PHI HoloMonitor M4 Setup And Operation Manual

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HoloMonitor

Setup and Operation Manual

February 2018, Hstudio 2.7.3, rev. 8

This manual is issued by:

Phase Holographic Imaging PHI AB

Scheelevägen 22 | 223 63 Lund | Sweden

www.phiab.se

support@phiab.se

P H A S E

H O L O G R A P H I C

I M A G ING

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Summary of Contents for PHI HoloMonitor M4

Page 2: Table Of Contents
View live images The theory of focusing Practical focusing Moving the motorized sample stage Change how live images are displayed Change default cell and medium refractive index © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 3 Analyze and export data Measure cells interactively Analyze results in plot Multiple analyses Display results in scatter plot Display results in histograms Export plots and cell data © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 4 Transfer data from one computer to the next Troubleshooting Live capture View images Identify cells Track cells Analyze data Export images Morphological parameters Holographic microscopy Cell morphology parameters References © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 5: The Manual
The symbol below is found throughout the manual. Clicking it will bring you back to the previous view. Interactive button for previous view. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 6: Instrument Safety
Instrument safety HoloMonitor M4 was designed and tested in compliance with low voltage directive 2006/95/EC and electromagnetic compatibility 2004/108/EC. References to relevant harmonized standards used, or references to the specification in relation to which conformity is declared, are EN61010- 1:2001 safety requirements for electrical equipment for measurement and EN60825-1:2007 safety for laser products.
Page 7: Warranty
With the exception of the procedures specified in this manual, no maintenance or repair of HoloMonitor may be performed by users. Repairs may only be performed by specially authorized personnel. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 8: Introduction To Holomonitor® M4
Introduction to HoloMonitor® M4 HoloMonitor M4 is a live cell imaging time-lapse cytometer, providing imaging and quantifi- cation of unstained living adherent cells directly in their culture vessels. It can be used for a wide range of applications, including: • Cell count and proliferation •...
Page 9: Computer Requirements
Display: Full HD (1920x1080) or higher The files associated with Hstudio are stored in the folder C:\PHI, which must not be used for anything else. User created data is stored under C:\PHI as well. However, the user data location can be changed at any time.
Page 10 Click Install to continue and wait for the installation to complete. 5 HSTUDIO INSTALLATION Progression of installation. Click Next to continue. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 11 Next Enter your information. Click continue. Click Install to start the installation process. Click Finish to complete the installation. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 12: Holomonitor Installation
11 HoloLid Petri 5 HoloLid 6-well 12 Cotton swabs 6 Transportation plate 13 USB cables 7 USB memory with Hstudio 14 Spacer plates for fixed stage (not shown) © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 13 • The top of the objective (A) and the object beam window (B) are cleaned with a fresh cotton swab dipped in 70% ethanol. • Use a dry cotton swab to remove ethanol residues. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 14 • Connect the laser unit power supply to a Laser unit power outlet, using one of the power adapters. 5 CONNECTING THE STAGE • Make sure the motorized stage is connected. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 16: Starting The Instrument For The First Time
If the calibration is in the green area, the instrument is ready to use. Follow the instructions in section View live images (page 26) If the calibration is in the red area, go to Troubleshooting (page 98). © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 17: Placing Holomonitor Inside A Cell Incubator
HoloMonitor is inside the incubator. Do not lift HoloMonitor by holding the motorized stage. Only lift holding the blue and white metal of the instrument. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 18: Cleaning Instructions
• The instrument can be wiped down with a damp cloth (ethanol or water). Cleaning must be performed so that no liquid enters the interior of the instrument. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se...
Page 19: Calibration Procedure
If any of the Diagnostic values remain outside the green range after several cleaning attempts and overnight drying (see Troubleshooting), contact the technical support at support@phiab.se for further assistance. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 20: Long-Term Storage
If the cables are left inside the incubator for a longer period of time, remember to wipe them off regularly with 70% ethanol to reduce the risk of incubator contamination. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se...
Page 21: Sample Requirements
• Sarstedts Lumox 24 or 96 well plates with HoloLid. Glass-bottom Petri dishes are highly recommended. For a complete list of recommended cell culture vessels, see www.phiab.se/support/vessel. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 22: Hololids
HoloLids™ The PHI HoloLids have been especially designed to eliminate image disturbances caused by surface vibrations and condensation inside the cell culture vessel. HoloLids are available in number of different formats. They are made of poly methyl meth- acrylate (PMMA, Plexiglas), a non-toxic material often used in medical surgery implants, dentures etc.
Page 23: Introduction To Hstudio 2.7
Analysis of cells in images and time-lapses, as well as display and export of the results. Cell count Counting of cells in images. Export images Visualization and export of images and time-lapse movies. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 24 Information concerning the different side panels can be found by clicking the Information which are placed in the side panel headers: button © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 26: View Live Images
The software focuses the images only when the cell culture vessel is at approximately the correct distance from the objective. The aim is to have the cell surface of the cell culture vessel approximately 1 mm above the object tip. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 27 A holographic phase image that is in focus A holographic phase image that is out of focus A holographic phase image that is totally out of focus © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 28: Practical Focusing
When placing a spacer plate on to the stage, make sure that the two pins on the table are positioned in the holes in the spacer plate. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 29 • Select the correct vessel template from the in the An image of the selected vessel will appear. The blue area represents the area of the vessel available for image capture: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 30 (outer wheel) and small (inner wheel) focus adjustment steps. The wheels are moved by left-clicking, holding and moving the red knobs. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 32: Moving The Motorized Sample Stage
• Additionally, the stage can be moved to an absolute position by entering the target xy-coordi- nates and pressing the button. Every time the stage is moved the image might need a second or two to stabilize. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 33: Change How Live Images Are Displayed
Either the phase or bright-field image can be displayed: Phase displays the phase image. Amplitude displays the less used bright-field image. displays the holographic image in 3D. Rotate automatically rotates the image. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 34 • To move the live image, click, hold and drag the image using the left mouse button. • To flip and move the live 3D image, click, hold and drag the image using the right mouse button. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se...
Page 35 A colored triangle representing the new color will appear beneath the histogram. Alternatively, right-click on the x-axis and select color. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 36: Change Default Cell And Medium Refractive Index
(page 106), Hstudio uses the difference in refractive between the cells and the medium to calculate cell thickness in µm. The default refractive index Calibration side panel: values are changed in the © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 37: Capture Images
• Make sure that you are satisfied with the quality of the live image. • Select or create a Project and Group. • Click Capture © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 38: Capture A Time-Lapse Sequence At One Position
The minimum interval between captures is given to the right of the interval time unit. • The number of time-points will be given when total time and interval are entered. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 39: Capture Time-Lapses At Several Positions In Parallel
CAPTURE TIME-LAPSES AT SEVERAL POSITIONS IN PARALLEL When HoloMonitor is equipped with a motorized stage, multiple time-lapses may be captured in parallel by combining a Timelapse with a Capture pattern: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 40 Capture pattern. A saved capture position is indicated by a small red dot. • Repeat selecting capture positions until all desired positions have been selected. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 41 Capture Once capture begins, the stage will go through the selected positions one by one and repeatedly capture an image at each position. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 42: Saving Time-Lapses In Separate Groups
The suggested new group names may be changed by changing the Base name or by individually changing the group names. By checking Existing it is possible to select an existing group from the drop-down list. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 43 Options for position panel, which appears when a capture position is highlighted in the Capture pattern window. Close To save all the settings, click Close © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 44: Automatically Generate Random Capture Positions
First the relevant wells need to be selected. This is done by clicking on the wells in the graphical representation of the wells, which appears to left of the Capture panel. In the example below, wells A1-A3 are selected. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 45 When positions are generated randomly, the current Z-position of the stage will be saved and used for imaging of all positions. It is therefore important to verify focus. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se...
Page 46: Add Capture Positions Manually
When positions are generated by clicking, the current Z-position of the stage will be saved and used for imaging of all positions. It is therefore important to verify focus. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se...
Page 47: Focus Verification
Z-position that will be used when images are captured. Verify a sufficient number of capture positions to ensure that all capture positions are in focus. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se...
Page 48: View And Adjust Captured Images
Select an image to view it in the View area. Click to show the image frames as a movie. Auto-scroll © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 49: View One Position Of Several In Parallel Time-Lapses
• After clicking OK, check the Show only checked found below the Image frame list. Now, only the checked images will be displayed, and they will belong to the same time-lapse. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 50: Change How Captured Images Are Displayed
Group. Date specifies the capture date and time of the image. Channel specifies if the image is holographic or phase contrast. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 51: Re-Focus A Captured Image
After image capture most images are well focused. Some images may need to be re-focused manually after capture. If an image is not correctly focused, as in the example below, re- calculating the software focus may restore focus. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 52 Automatic in the Software focus panel and then use the button to apply the update to either the current frame, all frames or all checked frames. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 53: Cell Identification
Choose the In the Image frame list panel, select the Project, Group and the image of interest to make it appear in the View area: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 54: Change How Identified Cells Are Displayed
The software will then automatically suggest a cell identification: CHANGE HOW IDENTIFIED CELLS ARE DISPLAYED Options panel, it is possible to control how cells are displayed and how identification In the adjustments are saved: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 55: Adjust Cell Identification
Minimum error sets the threshold level using the minimum error histogram-based threshold method. Otsu sets the threshold level using the Otsu method. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 56 In other words, the identification adjustments are performed in three steps: • Determine which threshold calculation method is optimal for the current cell culture. • If needed, use the adjustment slider to adjust the threshold setting. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 57 View area. To apply the same adjustments to other image the image frame display in the frames, use the buttons below the Image frame list: Apply © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 58: Make Adjustments For Individual Cells
Draw cell boundaries manually. Manual changes tab To the right in the there are buttons that enable the user to edit the changes: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 59 Note that settings made using the Manual changes tab are applied only to individual cells. These changes cannot be applied to other image frames. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 60: Cell Tracking
If tracking has not been performed previously, a tab will appear with the choices of starting a new analysis or opening a previous. After clicking a message will appear: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 61 Several images can be added simultaneously if they are all selected. The shift key can be used to select several consecutive images and the Ctrl-key to select non-consecutive image frames. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 62 The cell will be tracked from the image where it was added. If the identification is not satisfying, go to the Identify cells tab, and adjust the Cell identification (page 53). © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 63: Cell Tracking Warnings
If the warning is caused by a cell identification error, as in the above example, click to go to the cell Identification tab. After adjusting the identification, click the Track cells to return. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 64: Displaying Cell Tracking
To set the plot axis values manually, uncheck and then enter the desired x- and y-minimum and maximum values. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se...
Page 65: Adjusting Cell Tracking
• Activate in the • Click on the tracked cell to be adjusted. A new set of functions will then be available in the Change tracking panel: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 66 Undo for this frame Undo for all frames frame or in all frames, respectively. The tracking will be recalculated accordingly. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 67 . From then on, both daughter cells will have new names and will be Division tracked individually. If the mother cell is named e.g. Cell, the daughter cells will be named Cell-1 and Cell-2: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 68: Tracking Cell Morphology
Once the cell movement has been tracked, cell morphology can be followed over time by using the Plot features tab: Click to include/ exclude cell from the diagram Remove selected or all cells from All tracked tracking cells are plotted © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 69 Auto-scale sets the y-axis scale automatically. When unchecked, the y-axis scale can be set manually. saves an image of the tracking diagram. Save plot… © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 70: Multiple Analyses
EXPORT TRACKING DATA In the Tracking tab, selected tracking data may be exported. The data is exported to an XML-file which can be imported to Excel: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 71: Analyze And Export Data
View area while the measuring function is activated. The image is moved by clicking and dragging. When is clicked again, the measuring function is deactivated. Measure © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 72: Analyze Results In Plot
If data has not been analyzed previously, a tab will appear with the choices of starting a new analysis or opening a previous: After clicking a message will appear: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 73 Several images can be added simultaneously if they are all selected. The shift key can be used to select several consecutive images and the Ctrl-key to select non consecutive image frames. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 74: Multiple Analyses
Analyses are optionally saved when a Data analysis tab closed: Additional analyses are started by creating addi- tional Data analysis tabs: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 75: Display Results In Scatter Plot
Source frames tab, 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: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 76 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: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se...
Page 78 Repeated clicking in the diagram results in an irregular figure that outlines the region. To finish the region, close it off by clicking the starting point. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 79 Regions can be hidden by unchecking the region: Remove selected or all regions Regions can be copied from one analysis to the next by using Display from other in the Regions tab: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 80: Display Results In Histograms
The x-axis label of the histogram can be changed by using the Cell feature list below the histogram. A list of x-axis label parameters are found in Morphological parameters (page 106). © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 81: Export Plots And Cell Data
Excel. Images of the current plot and histogram can be saved as bitmap, GIF, JPEG, PNG or TIFF by using the buttons. Save plot… Save histogram… © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 82 • The file will automatically be named, however it is possible to rename the file. • Decide which image format you want (bitmap, gif, jpeg, png, tiff). • Save the file to an appropriate place. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 83: Cell Count
Image frame list Source frames panel Add buttons Fill in the correct cell culture vessel growth area and the volume of the cell culture vessel medium content: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 85: Remove Data From Plot
Save report… be saved as a pdf or as an xml-compatible file containing all relevant data. Histograms and image frame list may be excluded from the report: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 86: Export Images And Movies
All image frames can be added by clicking Add checked . Alternatively, the images can be dragged and dropped into the Source Frames window. Add all © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 87: Edit The Images
• To move the image to a desired location in the View area, click, hold and drag using the left mouse button. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 89 The colors that are applied will color the image relatively to Change the thickness of the cells. For additional information on how to set the coloring, see image coloring (page 35). © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 90: Create An Avi Movie
When image perspective and color looks good, click the buttons in both the Use for all Perspective panel and the Coloring panel: Preview the movie by clicking © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 91 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. When ready, click Export © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 92: Export Images
3D will be displayed in the exported image. When Transparent background is checked, TIFF-images can be exported with a transparent background. When ready, click Export © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 93: Database Handling And Backup
• In the lower part of the database handler window, information on the currently selected database will be displayed. Click to use the database in Hstudio. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 94: Switch To A New Database
Move database window will appear. • Browse to the new location and then click . The entire database will be moved to Move the new location. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 95: Copy And Move Individual Images, Groups Or Projects
• Click the little arrow beside the database that contains the data to backup. A list of Projects contained in the database will appear. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 96: Transfer Data From One Computer To The Next
The database data are now on the computer and can be accessed by Hstudio. TRANSFER DATA FROM ONE COMPUTER TO THE NEXT • Go to the top menu and click Database: © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 97 • Browse to find the transfer folder on the USB hard drive. • Click . The database data will be transferred to the database that is currently Start used by the Hstudio software. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 98: Troubleshooting
If the problem persists, try to run the installation program again. The driver files are located under C:\PHI\Drivers as sub folders. If a driver displays an exclamation mark in the device manager it may be possible to re-install it by right-clicking in the device manager and selecting Update driver.
Page 100 • The motorized stage cannot move fast enough to be able to capture the images on time. Instead it works as fast as possible. Set fewer capture points, or allow more time between captures. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se...
Page 101 The image dynamic display is off range • Click the R button in Coloring panel to reset the image dynamics display, see Change image coloring (page 35). © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 102 Capture Position window • Recreate the Capture setup, and ensure that is clicked. That will close the window Close and save the set up. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 103: View Images
Capture extra images and then discard the bad ones. • A passing disturbance, e.g. floating cell debris, caused the software auto-focus to miscalculate. Follow the instructions how to manually reset an unfocused image. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 104: Identify Cells
• The cell confluency is very high and the cells are difficult to separate out. Separate the two cells by using the button under the Manual changes tab in the Identify Add or remove cells tab. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 105: Track Cells
Uncheck Only checked. The cell image in the view area is white • The image dynamics is not optimal. Click the button in the Coloring panel. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 106: Morphological Parameters
Change default cell and medium refractive index (page 36). Small variations in the refractive index do not cause any significant changes in the calculations. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 108 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. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se...
Page 109 Perimeter length The length of the perimeter around the cell. The calculations are based on a threshold setting that distinguishes background from cell. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 110 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. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 111 Entropy is a statistical measure of randomness that can be used to characterize the texture of the cell image. Texture homogeneity Measures how many different gray levels the image holds. © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 112: References
Optics Express, Vol. 13: 9 Kemper et al (2010) Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy, Journal of Biomedical Optics 153 © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se support@phiab.se...
Page 114 P H A S E H O L O G R A P H I C I M A G ING...

PHI HoloMonitor M4 Setup And Operation Manual

The Manual

Instrument Safety

Warranty

Introduction to Holomonitor® M4

Computer Requirements

Hstudio Installation

Holomonitor Installation

Starting the Instrument for the First Time

Placing Holomonitor Inside a Cell Incubator

Cleaning Instructions

Calibration Procedure

Long-Term Storage

Sample Requirements

Hololids

Introduction to Hstudio 2.7

View Live Images

Capture Images

View and Adjust Captured Images

Cell Identification

Cell Tracking

Analyze and Export Data

Cell Count

Export Images and Movies

Database Handling and Backup

Troubleshooting

Morphological Parameters

Quick Links

Need help?

Questions and answers

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Summary of Contents for PHI HoloMonitor M4

Table of Contents