecking the box Figure 109 and then entering the desired X and Y minimum and maximum values Figure 110 7 Auto scale X D 826 Tof ie ie es ls y Pa a m o pm pm 2 O 13 JUL f Lil Figure 109 The Plot Area settings Eo E Figure 110 The Fixed Plot Area window Alternatively the plot area can be changed with the mouse scroll button Left click in the plot and scroll using the mouse scroll button To move the plot area in the scatter plot left click in the plot hold and drag 7 3 4 Create hide and delete plot regions The scatter plot diagram can be divided into regions to separate out data from certain cells or cell populations When clicking the right mouse button while hovering over the plot a menu will open When Define New Region is selected a region menu will appear Figure 112 Alternatively there is an Define Region button Figure 111 below the Scatter plot tab to the right of the Auto scale function D
60. from each frame is displayed when the corresponding image is highlighted in the Source Frames tab Figure 120 below the Scatter Plot tab El Only current frame Figure 119 The Only Current Frame button The data from each image frame will be represented in the histogram with the color shown in the color box in the Added Frames window To change the display color left click the color box beside the frame number in the Source Frames tab and select a different color Figure 121 Es Number Group Project Comment Me Lapsus Deierborg2 Take 7 2 245 2 245 Y Bo Lapsus Deierborg2 Take 9 2 245 2 245 I vi Lapsus Deierborg2 Take 17 2 245 2 245 mE Lapsus Deierborg2 Take 13 2 245 2 245 a Ml sa Lancue Naiarharn Taba 15 1 7 45 7 IAS Figure 120 The Source Frames tab MT m E nb m Grou p Dele Deie Deie Deie fel Mais Figure 121 Color options for histogram data display 7 4 1 Change the histogram axis units The X axis label of the histogram can be changed by using the Cell feature list below the Histogram Figure 122 A list of x axis label parameters are found i Addendum A Cell feature Area um Figure 122 The Histogram X axis label 7 4 2 Change the histogram X axis intervals manually The histogram X axis interval is set automatically Figure 123 to fit in all values from all added frames nge 39 3 1105 6
61. g There is a Timeline below the Cell Tracking window Figure 81 By moving the small gray slider the image frame sequence will be played through The tracks showing the cell movements movement will then be displayed Figure 82 Source Frames M Remove X Remove all Time index DateandTime Project Group Number Comment 1 2010 09 02 12 08 01 Deierborg2 Lapsus alla 8 Take 7 2 245 2 245 2 2010 09 02 12 13 01 Deierborg2 Lapsus alla 9 Take 8 2 245 2 245 3 2010 09 02 12 18 01 Deierborg2 Lapsus alla 10 Take 9 2 245 2 245 4 2010 09 02 12 23 01 Deierborg2 Lapsus alla 11 Take 10 2 245 2 245 Figure 78 The Source Frames window 40 Le JL 2 hrs 30 min Figure 81 Timeline for cell tracking Figure 82 Tracks showing cell movements The movements can also be seen in the Plot Movement tab which is found behind the cell tracking window 6 2 Cell tracking warnings When the cell tracking for some reason is deviating from the previous image the software will give warnings The warnings are listed in the Warnings tab Figure 83 which is found below the Timeline When the Show button is clicked Figure 83 a window opens which displays the previous current and next segmentation of the cell Figure 84 It is then possible to see if the warning is appropriate or if if can be ignored If the warning is caused by a normal cellular event such
62. g 1 The calculations are based on a threshold setting that distinguishes background from cell Hull convexity A measure on how much the 3D cell shape deviates from the perfect convex shape A cell is Usually rather smooth in 3D A higher value means less dips in cell thickness i e a more perfect shape Irregularity is a measure of how much the circumference of the cell deviates from the circumference of a perfect circle A value of O means the cell is circular and higher values mean a longer more irregular outline The calculations are based on a threshold setting that distinguishes background from cell Migration The shortest direct distance from the starting point to the end point um Migration speed The shortest direct distance from the starting point to the end point given per hour um h Motility The actual way traveled from the starting point to the end point um Motility speed The actual way traveled from the starting point to the end point given per hour um h Optical path length avg um How far the light has traveled through the cell in average for the entire cell The calculations are based on the phase shift of the light and the wavelength Phase shift P Wavelength Optical Path Length OPL P x A Optical path length max um How far the light has traveled through the cell as maximum for the cell The light path becomes longer when the cell rounds up Peak pos X pixel Position X in image for
63. hecking or unchecking the colored boxes displayed beside each cell name morphological parameters are shown for that cell Black always shows the average of the cell population Cell feature Area um o i y Figure 89 Switching cell features 6 4 2 Display different cell morphology parameters By using the Cell Feature function found below the diagram Figure 89 different morphological parameters can be displayed The list contains all parameters as well as distance and speed of movement For a full list of parameters go to Addendum A Track Export Figure 90 Track and Export buttons found to the right of the Cell Tracking window in the Tracking tab 6 5 Export the tracking results The raw data can be exported into an xml file by clicking the Export button Figure 104 which is found in the Tracking tab The xml file can be imported into e g Xcel The image currently displayed in the cell tracking window can be saved by clicking the Save button found below the Cell Tracking window Figure 91 which is found in the Tracking tab 2 Identify Save Center Figure 91 The Save button which is found below the Cell Tracking window 43 Chapter 7 Analyze and export data The main part of the cell data analysis is performed in the Analyze Data tab that contains tools for analyzing the identified cells A simple tool for measuring distances or objects in a currently viewed image is fo
64. ic phase representations can be captured If e g an FFT image is desired please use the snapshot button in the Viewer Options side window Figures 27 and 29 Figure 32 The Live Capture tab 3 1 Store captured images Select the Live Capture tab Figure 32 Before images can be captured a place of storage must be prepared The images must be stored in a Group within a Project Either open an existing project or create a new project in the Capture window Figure 33 Then open an existing group or create a new group where the images will be saved When a new project or group is created the date and time are automatically included in the name New Delete Rename 2015 04 28 13 17 Testar EX Group 2015 04 28 13 18 Control2 New Delete Rename _ Timelapse _ Capture pattern Capture 1 images Figure 33 The Capture window Capture ces Project PO F Group New Delete Rename New Delete Rename IS E _ Timelapse Capture pattern Capture 1 images Figure 34 The Capture window with an inactive Capture button 3 2 Capture a single image Put the cell sample on the stage A live image will appear in the Main Viewing window Ascertain that you are satisfied with the quality of the live image See Chapter 2 Click the Capture button The Capture button is inactive unless a project and a group have been selected Figure 34
65. ical cell thickness combined with the cell area is the base for several other morphological parameters They are described in the parameter list below Threshold settings The morphology calculations are also offen based on threshold settings that define what is cell and what is background The threshold is set by the computer and the user in collaboration Figure 161 Threshold settings yellow outline determine what is cell and what is background This step is important for both morphological determinations and cell tracking Note It is important to remember that the morphological data cannot be attributed to the inside or the surface of the cell but rather is the sum of both lf e g the roughness of the cell increases it is not possible to say whether it depends on changes of the cell surface or in the nucleus and cytoplasm as the phase shift for each pixel is given as the total for both the contents and the surface of the cell Cell morphology parameters Area um 2 is the surface area of the image that is occupied by the cell The calculations are based on a threshold setting that distinguishes background from cell Area pixel Surface area in image that the cell occupies The calculations are based on a threshold setting that distinguishes background from cell Boxed breadth and width In order to measure the length and the width of the cell a frame is fitted around the cell The frame that fits the cell wh
66. ight of the Main Viewing window Highlight the first frame in the Image Frame List side window Below the Image Frame list there is an Auto Scroll button Figure 48 Click that button to show the image frames as a movie 4 3 View one position of several in a timelapse 4 3 1 Capture pattern timelapse stored in single group When a timelapse has been captured at several different positions in parallel using the motorized stage and then stored in a single group it is possible to view the timelapse for each position separately The images in the Image Frame List will be arranged in order of capture If e g seven positions have been selected every seventh frame will belong to the timelapse of that position F lake 54 Check selected frames i Se Check all Delete Holograrr Uncheck selected frames a A 2012 07 punt 35 Uncheck all onfl 12 Take 457 Check every X frame Check f ith comment 1 z E aoga eck frames with com Ae l 2012 07 F igure 47 The Check function First highlight the first of the images at the chosen position Then right click in the Image Frame List select Check and thereafter Check every X frame Figure 47 In the above example 7 would then be entered in the Check Frames window Figure 49 After clicking OK check the Only Checked option found below the Image Frame List Figure 48 Now only the checked images will be displayed when Auto scrolling ES Hologram x20 2012 07 06 2
67. ile covering the smallest area is selected for measurements The parameter depends on the threshold setting that distinguishes background from cell Boxed breadth pixel Width of an area minimized aligned bounding box that encloses the cell Boxed breadth um Width of an area minimized aligned bounding box that encloses the cell Boxed center pos X pixel The geometric center position X of an area minimized aligned bounding box that encloses the cell Boxed center pos Y pixel The geometric center position Y of an area minimized aligned bounding box that encloses the cell Boxed length pixel Length of an area minimized aligned bounding box that encloses the cell Boxed length um Length of an area minimized aligned bounding box that encloses the cell Centroid The center of mass is the arithmetic mean of all points weighted by the thickness of the cell If a physical object has uniform density then its center of mass is the same as the centroid of its shape Usually but not always the mass center corresponds to the cell nucleus Centroid pos X pixel The X position of the mass center of the cell Centroid pos Y pixel The Y position of the mass center of the cell 63 Eccentricity is how elongated the cell is or how much the cell deviates from being a circle A value of O corresponds to a circle and the more elongated the cell is the higher the eccentricity value becomes approachin
68. image When the box for Transparent Background is checked the images will be exported without background S6 Settings Y Append frame comments to filename Image file format Bitmap bmp E Transparent background L Use plain image Bitmap bmp OF gif JPEG jpg PNG png TIFF tiff loring bounds Cancel Figure 151 The Export Images side window Chapter 10 Back up In order to make a safety back up of your data you need to either export or copy the database Fila View Database Figure 152 The Database menu Please select a destination directory Figure 153 The Browser window v lj mapp Browse for an empty folder to store the database export E Skrivbord gt a Bibliotek gt a Hemgrupp b JA kersti gt 1 Dator gt th N tverk gt Bi Kontrollpanelen Papperskorgen Skapa ny mapp igure 154 Create new folder 10 1 Back up of individual projects groups or frames Click Database in the top menu Figure 152 Then click Export A browser window will open Figure 153 Click Browse Create a new folder at the selected destination Figure 154 When a folder has been created a window displaying the groups and projects will appear Figure 155 Select the projects groups or images that you want to export back up Click Export Alla database information will now be copied to the create
69. in an incubator for at least three hours while switched on If the instrument is to be operated at room temperature start the HoloMonitor 30 minutes prior to use Start the computer Start HoloStudio M4 Image capture Open the Live Capture tab Put the cell sample on a spacer plate or vessel holder of the correct type Make sure the image is focused Choose capture positions in the sample s First select a suitable vessel in the Stage position box Find an interesting position for imaging Click on Remember in the Stage position box Repeat for as many positions as required Create a project and groups for the parallel timelapses First tick the Capture pattern checkbox in the Capture box Then click on Setup storage which will show the Configure destinations window Select a project and then tick the Multiple destination groups checkbox Click OK Press Capture Use the View Images tab to ensure that the captured imges correspond to your settings When image capture is complete proceed to the View Images tab Run through your images by clicking the Autoscroll button If needed recalculate the images Export individual images or a movie Go to the Export Images tab Highlight or check the image frames you want to include in the movie or image export Click the Add buttons in the lower right corner of the program to add the data from the highlighted or checked image frames Adjust the coloring
70. in gray scale and in 2 D unless artificial coloring has been chosen and the image display is set to 3 D Viewer options nis Ol Measure 3D IM Show ruler IF Rotate W Show color bar 2 Show image info E Light effect Shiny surface Background IM Figure 50 The Viewer Options side window for holographic images 4 5 1 Change the image display The holographic image display can be modulated using the Viewer Options window Figure 50 which is found to the left of the Main Viewing window By pressing the Center button Figure 51 the image will be centered in the Main Viewing window eS Figure 51 The Center button By pressing the Snapshot button Figure 52 an image of the current view will be saved lo Figure 52 The Snapshot button By checking the boxes different options can be activated Most boxes can be checked in parallel to combine options e Checking 3 D displays the holographic data as a 3 dimensional representation e Checking Rotate auto rotates the image Rotate is only active when 3 D has been checked e Checking Show Ruler displays a horizontal scale bar representative of the distance in X and Y e Checking Show Color bar displays a vertical scale bar representative of the height in Z e Checking Show Image Info displays additional information associated with the image such as Figure 53 o Project specifying in which project the image Is located o Group specifyi
71. ing oonnnnnnnnnnnnninninnc 40 6 2 Cell tracking WarningS cccccccccccsssssssscccessccsesces 41 6 3 Adjusting the cell tracking sssssssssssssssssssssrssresr 41 6 3 1 Select the cell to be aAdjust d ssssrrrssrrrrrrrrrrnna 41 6 3 2 Switch the tracking from one cell to another 42 6 3 3 Discontinue a cell tr CkiNlS ssssrrsrrrrrrrerrrrerrrrrr rna 42 6 3 4 Continue a discontinued cell tracking 42 6 3 5 Undo manual CHANGES sseerrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrn rna 42 6 4 Tracking cell MOrpholOQy cccccccsssccsssccssssccesscees 42 6 4 1 Display cell morphology parameters for individual A A NSA SAN 42 6 4 2 Display different cell morphology parameters 42 6 5 Export the tracking resullts scsssccsssscsssccceesees 43 7 Analyze and export dala viccoccocioniciconcionoci necacccnanacancncacici necas 44 7 1 Measure distances directly in the currently viewed IMA A de siden S NDE 44 7 2 Analyze results in plot cccssscccccssssssssccssscccescccesees 44 7 Did Start a NEW analysis ri iii 44 1 22 Remove data from plob ie 0 ans eee ees 45 7 3 Display results in scatter plOt cccccccssssssssssscccoseees 45 7 3 1 Identify data points as cells oooooooocnncccncccnnninnccnnnos 46 7 3 2 Change the scatter plot axis UNilS ooooonccnnnncccnnno 47 7 3 3 Change the scatter plot axis display manually 47
72. ions application notes Table of Contents Introduction to the HoloMonitorTM Ma4 ssscsssseeeees 5 Introduction to HoloStudioTM sccccccsssssssssesesees 5 AC ATIONS coiir eaaa aeaa aiia eae ieiet 5 Sotware Manuals ossessi eeoa asaite aaie Eens 8 HoloStudio M4 2 6 outline ssssssssssssssccscccccrroserrrorrrnnrrrsnnnrs 9 Main tabs OVErVICW sssssesssosssossssosspussusesesssssnssessssssssessssesness 9 AAA A Aer RA Asse ses 9 ANALYZE Data asnicar EAEE 9 Main Viewing WINDOW AAA kitin naaii sasina 9 SEAN AAA A oE sisa aen rS 9 Manual PART ONE QuickguidesS ssssssssssssssssssssosssrosrrersr 11 Start up and calibrate the instrument cccccccsseeeeeees 12 Time lapse Capture iiicccsctesssiceiscsacccseucseeesssensetsnessdsasdeasteniasas 13 Parallel timelapse Captures cccccccccsssssssssssccccssscoesscees 14 Cell counting and confluenCe sssssssssssssssssrsrsrsrsrrrnrrnr 15 Proliferation studies ccscsccsesccscccccssccssesccsesseesssscoseacesscsereessess 16 Morphology analysiS eeccoocossssscccoccssssseccceessecsssosesossessee 17 CON ra CKIN nrnna ai 18 Data DACKUPsc5ses rsodasssonsssevesesdensdsnsosssrsvsaserenssnsas sssensssskssesener 19 Manual PART TWO a user 2uide cccsscccccssssssssssssccceees 20 1 Start up and Close GOWN cccscccccccsssssssssssssccssscoesseoees 20 1 1 Start up at room temperature s
73. is usually more or less irregular and uneven This is a measure of how uneven the shape of the area is Texture is a measure of the variation of the cell 64 structure lt describes smoothness coarseness and regularity The texture of an imaged cell may differ depending on where the cell is in its life cycle or depending on different treatments The provided texture parameters are all different ways of presenting the texture The different ways to look at the texture may reveal different effects of cell treatments These texture features are reported in Haralick et al 1973 Texture clustershade Clustering is the process by which the natural groupings are determined such that the objects in each group exhibit more similarity to one another than to objects in other groups Cluster shade is a measure of skewness of the gray levels When the cluster shade is high the image is not symmetrical Texture contrast Measures the thickness contrast between a pixel and its neighbor Texture correlation Measures the joint probability occurrence of specified pixel pairs i e how likely it is that pixels with certain phase shifts occur together Texture correlation infol A different way of calculating the texture correlation Texture correlation info2 A different way of calculating the texture correlation Texture energy Provides the sum of squared elements in the gray level co occurrence matrix Also known as uniformity or the angul
74. ive Capture tab will be inactive 1 2 Start up in the incubator e Put the M4 instrument in the incubator and connect all cords The electricity must be connected when the instrument is in the incubator otherwise there will be a condensation problem e Wait 3 4 hours for the instrument temperature to stabilize e Switch on the laser lt needs 15 minutes of pre warming before the laser is stable e Start the computer e Open HoloStudio 4 Calibration L Please remove sample from the stage Continue ew Calibration is required periodically to ensure optimal performance of the instrument Please remove any sample and click continue 4 Calibrate background Close Figure 7 The Calibration Wizard 1 3 Calibration The first time the Live Capture tab is selected after starting the software with an instrument connected a 20 Calibration Wizard window will appear Figure 7 The wizard can also be accessed from the Help option in the Top Menu Figure 8 or from the Calibration side window Figure 9 When the instructions of the wizard are followed the instrument will be calibrated Do this every time the instrument is started or if it has been running for a long time E Hstudio Ma JS ag Calibration Wizard L Collect Diagnostics E About Hstudia l a ee SE Figure 8 The Calibration wizard in the top menu Calibration 32 Calibration Wizard Ca
75. l markers Show outline al aj a Show edge cells outline 5 3 Save the cell identification settings 9 View raw image All changes are saved automatically if the box for Fd Auto apply changes Auto apply Changes is checked This function is found in the Options side window to the left of the Main Viewing Window Figure 70 Figure 70 The Options side window If the box is not checked a warning will appear that the changes are not saved when the user switches from the Identify Cells tab to another main tab Figure 71 Add marker here Delete marker Grow or shrink Delete area Figure 68 The adjustments menu when clicking on a cell area Add marker here Figure 69 The adjustments menu when clicking outside identified cell areas Segmentation settings can be applied to a certain frame to checked frames or to all frames by using the Apply Current the Apply Checked or the Apply All buttons that are found below the Image Frame List Window Figure 72 Note that the changes performed using the Manual Changes window Figure 67 are applied only to individual cells These changes cannot be applied to other image frames 38 Save changes The segmentation for the current frame has been changed Do you wish to save the changes Figure 71 Warning message for changes that have not been saved No image Count 3 Confl Lal 7 No ima e i eam s LJ a n Ken alm F a i Count 3 C
76. l sample on a spacer plate or vessel holder of the correct type Select the correct vessel type in the vessel list in the stage position side window Ascertain that the live image looks well Press Capture Use the View Images tab to ensure that the captured images correspond to your settings Move the sample Press Capture Move the sample and capture again Continue capturing images until at least five images have been captured image capture is complete proceed to the View Images tab Make sure that the captured images look good Proceed to the Identify Cells tab Identify cells Adjust the cell identification in the first image frame using the Adjustments and Manual Changes tabs Make sure all your images are checked in the Image Frame List window on the right side Click the Apply checked button to the bottom right of the program in order to apply the cell identification adjustments on all the captured images Alternatively click apply All Quickly preview the rest of the images to make sure they are all well segmented Proceed to the Analyze data tab 17 20 21 22 23 24 ploy 26 27 Data analysis using HoloStudio Click New analysis to start a new project Highlight or check the image frames you want to include in the plot Click the Add buttons in the lower right corner of the program to add the data from the highlighted or checked image frames to the plot Create pl
77. lculations are based on the phase shift in each pixel of the thresholded cell A healthy cell has usually a low degree of roughness while a dying or dead cell usually has more roughness The roughness of cells may be differently distributed in the cell after different treatments The different ways to look at the Roughness may reveal different effects of cell treatments Roughness avg The average of the roughness distribution Roughness kurtosis How peaked the value distribution is A positive kurtosis distribution has a sharper peak and longer fatter tails e g a Student s t distribution while a negative kurtosis distribution has a more rounded peak and shorter thinner tails e g a Bernoulli distribution A neutral kurtosis distribution O corresponds to a normal distribution Roughness RMS The root mean square of the surface roughness This value is also known as the quadratic mean All the values are squared then averaged and thereafter the square root of the quadratic mean is presented Roughness skewness How symmetrically the roughness values are distributed around the mean A negative skew indicates that there are more values that are higher than the mean and a positive skew indicates that there are more values that are lower than the mean Zero skew indicates that the values are symmetrically distributed around the mean Shape convexity is a measure on how convex the outline of the cell is The area of a cell
78. librate background Cell refractive index 1 38 Medium refractive index Figure 9 The Calibration wizard in the Calibration side window 1 3 1 When everything Is well If the calibration is successful all parameters necessary to achieve good imaging are within their bounds A window will appear to inform about the successful calibration of objective and background Figure 10 Thereafter the Calibration Wizard will show the status of three important parameters Figure 11 Click the Close button to exit from the Calibration Wizard Values shown to be in the green are excellent and values in the yellow are acceptable Y Calibrated objective y Calibrated background we Calibration is now performed Please wait a few seconds Y Calibrate background Figure 10 The Calibration Wizard showing a successful calibration of objective and background Calibration finished Y Exposure time SL Pattern contrast 43 A Hologram noise 0 75 Diagnostic values above give an indication of image quality All values should be in the green range If image quality is not as expected please see further instructions on the support site You can now close this window Y Calibrate background T Recalibrate Figure 11 The Calibration Wizard after a successful calibration 1 3 2 If the calibration shows something amiss If there is a problem with the calibration a warning window will appear Figure 12 Whe
79. n the OK button has been clicked a window showing diagnostics values in the red will appear Figure 13 DP ra Calibration could not be performed under optimal conditions Please A check the diagnostic values and recalibrate if needed Figure 12 The Calibration Warning window Calibration finished PS Exposure time PS Pattern contrast PS Hologram noise Diagnostic values above give an indication of image quality All values should be in the green range If image quality is not as expected please see further instructions on the support site You can now close this window Close Y Calibrate background o Recalibrate Figure 13 The Calibration Wizard after an unsuccessful calibration If any values are in the red and the calibration could not be performed check the following e That all cables and connections are correctly attached e That there is laser light e That the blue laser fiber is not pinched down bent or pulled in any way 21 That the objective and the small laser window are clean otherwise clean with a cotton swab dipped in ethanol Then recalibrate using the Recalibrate button Figure 13 1 3 3 If the problems persist If problems persist collect diagnostics by selecting the Help option and then Collect Diagnostics in the top menu Figure 14 Follow the instructions of the Collect Diagnostics Wizard Figure 15 4 Calibration Wizard E Collect Diagnostics f Abou
80. name can be set to either a new group or to an existing one The default is to create new groups There will be an automatic suggestion of the group name but it is also possible to name the groups manually It is possible to mix new and existing groups Click OK to save all groups In short 1 Click in the vessel image or use numeric arrow keys to find an interesting position 2 Click on Remember in the Stage position box Every time you click Remember a capture position is saved at the current XYZ location 3 Repeat 1 2 for as many positions as required Tick the Capture pattern checkbox in the Capture box 4 Click on Setup storage which will show the Configure destinations window 5 Select a project and then tick the Multiple destination groups checkbox 6 Change group names if needed or click OK to accept The new groups will be created 7 Select Time lapse in the Capture box and enter duration and interval 8 Click Capture The capturing process will begin and the resulting time lapse images will be stored in the configured groups 425 Destination Project 2015 04 30 09 14 1 New Delete Rename ER Destination Group s L Single destination group 2015 04 30 09 15 5 Empty New Delete Rename Multiple destination groups Auto name base Capture 1 april 30 Autoall Setallnew Setall existing Capture Positions Well X pos Y pos Destination Group
81. ncita 51 cell culture vessel growth arar as 51 COMME SAIS Ute ESA E es re 49 COM TACT CAO AAA A A NEN 36 cell identification settings ooocnnncccccccnonnnnnnnnnnnnnnnnnnnnnnononccnnnnonos 38 Colo MON aaa 39 col rta da tl do ia 36 Cell TAC Rin trios 40 Center DONON rias 32 Cener IMA A A Sr TOR TORA SAST 25 COM bo 63 Controld POS Ai toto da 63 CONTO DOS Vonir a alicia 63 CHAM CS COLON rita tec 55 chanpe Me Col o A 33 chance The color spaim fina e alt tel ened SN 33 55 Change Trackin cascada Mebane tas 41 A A A INR TER 38 Clear Pateri DOON ios 29 CTOSE 00 0 PERE E E iden nee b bensen SNS E 21 color settings save the CUITON occcccccccncnnnnnnnnnnnnnnnnnonnnnnonanononos 26 color Spams Chane Meadas 26 COLOT a ANE tes 33 CA MA e Sateen ea 26 color chanse the plot display ciasne ren ste S ner sd ber re 45 66 COLO NEW aoatra a aa ut dan aE a E EON EENEN 26 A A A A SR SA SD colonne OSCR Casa e 33 A 33 55 COS ral tica 26 EOL den 33 55 E01 161 aot ot OR Fs EO iee Ras Br Om Oe RT SED ee Pee 26 COLOPSCES SAVE A TIC W onnie aea a aiea a A 26 Ms A A O E EEEE ET 33 condensa ONE oieee a E N E eee ene 59 Configure Destinations AA dd 30 COMMS NC Cn S 36 COUT CC MS a E ARA 51 AA II A A Nee 32 54 Paca A BL ASSA IANA ENS 47 delete a Colors turrarani ture net SONEN SERA NG RSA SAR 33 Delete tea id did aleta 37 delete Tepi OT aa dada 47 Double adaptive PAUSA iii 37 Double adapuy MA a osa iia 37 Double 37 CC CMU Lo 0 O
82. ng in which group the image Is located o Nbr Specifying which number the image has in the group o Type Specifying if the image is holographic or phase contrast o Date specifying the capture date and time of the image o Width specifying the image width in um and pixels o Height specifying the image height in um and pixels e Checking Light Effect applies an artificial light source to the image which may sometimes render an improved image Checking Shiny surface applies a change in the surface image display that sometimes renders a better image Shiny surface is only active when Light Effect is checked Figure 53 Hologram Image Information Colonng I Colorset gold red M 14 64 14 19 23 46 E AA U Figure 54 The Coloring side window Add color Change color Save colorset Save colorset As Delete colorset Apply to Figure 55 Additional functions found in the Coloring side window 33 4 5 2 cChange the image coloring All holographic images will appear in gray scale Use the Coloring side window Figure 54 which is found to the left of the Main Viewing window to add artificial coloring to the images The colors that are applied will be distributed in the image relatively to the thickness of the objects A set of colors that are saved together is called a colorset A previously saved colorset can be used with the current image by making a selection in the Colorset li
83. nnonccnnnonos 32 54 SI Erde een RN Ner 39 SHOWER UT iii ii 25 32 54 SHOW Titel 38 DIGS WDO Wis POR A a ter ace eereie cere hemala es 9 Snapshot DULON td a eatin 25 32 SOL Ware TOC o le ink elena nance 23 SOLLWArO FOCUS A A 34 solare Tocus distance nie 34 Source Frames abla tdi 45 Source Frames WINdOW coooccnnnccnnnccnnnnccnnnocnnuncnnnnccnnnoconaninnnns 40 51 A E NE eed 23 stage Movement aio 24 PT 24 Startup at room temperature a i 20 Start ap in the incabalon aiii 20 A ds E E Un ST FRANK AS RR R 64 MPG SUES CIS CCT SACS Sra tt lt dios 64 Texture COMPAS Gig tts den cacoeer a seinen upeeatmndtsaeeeatede te das 64 TEE CO ANON 25 25 ace toh testes et as settee ete 64 Texture correlation ato letal e td AO eet a 64 Texture correlation OL 64 Tex ure enero ysr E o E E es Cran SG USGA 64 Texture COTO PY EAO TAPAE Pa 64 Texture NOMOOGIILY jase cess a dad ennen uactaneste acters 64 Texture max proba Dili yss is a 64 TIC KHG SS avisa 39 64 TIC aerea ys OREA OR uo ren hace ete ae dn asus shia t nr ber 64 WAT A dd er A e ar SEE SENSE 39 64 threshold settings autoMatiC oooonnnnnccccccccnnnnnnnnononnnnnnnnnnnnnononnnonos 36 threshold settings MethodS oooooooooconoooooooccononononocccccnnnnnnnnnnonos 36 thresholds SC US TNC celled dd as 37 A RAT en ee 21 UNS FAIS CS aes eat rar E iaa 40 A A Sa elites RAA 27 Timeline esaeen aa 40 A E EATE 9 A E 40 Troubleshoonn Os lll cd ellos dls a ld 59 DP a did osc 32 54 LEDE oTe
84. nstall it by right clicking in device manager and selecting Update driver Then browse to driver folder under C PHI Drivers 11 1 2 It is impossible to focus the live image Why For M4 with distance plates the wrong distance plate might be used Fix Make sure that the correct soacer plate or vessel holder for that cell culture vessel is used The bottom of the vessel where the cells are should be approximately 1 mm from the objective Vessels with a thick bottom or a high socket need the thinnest soacer plate Vessels or slides which are thin should be put on the thickest or medium thick distance plate Why The cell culture vial is scratched or smudged Fix Even wiping the cell culture vessel clean will probably not help much as traces of dirt will still persist Instead avoid touching the top and bottom surfaces of the cell culture vessel when handling it even when wearing gloves Avoid anything that might scratch the surfaces Cell culture plastic is very easily scratched Why There is condensation on the inside of the cell culture vessel Fix 1 If the cell culture vessel is a flask tighten the cap and gently rinse the condensation away with the cell culture medium Avoid getting medium into the cap If the cell culture vessel is a multiwell plate or a petri dish allow the vessel to reach room temperature alternatively switch lids to a new lid or try working without a lid NOTE Working without the lid is a non steril
85. ocus distance can be adjusted in large steps by left clicking holding and moving the black bar with the mouse cursor This allows the stage to be moved up and down The distance between the objective and the sample is shown in the Focus text Dox To the right in the Microscope Settings side window there is a double gray wheel with red knobs which is used for intermediate outer wheel and small inner wheel focus adjustment steps The wheels are moved by left clicking holding and moving the red knobs Keep adjusting the focus until the white rectangle in the color bar in the Software Focus side window Figurel7 is placed in the center of the green area of the color bar The hardware focus is now set to allow the software focus to operate optimally When using the 20x objective the aim is to have the cell culture surface 1 mm above the objective tip Figure 18 Microscope settings Delete 4 5714 Save Focus o O Figure 23 The Microscope Settings side window 2 4 2 Focus the image manually Automatic focusing mostly results in well focused images Some cell samples are more demanding and need to be focused manually Check Manual in the Software Focus side window Figure 24 The text box next to the Manual button shows the current software focus distance in mm That distance can be set to a different value either by entering a value manually or by using the colored slide bar beneath the text box
86. ologram can be displayed showing either the phase or amplitude information of the light wave e Checking Phase displays the light wave phase information in the hologram e Checking Amplitude displays the light wave amplitude information in the hologram e Checking 3 D displays the holographic data as a 3 dimensional representation e Checking Rotate auto rotates the image e Checking Show Ruler displays a horizontal scale bar representative of the distance in X and Y e Checking Show Color Bar Figure 27 displays a vertical scale bar representative of the height in Z e Checking Light Effect applies an artificial light source to the image which may sometimes render an improved image e Checking Shiny Surface applies an artificial light source to the image which may sometimes render an improved image Shiny Surface is only visible when Light Effect is checked 2 9 Change the holographic image coloring All images originally appear in gray scale If colors are needed or wanted use the Coloring side window Figure 30 fo the left of the Main Viewing window Figure 5 A set of colors that are saved together is called a Colorset A previously saved Colorset can be used with the current image by making a selection in the Colorset list which is found at the top of the Coloring side window Figure 30 By left clicking the R button Figure 31 the coloring in the image is rescaled to better E utilize the optimal dynamic
87. olume is the optical cell volume in um The volume measurements are based on the phase shift of the light and are therefore called optical e Thickness max is the optical thickness of the cell is at its thickest in um e Thickness avg is the average optical thickness of the cell in um Perimeter length is the circumference of the cell in um e Eccentricity is how elongated the cell is or in how much the cell deviates from being a circle with O corresponding to the perfect circle and 1 being the maximum eccentricity e Iregularity is how much the circumference of the cell deviates from the circumference of a perfect circle with 0 corresponding to the perfect circumference and 1 being the maximum deviation 39 Chapter 6 Cell Tracking Cells can be tracked through a timelapse sequence and analyzed both for movement and for morphology changes over time Beer z n 5 r al fa Live Capture 2 Identify cells Track cells Analyze data B Cell coun Figure 74 The Cell Tracking tab 6 1 Start tracking cell movement Go to the Cell Tracking tab Fig 74 A window will appear where a new analysis or a stored analysis can be selected Figure 75 New analysis Start a new data analysis FS Open Load a previous analysis Figure 75 The Select Analysis buttons After clicking the New Analysis button a text will appear which says that frames must be added in order to analyze images for cell
88. onf 7 Hologram Phase cont Only checked Apply current Apply checked Apply all Figure 72 The Apply buttons 5 4 Change the image display In the Options side window Figure 70 which is found to the left of the Main Viewing window it is possible to change how the image is displayed The different display functions can be activated by checking the boxes The boxes can be checked in parallel enabling the user to combine functions e Checking Show Threshold displays a red coloring that distinguishes cells from the background e Checking Show Cell Markers displays a blue coloring that indicates the cell core e Checking Show Outline displays yellow lines that indicate the border of the cells rea 962 7 um Volume 2 30E3 pm e Checking Show Edge Cells Outline displays yellow lines that indicates the border of the cells touching the image edges E Thickness max 6 90 pm Ch Thickness avg 2 39 pm we Perimeter length 160 6 pm e Checking Raw Image causes the image to be Pau Eccentricity 0 64 displayed as the unadulterated gray scale Irregularity 0 54 image oe z Information pertaining to a e Using Auto apply Changes allows the user to single identified cell implement all changes immediately 5 5 Image information When the mouse cursor hovers over one of the segmented cells information concerning that cell will be displayed Figure 73 e Area is the cell area in um e V
89. ononos 34 recalculate software 1OCUS tios elisa 34 Recalibrate DUO Hae rar eas AN Lone 35 O ass 38 Re VOM TY D sete Spear ten o EE E ETE EE hase eee 47 RE CIONS Ia Dase 5i tee nacet et tole iene fansens eae amen oats 47 REMOS a id celda 53 Remove All command ii A tn ida 52 Remove data Hon eo 45 52 Remove Highlighted command oooooonnnnnnnnccnnnnnnnnnnnnnnnoncnnnonccnnnnos 52 remove single Capture POINMtS ssssserersrrrsrerrrrrrerrerrerrrrrr enn rra 29 FRO Cate tees hares PEE A II 25 32 ROC MIC SS ca 64 IRA a leser er bee asset 64 Rous ness KUrtOSi becin 64 PRO UO ness MS e saker Mig skur EA Ri 64 ROUSHNESS SKEWHESS etoen ii 64 SIVE COLO ici eS 33 savo a HOWCOLOTSC onssas a tisk 55 save images with the current colorset ooooooooooooccnnnnonccnnnono 33 Save tepon DUO a le laca le 52 save the current color SCtUIN GS acia 33 55 SS A eee ee er 45 scatter plot display COlO sssssssssssrsreerereerenrrrrrrnrrrrrrrr rr rr rna 45 A A A AN moa T 59 Select Mode zorserien 41 SClECE POSINONSDULON 4 add 28 ARSS tacts E 28 DElECHOM acti tasten dio 28 DEP LOCAL T ist aia ll cs 42 67 Settle Time After Each Stage MOVemMent occcccccccncnnnncnnnnonccnnnnons 29 Shape COVES tdt 64 A N 53500 SOU a 23 SKOWE Marker n tact ee ni ana 39 SHOW COLOR a a e a 32 SHOW Coltrane 25 54 show Pase Celli OulNE tenere a einanie atacsdead Cae 39 Show Image Info ooooooconnonononccccccococonoconnnnnnnnnnncnncn
90. ots showing the cell morphology by using the X and Y axis labels of interest If necessary add regions to gain separate data for the included cells in addition to the complete data set External data analysis Go to the Export tab below the Main Viewing window Click the Export data button to save the cell data of all added image frames to an xml file at a location of your choice Open the data sheet in a spreadsheet program of choice Analyze the parameters of interest Quick guide Cell tracking in a timelapse 11 12 20 21 When the Startup Make sure that the placed in an incubator for at least three hours instrument has been while switched on If the instrument is to be operated at room temperature start the HoloMonitor 30 minutes prior to use Start the computer Start HoloStudio Image capture Go to the Live Capture tab Choose create a project and a group Put the cell sample on a spacer plate or vessel holder of the correct type Select the correct vessel type in the vessel list in the stage position side window Ascertain that the live image looks well Press Capture Use the View Images tab to ensure that the captured images correspond to your settings Move the sample Press Capture Move the sample and capture again Continue capturing images until at least five images have been captured image capture is complete proceed to the View Images tab Make sure that th
91. ould be added to the analysis The more unevenly the cells grow the more images are needed for the cell count Area histogram W Auto Volume histogram Auto 126 7 4674 5 um Nbr of bins Figure 136 The histogram adjustment functions 8 2 Adjust the histogram proportions The X axis of the Area and Volume histograms can be set either automatically or manually For each axis the lowest and the highest value can be set as well as the number of bins that present the data The adjustment text boxes are found below the cell count report window Fig 136 8 3 Remove data from plot To clear the plot from all data from all image frames use the Remove All command which is found with an X to the right in the Source Frames window Figure 137 To remove data belonging to a single frame from a plot highlight that frame in the Source Frames window and then use the Remove Highlighted command which is found with an X to the right in the Source Frames window Figure 137 Nbr Group Project Commen t eierborg2 Take 7 2 245 2 245 D Deierborg2 Take 8 2 245 2 245 10 Lapsus Deierborg2 Take 9 2 245 2 245 D D D 11 Lapsus eierborg2 Take 10 2 245 2 245 12 Lapsus eierborg2 Take 11 2 245 2 245 13 Lapsus eierborg2 Take 12 2 245 2 245 Figure 137 The Remove functions 8 4 Export results Below the Cell Count Report there is a Save report but
92. ow the image has been moved flipped or zoomed To zoom left click the cell image in the Main Viewing window and then use the mouse scroll button To move the image to a desired location in the Main Viewing window click hold and drag using the left mouse button To flip and move the 3 D image click hold and drag using the right mouse button A rotation 40 rotation O rotation O X offset O Y offset O Zoom 0 30 Use for selected Use for all Figure 143 The Perspective side window Click one of the Use buttons to apply the changes to the added images that are highlighted or to all added Images Viewer options 3D Show ruler Show color bar E Show image info El Light effect Shiny surface Background IN Figure 144 Viewer Options side window a Viewer options 3D Show ruler Show color bar E Show image info El Light effect Shiny surface Background CEE Figure 145 The Background color setting 9 2 2 Adjust the image display The Viewer Options side window Figure 144 which is found to the left of the Main Viewing window is used to adjust the image display Some of the options are inactive when a phase contrast image is viewed e Checking 3 D displays the image as a 3 dimensional representation e Checking Show Ruler displays a horizontal scale bar representative of the distance in X and Y e Checking Show Color Bar displays a vertical scale ba
93. pm Auto settings er Figure 123 The Auto Setting function __39 3 _1105 6 um Range E Auto settings a No of bins Figure 124 Making the settings manually 49 To set the histogram X axis interval manually Auto settings must be unchecked Figure 124 Enter the minimum and maximum values 7 4 3 Change the number of bins The number of bins in the histogram is set automatically To set the number of bins manually uncheck the Auto settings Figure 124 which is found below the Histogram By adjusting the slide bar few Figure 125 or several bins Figure 126 can be displayed in the histogram 0 39 3 130 0 220 6 3113 4019 4925 _ 5832 6738 764 5 85 Area um Figure 125 A histogram with few bins 30 0 39 3 617 953 1289 162 5 218 6 2522 285 8 3418 3754 409 0 465 0 498 7 5323 Area um 588 3 6219 655 5 7115 7451 7788 834 Figure 126 A histogram with several bins 30 0 393 61 7 953 1289 162 5 218 6 2522 285 8 3418 3754 409 0 465 0 4987 5323 r Area um 588 3 621 9 655 5 711 5 745 1 778 8 8344 Figure 127 A histogram with several bins 7 5 Export plots and cell data In the Export tab Figure 128 cell data from the current plot can be exported to an XML file The xml file can be imported into e g Xcel Images of the current plot and histogram can be saved as bitmap GIF JPEG PNG or TIFF by using the Save buttons Fig
94. r Clear iI Figure 41 Adding a capture point located at the current position to a capture pattern 3 4 6 Clearing the capture pattern To delete the created pattern click the Clear Pattern button Figure 39 or the Clear button Figure 41 To remove single capture points allow the cursor to hover over a red dot When a blue appears the dot can be removed by right clicking Figure 42 Pinpointing the dots can be made easier by zooming in the vessel representation in the Capture Pattern window The image is zoomed in by left clicking in the window and then using the mouse scroll wheel Figure 42 Removing single capture point from a capture pattern 3 4 7 Settle time after stage movement The function Settle Time After Each Stage Movement Figure 36 allows medium movement to quiet down after moving the stage to the next capture point The more the medium moves the longer settling time is necessary For a tissue culture flask 5 seconds are usually enough 3 4 8 Capture the pattern After setting the Capture Pattern click the Capture button Figure 36 Once capture begins with Capture pattern selected the stage will go through the remembered positions one by one and take an image at each location The Capture Pattern window is closed by clicking the Selection button 3 5 Capture timelapses at several locations in parallel When combining the Timelapse function and the Capture Pattern function
95. r representative of the height in Z e Checking Show Image Info displays additional information associated with the image such as o First row specifies in which project the image is located o Second row specifies in which group the image is located o Nbr specifies which number the image has in the group o Type specifies if the image is holographic or phase contrast o Date specifies the capture date and time of the image e Checking Light Effect applies an artificial light source to the image which may sometimes render an improved image e Checking Shiny surface applies a change in the surface image display that sometimes renders a better image Shiny surface is only active when Light Effect is checked e To change the Background color left click the Background color box and select a new color Figure 145 9 2 3 Holographic image coloring Adjust the holographic image color display and the image dynamics with the Coloring side window Figure 146 A set of colors that are saved together is called a colorset A previously saved colorset can be used with the current image by making a selection in the Colorset list which is found at the top of the Coloring side window Figure 146 Click one of the Use buttons to apply the changes to the added images that are highlighted or to all added images coore Tr 1111 14 64 2346 d 4 DAA I Use for selected Use for all Figure 146 The
96. s being counted as one when they are very close to each other 5 2 Make adjustments for single cells It is possible to make manual changes to the cell identification for individual cells It is possible to add remove and delete as well as enlarge or shrink identified cells These functions are found in the Manual Changes window Figure 67 below the Main Viewing window e Add or Remove Figure 67 allows the user to add or remove the blue cell markers that identify the cell core This results in mergers or splits of identified cell areas e Grow or Shrink allows the user to enlarge or shrink the identified cell regions e Delete Area allows the user to identified cell areas remove Figure 67 The Manual Changes window Add or remove existing ones Use left mouse button to add a marker and right button remow Iza Ha Io ce The e Grow or shrink to select a cell and then use the mouse wheel Finish by left clicking Hold down CTR Delete area small objects that could not be removed by the Min object size adjustment 37 Edit manual changes 9 manual changes mouse b ii n i Undo Redo M Remove all Several of these functions are also found as a menu when clicking the right mouse button in the image Figures 68 and 69 Show threshold To the right in this window there are buttons that enable the user to undo the last adjustment step to redo the undone step and to clear all adjustments Show cel
97. scceeesees 32 4 5 1 Change the image disPlQa ssrrsssrsrrrrrrrerrrnrrnn 32 4 5 2 Change the image COlOYING sseerssrsrrrrrrrerrrrrrrrrrnnn 33 4 6 Move flip or zoom the cell image cccccssscsssseoees 34 4 7 Recalculate a holographic 1mage cccccsssscsssscoees 34 4 7 2 Recalculate the focus manudlly ccccccccceeccee ees 34 4 7 1 Recalculate the focus automatically 34 4 7 3 Recalibrate a holographic iMA2C ssrrrsrrrrsrrrrrn 33 4 7 4 Using a background holOograM rmmmmrrsrrrre 35 S CellidentificatiO Mses eric diia 36 5 1 Identify CellS ssscsssssovsss cessbvasdssv ssndosssesvnsessossssssseesusssansns 36 Ihse A iden e tres 36 5 1 2 Automatic threshold SettiNQSS sssserrrrrsrrrrrrrrerrrrrnnn 36 5 1 3 Adjust the cell identifiCatiON ssrersrrrrrrrrrrrrrrrran 37 5 2 Make adjustments for single Cells sssssccssssessees 37 5 3 Save the cell identification settingS 38 5 4 Change the image display cccccccssssscssssccssssceessees 38 5 5 I mage informatiOnN sssssssssssssssssssssersrsrrossserrrsrrsnrrnr 39 6 Cell Tracking isis 40 6 1 Start tracking cell movement sssssssssssssssccrsrrsr 40 6 1 1 Add image frames to the analysiS ooonnnnnnnnnnnc 40 6 1 2 Select cells to be ANAaLlyZeA sssserrrrrrrrrrrrrrrrrrrrrrrr rna 40 6 1 3 Displaying the cell track
98. slets 14 Cell counting and confluence measurements ossono0 15 PROMMCFA MORES IM CI CS us a 16 Morphology analysis cad 17 Cell tracking ina tmp ind 18 Data Dacia ae Merce ner re Or aD UPPED eM A SOS Re 19 11 Quick guide Start up and calibrate the instrument 12 15 14 Los 16 Putting the instrument in the incubator Put the HoloMonitor M4 in the incubator and immediately attach all cords The instrument must be connected to the wall socket at all times when placed in the incubator Wait 3 4 temperature to stabilize hours for the instrument Startup Start the laser 30 minutes prior to use Start the computer Start HoloStudio Calibration at start up Go to the Live Capture tab The Calibration Wizard window will appear Follow the instructions of the wizard to perform a calibration If all values are in the green everything is ready to go Calibration at other timepoints To calibrate at other timepoints select the Help option and then Calibration wizard in the top menu Proceed as above Troubleshooting If any values are in the red check the following All cables and connections are correctly attached There is laser light The objective and the small laser window are clean Recalibrate 12 Collect diagnostics If problems persist collect diagnostics by selecting the Help option and then Collect Diagnostics in the top menu Follow the
99. sssssssssscssccrscnr 20 1 2 Start up in the incubator ssssssssssssssssssrererssrrnnrnr 20 AA TI 20 1 3 1 When everything is Wells sssssssrrrrrrrrerrrrrrrrrrrrrrrr rn na 20 1 3 2 If the calibration shows something amiss 21 1 3 3 If the problems PerSlStusnusssssssssrsrrrssrrrrrrrrrrrer renen nn na 21 14 Close COW sccsvessdessasseswissnecad sess scodsvessiesssiesdesteeusevsbavetass 21 Ze View LIVE IMAGES siii lada Qeeseesadeciis 22 Z1 FOCUSING rad ic 22 2 1 1 Automatic versus manual fOCUSING rrrerrrrrrerrrna 22 2 2 Focus a live holographic image using an M4 with a standard sample St gZ sssssssssssossrsrrorsserserernsrrnsrrrerrer 23 2 3 Focus a live holographic image using an M4 with a Manual AN iaa 23 2 4 Focus a live holographic image using an M4 with a motorized AY Z StADCisorsos pensssssssrunssss rs sssssvesskadensss nst snterd 23 2 4 1 Focus the image semi automaticall 23 2 4 2 Focus the image Manual sssrrrrrrrrrrrrrrrrrrrrrr rna 23 2 5 Improve calibrate the holographic image 24 2 6 Move the sample stage ccccccccssssssssscccccccsssessssssscees 24 2 7 Move flip or zoom the holographic image in the Main VICWING VV INO OWisssbocsssacesenesssssb nessessesesderoedersebereskesdersnenessdens 24 2 8 Change the holographic image display 25 2 9 Change the holographic image coloring
100. st which is found at the top of the Coloring side window Figure 54 e By left clicking the R button Figure 55 the coloring in the image is rescaled to better utilize the optimal dynamic range of the image This button needs to be operated every time the image coloring is off e To add a new color left click the plus button Figure 55 and click Add Color Select a color A colored triangle representing the new color will appear beneath the histogram Figure 54 Alternatively right click on the X axis and select Add Color e Change the color by using the right mouse button to click on a colored triangle beneath the histogram Figure 54 and select a new color Alternatively left click the arrow button Figure 55 and select Change Color e To change the color span left click and move the desired colored triangle beneath the histogram Figure 54 using the cursor To save the current color settings to a new colorset left click the arrow button and choose Save As Figure 55 e TO save the current color settings to an already existing colorset left click the arrow button and click save Figure 55 Note that this will overwrite the settings previously saved to this colorset e To delete a colorset select it in the Colorset list Then left click the arrow button Figure 55 and click Delete Colorset e To save a colorset to an image left click the arrow button Figure 55 and choose Apply To followed by Frame The current
101. t Hstudio a ne Figure 14 The Collect Diagnostics Wizard in the top menu Collect Diagnostics This wizard will collect essential information about the 8 performance of your HoloMonitor The information is saved in a compressed file that you can send to assist in support Click Begin to start the wizard Begin Cancel Figure 15 The Collect Diagnostics Wizard 1 4 Close down by using the on off switch on the instrument and by closing the computer program For further information concerning the handling of the HoloMonitor M4 please use the Getting Started guide Chapter 2 View live images The software focuses the images only when the cell culture vessel is approximately the correct distance from the objective The aim is to have the cell surface of the cell culture vessel approximately 1 mm above Select the Live Capture tab Figure 16 the object tip Figure 18 Below three holographic phase images are shown that are in focus out of focus and completely out of focus Figures 19 20 and 21 ile View Databas e t _ ive Live Capture rp D images 2o Identify cells Qi Track cells La Analyze data hb Cell count Figure 16 The Live Capture tab 2 1 Focusing There are two different focusing mechanisms that need to cooperate in order for the image to be in focus With the hardware focus the user places the cells to be viewed at the approximately correct distance from th
102. the background to be set at an incorrect level Fix 1 Make sure that background and laser are calibrated prior to analysis Fix 2 Capture extra images and then discard the bad ones 11 4 4 Some cells are incorrectly segmented as two or more Why The minimum object size is set too low Fix Adjust the Min object size using the slider in the Object definition box under the Adjustment tab so that each cell has one blue marker 11 4 5 Two cells are segmented as one Why The minimum object size is set too high Fix Adjust the Min object size using the slider in the Object definition box under the Adjustment tab so that each cell has one blue marker Why The cell confluency is very high and the cells are difficult to separate out Fix Separate the two cells by using the Add or remove button under the Manual changes tab in the Identify cells tab 11 5 Analyze data 11 5 1 No image frames are visible in the Image Frame List window Why No Project and Group has been selected Fix Select a Project and a Group Why If the Only Checked box is checked only checked images will be displayed If no image frames in the current Group has been checked no frames will be visible Fix Uncheck the Only Checked box 11 5 2 The cell image in the Main Viewing window is white Why The image dynamics is not optimal Fix Click the R button in the Histogram side window 11 5 3 The dots in the dot plot disappeared Why The
103. to be operated at room temperature start the HoloMonitor 30 minutes prior to use Start the computer Start HoloStudio M4 Image capture Go to the Live Capture tab Choose create a project and a group Put the cell sample on a spacer plate or vessel holder of the correct type Ascertain that the live image looks well Press Capture Use the View Images tab to ensure that the captured imges correspond to your settings Move the sample Press Capture Continue until at least five images have been captured image capture is complete proceed to the View Images tab Make sure that the captured images look good Proceed to the Identify Cells tab Identify cells Adjust the cell identification in the first image frame using the Adjustments and Manual Changes tabs Make sure all your images are checked in the Image Frame list on the right side Click the Apply checked button which is found to the bottom right of the window in order to apply the cell identification adjustments on all the captured images Quickly preview the rest of the images to make sure they are all well segmented Note that for cell counting the cell area identification does not need to be exact but need to be the cell markers blue dots 16 20 21 22 23 24 correctly placed For confluence measurements the threshold setting needs to be correct Proceed to the Cell Count t
104. ton Figure 138 Clicking the button generates a pdf containing all relevant data including the area and volume as histograms and a list of the included image frames When the boxes are un checked histograms and frame list can be excluded from the report Save report 7 Include histograms W Include source frames list Figure 138 The Save Report button 52 Chapter 9 Export images and movies In the Export Images tab image frames can be edited and exported either as individual images in several standard formats or as AVI movies Figure 139 File View Database About a Live Capture Identify cells a Track cells lu Analyze data EET g Eme Figure 139 The Export Images tab Export images Project New Delete Rename 2012 10 12 10 08 Deierborg2 New Delete Rename Hologram x20 2010 09 02 12 08 01 Count 126 Confl 20 Take 7 2 245 2 24 3 ES Count 127 Confl 19 SEL 10 Count 128 Confl 18 11 _ Count 127 Confl 18 12 Count 128 Confl 19 Hologram x20 2010 09 02 12 13 01 Hologram KE Take 9 2 245 2 24 Hologram x20 2010 09 02 12 73 01 Take 10 2 245 2 2 Hologram x20 2010 09 02 12 28 01 Take 11 2 245 2 2 Hologram x20 2010 09 02 12 33 01 Count 131 Confl 19 Take 12 2 245 2 2 E Hologram 7 Phase cont E Only checked
105. tracking Figure76 Start by adding frames from the frame list to the right to create a timeline Figure 76 The Cell Tracking instruction text Conf 18 C o Hologram Phase cont E Only checked 359 frames Clear all Add highlighted Add checked Add all Figure 77 The Add buttons Tracked cells Warnings 6 1 1 Add image frames to the analysis Highlight or check the images to be analyzed in the Image Frame List to the right Click the appropriate Add button Figure 77 The Add buttons are found below the Image Frame List The added image frames will be shown in the Source Frames window Figure 78 below the Cell Tracking window Figure 91 6 1 2 Select cells to be analyzed In order to follow a cell through a timelapse the cell needs to be added Go to the Select Mode side window Figure 79 and activate Add Cells a select mode Add cells Select Figure 79 The Select Mode side window In the Cell Tracking window the center of each identified cell is marked with a small orange Figure 80 If the identification is not satisfying go to the Identify Cells tab and adjust the cell identification Chapter 5 Click each cell to be followed Figure 80 The cell will be followed from the frame where it is added A Click to start tracking this cell from now on Figure 80 Clicking to add a cell to the tracking results 6 1 3 Displaying the cell trackin
106. ult is a cleaner threshold mask The Double Otsu uses double thresholding with Otsu global threshold as mid level threshold Double adaptive mean same as Double Otsu but with two adaptive mean threshold masks Double adaptive gaussian same as Double Otsu but with two adaptive gaussian threshold masks Goa Manual changes Background threshold Miscellaneous Method Auto Otsu in blocks N I Use pre smoothing l Y Join nearby markers Adjustment 107 Object definition Min object size 19 Figure 66 The Adjustments window Adjustments Manual changes 5 1 3 Adjust the cell identification In the Adjustments window which is found below the Main Viewing window Figure 66 the cell identification settings can be adjusted in several ways In addition to selecting the method to calculate the threshold as described above adjustments can be made for each method The slide bar labeled Adjustment Figure 66 is used to manually adjust the threshold that is set between cells and background thus adjusting the area of the segmented cells The slide bar labeled Minimum Object Size Figure 66 is used to manually adjust the size of the cell core thus adjusting which identified areas that are cells e Checking Presmoothing Figure 66 activates a noise reduction function that will smooth the edges of the cells e Checking Join Nearby Markers Figure 66 results in two distinct cell marker
107. und in the View Images tab 7 1 Measure distances directly in the currently viewed image Choose the View Images tab Figure 92 File View Database View ima Ident Track cells Analyze data Cell count Aa Export images ges o yz p g Figure 92 The View Images tab Viewer options ite Measure W 3D Show ruler E Rotate Show color bar Show image info El Light effect Shiny surface Background IN Figure 93 The Viewer Options side window for holographic images Line measure 27 5 um Profile Analysis Figure 94 The blue measuring bar and the results window 44 Click the Measure button in the Viewer Options side window Figure 93 to make manual measurements of objects in the current image When the measuring function is activated the measurement is started by left clicking anywhere in the image The measurement is finished by left clicking at a new point in the image The measured distance is shown with a blue bar Figure 94 A window displaying the results appear to the bottom right of the main viewing window The results include a profile of the measured object The data are saved as an image when the Snapshot button Figure 95 in the Viewer Options window is clicked Lo Figure 95 The Snapshot button It is possible to move the image in the Main Viewing window while the measuring function is activated The image is moved by clicking and dragging When the
108. ure 129 7 5 1 Export cell data The Export tab is found below the Scatter plot Figure 128 By clicking the buttons cell data can be exported to XML or ICE files Source frames Filter Regions Export Export to Excel Exports analysis data to an Excel compatible XML file H Export to ICEFormat Exports analysis data to an Image Cytometry Experiment Format ICEFormat data directory Figure 128 The Export tab Click the Export buttons browser window will open to export all cell data A The file will automatically be named with date and a code but it is possible to rename the file Save the file to an appropriate place In this step all data pertaining to the image frames added to the current plot are exported Save plot Save histogram Figure 129 The Save Plot and Histogram buttons 7 5 2 Save plots and histograms The Save Plot and Save Histogram buttons are found below the Scatter plot and the Histogram Click the Save Plot button Figure 129 A browser window will open The file will automatically be named Scatter Plot but it is possible to rename the file Decide which image format you want Save the file to an appropriate place 50 Alternatively plots and histograms can be saved by right clicking the diagrams and then choose Save Chapter 8 Cell Count Cells are counted and analyzed for confluence cell area and volume in the Cell Count tab
109. w Figure 25 Remove the sample from the stage and click OK 2 Use the calibration wizard as described in Chapter 1 Stage position Vessels Microstide fr X 56 288 mm Y 25 038 mm Remember Clear Petare Figure 26 The Stage Position window 2 6 Move the sample stage The Stage Position window Figure 26 which is found to the right of the Main Viewing window is used to move the sample stage First select the kind of cell culture vessel currently used by making a selection in the Vessels list An image of the selected vessel will appear The blue area represents the area of the vessel available for image capture In Figure 26 the vessel representation shows microslides e The stage can be moved in large increments by clicking and dragging the objective representation i e the small double circle in the Stage Position window e The stage can be moved in very small increments by using the arrow keys on the numerical pad e The stage can be moved in intermediary increments by pressing the shift key simultaneously with using the arrow keys on the numerical pad Every time the stage is moved the image might need a second or two to stabilize 2 7 Move flip or zoom the holographic image in the Main Viewing Window To zoom the live image left click the image in the Main Viewing window Figure 5 and then use the mouse scroll button To move the live image to a desired location in the Main Viewing win

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