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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

[email protected]

P H A S E

H O L O G R A P H I C

I MAG ING

Содержание HoloMonitor M4

Страница 1: ...uary 2018 Hstudio 2 7 3 rev 8 This manual is issued by Phase Holographic Imaging PHI AB Scheelev gen 22 223 63 Lund Sweden www phiab se support phiab se 2018 Phase Holographic Imaging PHI AB All right...

Страница 2: ...tallation 12 Starting the instrument for the first time 16 Placing HoloMonitor inside a cell incubator 17 Cleaning instructions 18 Calibration procedure 19 Long term storage 20 Sample requirements 21...

Страница 3: ...everal in parallel time lapses 49 Change how captured images are displayed 50 Re focus a captured image 51 Cell identification 53 Change how identified cells are displayed 54 Adjust cell identificatio...

Страница 4: ...e 90 Export images 92 Database handling and backup 93 Switch to existing database 93 Switch to a new database 94 Move a database 94 Copy and move individual images groups or projects 95 Backup restore...

Страница 5: ...m different procedures using Hstudio such as how to capture an image and how to analyze the images In the third part of the manual there are trouble shooting guides for the software and the hardware T...

Страница 6: ...ctrical equipment for measurement and EN60825 1 2007 safety for laser products This manual includes information and warnings which must be observed by the user It contains information notes as well as...

Страница 7: ...rument or replace the instrument with an instrument free of defects No guarantee is provided for defects caused by natural wear wearing parts in particular or improper use The instrument manufacturer...

Страница 8: ...ue is based on measure ments of how the cells shift the phase of light that passes through the cells and does not require any kind of labeling or staining HoloMonitor is incubator compatible allowing...

Страница 9: ...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 loca...

Страница 10: ...llation will start with prerequi sites installation which launches separate installation programs from third party vendors Depending on the computer configuration this step may not occur Click Install...

Страница 11: ...Holographic Imaging PHI AB All rights reserved www phiab se support phiab se Enter your information Click Next to continue Click Install to start the installation process Click Finish to complete the...

Страница 12: ...page 23 9 8 1 10 13 12 3 4 5 6 7 2 11 HoloMonitor M4 with a motorized stage contains the following parts 1 HoloMonitor with motorized stage 8 Main unit power supply 2 Objective protective cap 9 Laser...

Страница 13: ...e in Remove the six screws holding the plate Turn the instrument back right side up and remove the tape and protective foam 2 REMOVING OBJECTIVE PROTECTIVE CAP Remove the cap protecting the objective...

Страница 14: ...er to avoid any unnecessary discomfort when handling the disconnected optical fiber make sure that the laser beam emerging from the fiber does not radiate directly into somebody s eye Carefully remove...

Страница 15: ...he correct position before tightening the lock ring Connect the power cable to a power outlet 8 ATTACHING THE VESSEL HOLDER HoloMonitor with motorized stage is deliv ered with three different vessel h...

Страница 16: ...capture tab is selected for the first time after starting Hstudio it will automatically suggest a recalibration Select the Live capture tab remove any sample from the stage and click Continue to ente...

Страница 17: ...ptical fiber the USB cable and the Main unit power supply cable through the access port of the incubator and reconnect them to the instrument Finally reconnect the Laser unit power supply to the power...

Страница 18: ...that the optical surfaces are clean calibrate HoloMonitor according to the Calibration procedure page 19 to ensure that the Diag nostic Values are within the green ranges Repeat cleaning if the Diagn...

Страница 19: ...the Live capture tab in Hstudio 2 CALIBRATION WIZARD The three Diagnostic values measure the image quality All values should be in the green range If this is not the case please follow the Cleaning i...

Страница 20: ...and the laser fiber can be kept inside the incubator at all times if it is inconvenient to remove them When detaching the laser fiber use the protective cap of the laser fiber Before re installing Hol...

Страница 21: ...m counting chamber a single use hemocytometer or a cell culture vessel similar to IBIDI slides Cell culture vessels For the best imaging quality use 35 mm Sarstedt Petri dishes with HoloLid IBIDI plas...

Страница 22: ...o 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 acry...

Страница 23: ...built in analysis functions can be used or data can be exported as XML files for further work in Excel or similar programs Cell movement and morphology can be tracked over time through a time lapse se...

Страница 24: ...sic functions controlling the View area are found in the Side panels to the left and right of it Main tabs Side panels Side panels View area If the side panels are collapsed they can be expanded by cl...

Страница 25: ...the right side panel is the Image frame list It controls which Group of images that is currently being viewed or analyzed A group of images may either be images captured at various cell culture posit...

Страница 26: ...ely correct distance from the objective The software focus then finds and calculates the cell images The Software focus panel indicates how well the computer is able to focus an image based on the lig...

Страница 27: ...se support phiab se Below three holographic phase images are shown that are in focus out of focus or completely out of focus A holographic phase image that is in focus A holographic phase image that...

Страница 28: ...image is not correctly focused the white bar will be in the red In that case the mechanical focus needs to be adjusted in order for the software focus to be able to work Focus with a fixed sample stag...

Страница 29: ...late or a different combination of plates until the software focus is in the middle of the green The thicker the plate the further away from the objective the sample will be and the thinner the plate...

Страница 30: ...the left in the Microscope settings panel there is an objective representation and a black horizontal bar that represents the sample stage The mechanical focus distance can be adjusted in large steps...

Страница 31: ...y need to be focused manually Activate Manual in the Software focus panel First the software focus settings must be adjusted The text box next to the Manual button shows the current software focus dis...

Страница 32: ...he stage is moved in large increments by click ing and dragging the objective represen tation i e the small double circle in the Stage position panel Alternatively place the mouse cursor on the desire...

Страница 33: ...ns panel can be activated by checking the boxes Some boxes can be checked in combination thus making it possible to combine functions FFT displays the Fourier transform Uncut displays the image as it...

Страница 34: ...lective visual effect Shiny surface is only visible when Light effect is checked The image below shows a holographic image with cells displayed in 2D and 3D The color bar represents the height in Z wi...

Страница 35: ...on in the drop down Colorset list which is found at the top of the Coloring panel Additional functions are found by clicking the button in the Coloring panel By left clicking the R button the coloring...

Страница 36: ...left click the button and choose Save as To save the current color settings to a previously saved Colorset left click the button and choose Save Note that this will overwrite the settings previously s...

Страница 37: ...p down Project list or create a new project by using the button 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 tim...

Страница 38: ...on the stage A live image will appear in the View area Make sure that you are satisfied with the quality of the live image Select or create a Project and Group Enter the total time for the time lapse...

Страница 39: ...ed or canceled at any time Imaging speed HoloMonitor can take a maximum of 240 images per hour HoloMonitor does not impair the normal growth of your cells as long as the maximum speed is not exceeded...

Страница 40: ...in focus If the cells are not in focus adjust the focus as described in The theory of focusing page 26 When a capture position is selected the current Z position of the stage will be saved and used w...

Страница 41: ...Clearing capture positions To delete a created Capture pattern click Clear pattern Capture images Click the Capture button to start the image capturing sequence Once capture begins the stage will go...

Страница 42: ...wo time lapses captured in each well will be saved in the same group Well A1 and A2 are saved in new groups while the other four wells are saved in existing groups When checking Multiple destination g...

Страница 43: ...e Surfacevibrationsdegradeimagequality Afterstagemovement themedia surface is therefore allowed to settle down before image capturing The needed time depends on the cell culture vessel type Typically...

Страница 44: ...individually it is possible to automatically generate Capture positions by clicking Selection and activating Select wells in the Capture panel Select wells First the relevant wells need to be selecte...

Страница 45: ...enerate the specified number of random positions in the selected wells When Add identical positions to selected wells is checked the same random positions will be generatedineachwell Thegraphicalrepre...

Страница 46: ...e Select positions has been activated capture positions can be generated by simply clicking in the blue areas in the graphical representation of the wells which appears to the left of the Capture pane...

Страница 47: ...o verify focus When hovering over a capture position the dot expands to a square When the square is clicked the stage moves to that position It will be imaged and displayed in the View area at the Z p...

Страница 48: ...he View images tab Select a Project and a Group in the Image frame list found to the right side of the screen An image frame list for the selected group will appear Images as well as information perta...

Страница 49: ...frame list will be presented in order of capture If for example 7 positions have been selected every seventh frame will belong to the time lapse of one of the positions Select the first of the images...

Страница 50: ...displays the image in 3D Rotate automatically rotates the image Rotate is only active when 3D has been checked Show ruler displays a horizontal scale bar representative of the distance in X and Y Sho...

Страница 51: ...ill color the image relatively to the thickness of the cells For additional information on how to set the coloring see Change image coloring page 35 Move flip or zoom the cell image To zoom the cell i...

Страница 52: ...well focused and note the focal distance of that image Select the image that needs to be re focused in the Image frame list Activate Manual in the Software focus side panel Focus the image using the...

Страница 53: ...aging PHI AB All rights reserved www phiab se support phiab se Cell identification Choose the Identify cells tab In the Image frame list panel select the Project Group and the image of interest to mak...

Страница 54: ...ed www phiab se support phiab se The software will then automatically suggest a cell identification CHANGE HOW IDENTIFIED CELLS ARE DISPLAYED In the Options panel it is possible to control how cells a...

Страница 55: ...n the Image frame list ADJUST CELL IDENTIFICATION There are several methods to automatically calculate the Background threshold in the Adjustments tab The background threshold determines which objects...

Страница 56: ...tive mean threshold masks Double adaptive gaussian is the same as Double Otsu but with two adaptive Gaussian threshold masks In addition to the different automatic calculation methods for cell identif...

Страница 57: ...every cell has a cell marker Every identified cell has a border and a blue cell marker Save cell identification adjustments When Auto apply changes is activated adjustments will be saved automatically...

Страница 58: ...well as enlarge or shrink identified cells These functions are found in the Manual changes tab below the View area Add or remove the blue cell markers that identify a cell This results in mergers or s...

Страница 59: ...ab se support phiab se It is possible to undo the last adjustment step to redo the undone step and to remove all adjustments Note that settings made using the Manual changes tab are applied only to in...

Страница 60: ...gh a time lapse sequence and analyzed both for movement and for morp hology changes over time Go to the Cell Tracking tab START TRACKING Start a new tracking Choose the Track cells tab If tracking has...

Страница 61: ...ame list found to the right of the View area To track cells select and add a series of image frames by using the Add selected or the Add all button found below the Image frame list Several images can...

Страница 62: ...h the added images the cell needs to be added to the tracking Activate Add cell in the Select mode panel The center of each identified cell is marked with a small orange Click the to add the cell to t...

Страница 63: ...Show button is clicked in the Warnings tab the previous current and next identification of the cell are shown It is then possible to see if the warning is appropriate or if it can be ignored If the w...

Страница 64: ...om the diagram Remove selected or all cells from tracking The cell movements are given as x and y coordinates over time Initially only the average movement is displayed but by checking the boxes next...

Страница 65: ...lose 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 time lapse Select the cell...

Страница 66: ...in the Change tracking panel The tracking will be discontinued from the present frame A new button Set location will appear in the Change tracking panel Continue a discontinued cell tracking When a c...

Страница 67: ...the tracking will follow one of the daughter cells In order to track both daughter cells select the cell in the frame where it divides and then click Division From then on both daughter cells will hav...

Страница 68: ...the Go to the Tracked cells tab Select the added cell by left clicking on it Select the mother cell in the Child of drop down list The cell will automatically receive a daughter name TRACKING CELL MO...

Страница 69: ...rphological parameters can be tracked For the full list of parameters and their description see Morphological parameters page 106 The following diagram options are found below the diagram Show guide l...

Страница 70: ...a saved cell tracking analysis Analyses are optionally saved when a Tracking tab is closed Additional analyses are started by creating addi tional Cell tracking tabs EXPORT TRACKING DATA In the Track...

Страница 71: ...e measurement is started by left clicking and releasing 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 A...

Страница 72: ...LYZE RESULTS IN PLOT Cell population analysis is performed in the Analyze data tab Start a new analysis Choose the Analyze data tab If data has not been analyzed previously a tab will appear with the...

Страница 73: ...ound to the right of the View area To add data select image frames and transfer to the plot by using the Add selected button found below the Image frame list Several images can be added simultaneously...

Страница 74: ...the plot the data from all images will be displayed in the same plot unless Only current frame is checked When Only current frame is checked only the data from the current frame is displayed Data fro...

Страница 75: ...presented as a colored box If several frames are represented in the plot the data points from all images will be dis played in the plot simultaneously while the frame that is selected in the Image fra...

Страница 76: ...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 Data for that cell will be displayed simultaneously When hovering o...

Страница 77: ...er plot axis range manually The scatter plot settings 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 all values from all a...

Страница 78: ...gion menu will appear Alternatively there is a Define region button below the Scatter plot tab to the right of Auto scale There are four region types X axis region The x axis values are the region cut...

Страница 79: ...lot 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...

Страница 80: ...is offset against the cell count If several regions have been added to the scatter plot they will show up with the appropriate color in the histogram as well Histogram with two arbitrary regions Chang...

Страница 81: ...added frames To set the histogram x axis interval manually Auto settings must be unchecked Enter the minimum maximum values and the number of bins EXPORT PLOTS AND CELL DATA In the Export tab cell da...

Страница 82: ...ly be named with date and a code but it is possible to rename the file Save the file to an appropriate place All data pertaining to the image frames added to the current plot are exported Save plots a...

Страница 83: ...ages for cell count and confluence The frames should be captured at random positions in a cell culture vessel Select or check the images to be analyzed in the Image frame list to the right Click the a...

Страница 84: ...0 15 more images should be added to the analysis The more unevenly the cells are distributed in the cell culture vessel the more images are needed for the cell count ADJUST THE HISTOGRAM PROPORTIONS T...

Страница 85: ...all button To remove data belonging to a single frame from a plot select that frame in the Source frames panel and then use the Remove button EXPORT RESULTS Clicking Save report below the Cell count r...

Страница 86: ...hen one or several images have been added By clicking the Add selected button found below the Image frame list the data from a single image frame or from several frames can be added when the frames ar...

Страница 87: ...yed in the View area Active image Remove buttons Clicking the Remove buttons will either remove the selected or all of the added images EDIT THE IMAGES Zoom move or flip the image The Perspective side...

Страница 88: ...changes to either the added images that are selected or to all added images Adjust the image display The Viewer options panel which is found to the left of the View area is used to adjust the image d...

Страница 89: ...width and height in m Light Effect applies an artificial light source to the image which may sometimes render an improved image Shiny Surface shows the rendered surface with a reflective visual effec...

Страница 90: ...PHI AB All rights reserved www phiab se support phiab se CREATE AN AVI MOVIE When image perspective and color looks good click the Use for all buttons in both the Perspective panel and the Coloring pa...

Страница 91: ...as for the saved movie as the movie can be saved at any speed If the preview looks good click Export movie in the Export panel This will open the Export movie window Select the destination folder by...

Страница 92: ...open the Export images window 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 Use plain Images is...

Страница 93: ...ta go to the Image database handler It can be reached from Database menu or by clicking the Switch button above the Project and Group The Image database handler SWITCH TO EXISTING DATABASE Databases t...

Страница 94: ...Browse to the desired location Name the new database Click Create The software will now use the new database MOVE A DATABASE Select a database in the Database Handler list of databases Click at the bo...

Страница 95: ...of Groups will appear Click the little arrow beside the Group and a list of images will appear Right click on the Project or Group or image you wish move and select Copy Go to the desired database and...

Страница 96: ...ied Project or Group into the backup database To restore a database to the computer Go to the Image database handler Click to create a new database on the computer Browse and find the backup database...

Страница 97: ...te a new folder at e g a USB hard drive Click Start When the process is finished connect the USB hard drive to the second computer Start Hstudio Go to the top menu and click Database Select Import The...

Страница 98: ...he 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 spacer plate Vessels or slides which are thin should be put...

Страница 99: ...c software focus Follow the instructions in Re focus a captured image page 51 Something causes the instrument to vibrate Check that the cables connected to HoloMonitor are slack and not touching anyth...

Страница 100: ...position is unstable The time lapse is drifting in X Y or Z Sometimes there will be a slight drift the first half hour The sample holder is not properly attached Ensure that the sample holder is firm...

Страница 101: ...objective will affect the image negatively Try moving around the culture vessel to different positions Make sure that the laser light has a free path through the vessel Clean the instrument as descri...

Страница 102: ...s been unplugged Remove the instrument from the incubator and let it dry over night in room temperature After drying calibrate HoloMonitor according to the Calibration procedure page 19 to ensure that...

Страница 103: ...been checked no frames will be visible Uncheck Only checked The cell image in the view area is white The image dynamics are not optimal Click the R button in the Coloring panel In a series of capture...

Страница 104: ...djustment tab 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...

Страница 105: ...e Only checked is checked only checked images will be displayed If no image frames in the current Group have been checked no frames will be visible Uncheck Only checked The dots in the scatter plot di...

Страница 106: ...displays the phase delay i e the phase 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 d...

Страница 107: ...e in cellular thickness or by a refractive index change in the cellular area imaged by a pixel CELL MORPHOLOGY PARAMETERS Area The cell area A is calculated in the same way as when using conventional...

Страница 108: ...l a frame is fitted around the cell The frame that fits the cell while covering the smallest area is selected for measurements The parameter depends on the threshold setting that distinguishes backgro...

Страница 109: ...ns are based on a threshold setting that distinguishes background from cell Migration distance The migration distance is the shortest distance between the starting point and the end point of the cell...

Страница 110: ...i divided by 2 Roughness 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...

Страница 111: ...e 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 d...

Страница 112: ...ed by Interferometry Biophysical J Vol 71 3511 3522 Haralick et al 1973 Textural Features for Image Classification IEEE Transactions on Sys tems Man and Cybernetics Vol SMC 3 610 621 M lder et al 2008...

Страница 114: ...P H A S E H OL O G R APHIC I MAGING...

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