FloMax
Software for Cytometry
Operating Manual
- Data Analysis
Contents
General Information ...4
Conventions Used in this Manual ...5
Introduction to FloMax Software ...6
Software Installation ...8
Software Registration ...9
Screen Elements ... 10
Introduction – One-Parameter Histograms... 11
Introduction - Correlated 2 Parameter Dotplots... 13
Introduction – FloMax Start and Exit ... 15
Introduction – Typical Steps for a Immunophenotype Analysis ... 16
Introduction – Typical Steps for a Kinetic Analysis... 17
Introduction – Typical Steps for DNA Cell Cycle Analysis... 18
Introduction – Typical Steps for Microorganism Analysis ... 19
Opening a Flow Cytometry Data File ... 20
Saving an Analyzed Flow Cytometric File... 21
Printing the Histogram Page ... 22
Printer Setup... 23
Selecting the Page Layout ... 24
Selecting Histogram Properties ... 25
Histogram Properties ... 26
Analysing Channel Contents... 28
Absolute Cell Counting / Analysing the Particle Concentration ... 29
Rerun From File Function ... 30
Zoom Function... 31
Gating - Defining Regions... 32
Gating - Applying Gates... 33
Gating - Logical Gates ... 34
Gating - Saving and Loading Gates and Regions ... 35
Gating - Moving, Resizing, and Deleting Regions... 36
Gating - Obtaining Statistics ... 37
Gating – Saving Region Statistics to a File ... 38
Gating – Exporting a Gated File... 39
Gating - Color Gating... 40
Crosstalk Compensation - Overview... 42
Crosstalk Compensation - Setup ... 43
Crosstalk Compensation – Saving and Loading ... 44
Crosstalk Compensation – Exporting a Compensated File ... 45
Crosstalk Compensation - Notes ... 46
Cell Cycle Analysis ... 48
Peak Analysis – Numerical Fit Method ... 49
Peak Analysis – Find Peaks Range Method & Batch Analysis... 50
Peak Analysis – Histogram Export... 51
Ratio Analysis... 52
Calculated Parameters ... 53
Calculated Parameters – Examples... 54
Calculated Parameters – Examples... 55
Calculated Parameters – Function Overview ... 56
Panels - Overview... 57
Panels- Definition... 58
Panels - Optimizing... 59
Generating Reports – Copy & Paste... 60
Generating Reports ... 61
Appendix – Software Support ... 62
Appendix - Mathematical Formulas... 63
Appendix – Computer Recommendations ... 74
Appendix – Software Specifications... 75
FloMax
Software for Cytometry
Operating Manual – Data Analysis
Version 2.3
General Information
QuestionsDisclaimer
Trademarks and Copyrights
FloMax is made for you! New software versions and you profit from your requests for new features or software changes. Partec is continuously working on software to fulfill your demands. If you have questions concerning this manual or the software, if you find problems associated with FloMax, or if you have a good suggestion to be included in a new version, please let us know by sending an email or a note to Partec.
FloMax software and this manual are intended to be used by experienced flow cytometrists. FloMax was developed and tested to make flow cytometry data analysis more comfortable. Complex software like FloMax and this manual however do not claim to be completely error-free. As with all software, results obtained with FloMax should be checked and verified critically by the user. Partec recommends to test FloMax for a specific application before running large scale sample series. Partec provides FloMax “as is”. Partec does not take responsibility that FloMax is suited for a specific application. Partec also does not take responsibility for any direct or indirect damage that could be caused by FloMax or based on results obtained by FloMax. Especially no responsibility can be claimed for data or reagent loss due to the use of FloMax.
This manual contains references to names and products from Partec and other companies which are registered trademarks or protected by copyright.
Partec GmbH: PAS, PA, CCA, Robby®. Quantum Analysis GmbH: FloMax®. Cytecs GmbH: CyFlow®.
Microsoft® Corp.: Windows, Word, Excel, PowerPoint, Paint.
Hewlett Packard®: Deskjet, Laserjet.
Conventions Used in this Manual
Selection of Menu FunctionsNotes and Tips
Mouse Operation
The location of menu selections is frequently noted in a short form as in the following example:
Analysis – Calculate Parameters – Formula…
denotes the following menu selection:
Notes and tips are denoted by this symbol.
click – single click with the left mouse key.
doubleclick – two consecutive clicks with the left
mouse key.
Introduction to FloMax Software
What...
... is Partec FloMax?
… topics are covered by this manual?
… other manuals are available?
… if my cytometer isn’t a Partec?
… are the applications for which FloMax can be used?
Partec FloMax software is an all-in-one solution for acquisition and analysis of flow cytometric data. FloMax data analysis works with data from flow cytometers that support the flow cytometry file standard (FCS). FloMax operates on computers with Windows 95, 98, and 2000. Data acquisition and instrument control by FloMax is provided for Partec Flow Cytometers.
FloMax – Data Analysis (this manual) covers all
aspects concerning the flow cytometric data analysis. Most of the functions are available online during acquisition as well as offline for files already stored.
FloMax - Instrument Control and Data
Acquisition covers additional functions for use on
a Partec flow cytometer.
Please consult the Instrument Operating
Manuals for your particular flow cytometer for
details on the instrument operation.
Application Notes and Tutorials are available to
get started and contain hints to achieve the best results.
FloMax supports the FCS (flow cytometry standard) data format. This makes FloMax suitable for data analysis from any flow cytometer, offering unique functionality and automation. FloMax offers automation for routine use and flexibility for research use for practically any flow cytometric application. The applications cover:
• Routine Immunophenotype Blood Cell Analysis
• Leukocyte Counting
• Rare Event Analysis
• DNA Cell Cycle Analysis
• Ploidy Analysis
• Microorganism Analysis (live/dead)
• Fermentation Control
• Particle Concentration Analysis
• True Volumetric Absolute Counting
• Particle Size and Fluorescence Distribution Analysis
Introduction to FloMax Software
What...
… functions are offered by FloMax?
… additional features offers FloMax in
conjunction with Partec flow cytometers and sample automates?
Instrument control and data acquisition functions are covered in a separate manual.
... should I know before operating FloMax?
... can I do in case I have questions or problems?
FCS Data Analysis and Report Generation
• Flexible data display on pages with 1-8 one- and two-parameter histograms and dotplots
• Multiparameter Gating with logical gates
• MultiColor Gating
• Colour Crosstalk Compensation by Software
• DNA Cell Cycle and Peak Analysis
• Calculated Parameters
• Panel System
• Automated Report Generation in conjunction with MS Word® and Excel®.
Instrument Control and Data Acquisition
• Flow cytometer instrument control
• Realtime Acquisition
• True Volumetric Absolute Counting
• Complete walk-away sample preparation and analysis
This manual assumes you have basic knowledge about flow cytometry. In the best case a well experienced "flower" is around - so let her/him help you. Many basic books are available about flow cytometry which may help you as well. FloMax makes operation as simple as possible. When you are familiar with Windows and other Windows software, you will find it quite easy to operate FloMax. We at Partec are proud if we may assist you with any question you have with your application. Don’t hesitate to contact us! Partec continuously publishes application notes which might contain important hints not covered by this manual. Please inquire for the latest list of application notes.
Partec is available for your support. Don’t hesitate to contact us by email, fax or telephone.
Software Installation
FloMax Installation CD.
If automatic launching of installation programs is disabled on your computer, the installation program does not start automatically. In this case, select Start-Run… in the Windows toolbar and type D:\setup.exe. Press OK and proceed with step 3.
Shortguide for FloMax Installation
1. Archivate existing FloMax folder: E. g. rename C:\FloMax to C:\FloMax20.
IMPORTANT: If you don’t archivate your existing version it will be overwritten.
2. Insert the FloMax Installation CD
– after some seconds, the FloMax installation programm starts.
3. Follow the instructions given by the installation program. Use the defaults whenever possible. - FloMax will be completely installed.
- The FloMax icon appears on the desktop (and under Start - Programs).
Software Registration
About FloMax box.
Software Registration Dialog.
Software Registration Form.
Each computer requires a separate registration. For each laboratory computer, a separate registration form should be submitted to Partec. With a full license, you may inquire registration codes for one or more computers in your laboratory you are using.
Software Registration
In order to offer you the best possible support for FloMax, you should register for a full license. A full license allows you to use FloMax on one or more computers in your laboratory which are under your responsibility.
Without software registration, FloMax can be run in a “demo mode” with limited options.
How to get the registration code
If you purchased FloMax or a flow cytometer together with FloMax, in order to receive your personal registration code:
1. Make sure FloMax is installed on your computer and start FloMax.
- When FloMax is not registered yet, the Software Registration dialog appears asking you for registration.
or
Select Help-About FloMax… in the menu - the About FloMax box appears.
Click Register…
- the Registration dialog appears. 3. Click Get Registration Code… – the Software Registration Form appears.
4. Fill out the form completely with your name, address, and all other information.
5. Click “Print Registration Form”, print the form on a printer.
Fax the form to Partec to the fax no. given below.
or
Call Partec at the phone no. below.
or
Click Editor… and cut and paste the registration form to an e-mail. Send the e-mail to the address below. This is the preferred way.
- Within a short perioud, you will receive the FloMax registration code by Partec.
FloMax Registration
Partec GmbH, Münster, Germany Phone +49 2534 8008-0
Fax +49 2534 8008-90 E-Mail [email protected]
Screen Elements
Elements on the FloMax software screen. Selection
Frame with Trackers Menu Bar
Plot Selection Marks
Dialog
Status Bar Toolbar Tooltip Title Bar
Buttons Plots
Scrollbar Plot Property Box
Results Field
Introduction – One-Parameter Histograms
What is...
… a parameter?
... a one-parameter histogram?
In flow cytometry, parameter denotes a measured property of the particles. Frequently, a parameter
is synonymeous to an optical channel. E.g. an instrument with 6 parameters is equipped with 6 optical detectors. Parameter no. 6 could e.g. be the blue fluorescence parameter that could be called FL4 and mainly be used for DNA analysis. A one- parameter histogram displays the
distribution of cell contents, e.g. how many
cells contain a given quantity of DNA or bind a given number of antibody molecules.
... a histogram channel?
... the count in a histogram?
... a peak?
... background in a histogram?
... the lower level (L-L)?
The cell content is assigned to one of many (e.g. 65536) quantity classes or channels. In a one-parameter histogram the channels are represented on the x-axis.
The number of cells being assigned to a given
channel is referred to as channel content or
simply count. In a one-parameter histogram, the count is shown on the y-axis.
All cells having about equal quantity of the cell
content, e.g. DNA, form a peak. In the case a of
typical DNA histogram e.g., one peak represents the G1 and another (with twice the channel value) represents the G2/M phase of the cell cycle. In the case of immunolabelled cells, there is usually one peak for unlabelled (negative) and one peak for labelled (positive) cells.
Peaks can be analyzed by drawing ranges or by numerical fits, e.g. in order to know the mean intensity or number of cells in a peak.
Histograms sometimes show undesired signals in the lower channels, frequently called noise or
background. These signals can originate from
cell fragments or other particles resulting from
sample preparation. In case of high gains, background can also be caused by not sufficiently clean sheath fluid or by background light.
The lower level (L-L) threshold is a means to
suppress background signals. Signals below the
lower level are rejected from the histogram during acquisition. To exclude noise from a histogram already acquired, a range-gate can be used.
Introduction – One-Parameter Histograms
One-parameter histogram with 512 channels. EAT cells (DAPI-staining, HBO-lamp excitation). Background due to cell fragments.
One parameter histogram of immunolabelled lymphocytes (green fluorescence excited by Ar-laser). The unlabelled cells (“neg.”) appear in the left peak (autofluorescence and unspecific antibody binding); brightly
background lower level threshold
peak of normal G1-cells
peak of tumor G1-cells
peak of tumor (G2+M)-cells
channels counts
Introduction - Correlated 2 Parameter Dotplots
What is ...
... a correlated 2 parameter dotplot?
... a cluster?
In a correlated 2 parameter dotplot, quantities of the cell properties, e.g. FSC (forward scatter) and the SSC (side scatter) intensity, are assigned to channels on the x- and y-axis.
Each cell with a given FSC and SSC intensity is represented by one dot in the dotplot. Several cells with the same FSC/SSC combination share the same dot location. The number of cells in a channel can be represented by a color, according to a color scale.
In a dotplot, subpopulations of cells with about
equal properties appear as clusters. These
clusters can be circled by drawing lines around them for more detailled analysis, e.g. in order to analyse the number of cells in this cluster. The process of analysing subpoulations is called "gating".
2 parameter dotplot of leukocytes with 256 x 256 channels. Side scatter intensity (SSC) is plotted versus the forward scatter intensity (FSC) in a linear scale. Colors (or grey levels) are a measure of the channel content. The dotplot shows the analysis of a blood cell sample. Preparation with Partec CyLyse. The circled cluster originates from the lymphocyte-subpopulation.
Introduction - Correlated 2 Parameter Dotplots
What ...
... if data falls outside a histogram range? If parameter values fall outside of the histogram
scale, e.g values are < 0.1 on a 4 decade logarithmic scale, FloMax will put them onto the histogram axis. These values will be taken into account e.g. in quadrant statistics.
2 parameter dotplot of a CD3/CD8 fluorescence analysis of lymphocytes with 256 x 256 channels. Note the logarithmic scale spanning a range of 4 decades. Data points below a relative value of 0.1 (circled) will reside on the axis. They are taken into account for quadrant statistics.
Introduction – FloMax Start and Exit
FloMax Icon on the desktop.
Welcome dialog.
Initial histogram page.
Start FloMax
1. Doubleclick on Partec FloMax Icon on the desktop
or
select Start-Programs - Partec FloMax from the Windows Start-button
- FloMax starts and displays the Welcome dialog. 2. Press OK
- FloMax shows an initial page with histograms.
Exit FloMax
Make sure the actual document is saved. Click on application close button
or
select File-Exit - FloMax is finished.
If changes to the actual open document have been made and not saved yet, FloMax asks to save the document before finishing. Refer to page 21 on how to save files.
Introduction – Typical Steps for a Immunophenotype Analysis
Open button
View – Page Layout…
Right click on histograms
or Analysis – Create Report…
1. Open FCS file.
2. Select proper display.
3. Select parameters to display.
4. Define lymphocyte gate.
5. Apply gate to histograms.
6. Compensate crosstalk.
7. Set quadrants.
8. Save FCS file.
Introduction – Typical Steps for a Kinetic Analysis
Open button
View – Page Layout…
Right click on histogram
Kinetics dotplot with time slice regions.
Analysis – Region Statistics…
Region Statistics dialog.
Start – Programs - Excel
Kinetics plot generated with Microsoft Excel.
1. Load FCS file.
2. Select proper display.
3. Select a kinetic dotplot with the dynamic parameter (usually a fluorescence parameter) vs. the time parameter.
4. Enter square regions in equidistant positions to define a number of time slices.
5. Select Analysis-Region Statistics…
- the region statistics displays the mean fluorescence values for each time slice.
6. Click Save to File… in the Region Statistics dialog
- the statistics results are saved to an ASCII text file.
7. Invoke e.g. Excel and open the text file. 8. Select Insert Graph… to generate a graph
from the mean values column. 9. Save/Print the Excel document.
Once the time slice regions are defined, click
Save… in the Gating dialog
- time slice regions can then be reload to analyse other flow files.
It might be of help to use the Zoom function before defining the time slice regions and possibly to select higher resolution data display for the dotplot for more precise entry.
If data was acquired without a time parameter,
the Analysis - Calculate Parameters… feature
of FloMax allows to generate an event number or “pseudo time” parameter that can be used for kinetic analysis, if the acquisition took place with a constant particle rate.
Introduction – Typical Steps for DNA Cell Cycle Analysis
Typical cell cycle analysis analysis based on a numerical fit: G1, S, and G2/M phase of a CHO cell line.
1. Load FCS file.
2. Perform Cell Cycle Analysis.
Introduction – Typical Steps for Microorganism Analysis
Typical microorganism analysis example: Bacillus subtilis spores (red) and vegetative cells (blue).
1. Load file.
2. Define microorganism regions.
3. Save file.
Opening a Flow Cytometry Data File
Open button
Open dialog box
Previous/Next File
In case you forgot where the file was located, you may use the Windows search function in the Start menu.
The file data format must be according to flow cytometry data standard FCS 2.0.
Open function
The open function reloads the results which have been saved in a file before.
Click on Open button in the toolbar - the Open
dialog box appears.
or
Click File in the menu bar - the file menu
appears.
Click Open... in the file menu - the Open dialog box appears.
In the Look in field, select the folder where the file is located.
Click on the file to be opened. The standard extension is .fcs.
Click Open button - the file is opened and the results are displayed.
or
Click the Previous File or Next File button in the toolbar - the actual file will be closed and the previous/next file in the current folder in alphabetical order will be opened.
You can also use the optional barcode reader to facilitate the selection of files, e.g. by using the sample barcode as filename.
Saving an Analyzed Flow Cytometric File
Save button
A previously opened file is overwritten by the
Save function. Generally, it is recommended to
use Save As function which always asks for a file
name.
Save As dialog box
Make use of different folders. E.g. use a different folder for each month or for each series of samples. This will help you when later locating your data.
Save function
The save function saves the results in a file with the name given in the title bar:
Click on Save button in the toolbar - the file is saved.
or
Click File in the menu bar - the file menu
appears.
Click Save in the file menu - the file is saved. In case no file name was specified yet, e.g. for a new document, the Save As dialog box appears (see below).
Save As function
The Save As function will ask for a file name before saving:
Click File in the menu bar - the file menu
appears.
Click Save As... in the file menu - the Save As
dialog box appears.
In the Save in field, select the folder where the file is to be saved.
In the File name field, enter a file name.
Do not enter the extension - the file automatically will get the extension .fcs used for flow cytometry standard files.
Click Save button - the file is saved.
You may use long file names as supported by Windows (up to 256 characters).
However, if you plan to use other software for further analysis (e.g. WinMDI), it might be possible only to recognize 8 character long filenames.
You can directly save a file to another computer in your computer network by selecting the appropriate network folder. Refer to Windows manuals for details.
Using Sample Barcodes
You can also use the (optional) barcode reader to specify e.g. sample bacodes as file names. Barcodes can be generated by optional barcode software.
Printing the Histogram Page
Print Now button.
Print dialog.
Printing status indication.
You can select a portrait (smaller) or
landscape (larger) printout by selecting a paper
orientation after clicking on the Properties button in the Print dialog box.
Wait for the printing to complete before starting a new acquisition. Acquisition during printing may cause reduced counting accuracy.
You may also use the report generator to print data according to your report form. In the reports, you can use annotations and your laboratory logo.
Printing the histogram page
To print the actual histogram page: Make sure the file is saved.
Click the PrintNow button in the toolbar
or
Click File in the menu bar - the file menu
appears.
Click Print Now... in the file menu toolbar - the histogram page is printed
or
Click File in the menu bar - the file menu
appears.
Click Print... in the file menu - the Print dialog box appears.
Check the printer settings and click OK - the histogram page is printed.
The status indication in the taskbar notifies an ongoing printing process.
Print Preview
In order to display a preview of the printed page on the computer display, a print preview can be selected.
Click File in the menu bar - the file menu
appears.
Click Print Preview... in the file menu - the print preview appears.
Move the Zoom glass by the mouse and click on regions of interest to see more details.
Click on Close button to finish the preview.
Printing is performed with higher resolution than the display screen. Printing allows to achieve high resolution histograms and dotplots, e.g. with more than 1000 channels.
Printer Setup
Printing is tested and guaranteed to work well for a series of printers (see appendix). Therefore, all these printers are recommended.
However, printing works on almost any printer supporting Windows. Most printers require the installation of a printer driver software. The printer driver usually can be found either a) on disks provided by the printer manufacturer or b) on the Windows CD.
Change printer settings
For permanently change of the printer settings, e.g. to select another paper orientation, click File
in the menu bar - the file menu appears.
Click Print Setup... in the file menu - the print
dialog box appears.
Make sure the selected the printer is the one which is actually connected.
Click the Properties button. Change the printer settings. Click OK.
Selecting the Page Layout
After selection of the basic layout, the type of the histograms (e.g. 1 parameter / 2 parameter) and parameters on the histogram axis can be selected by right-clicking onto the histograms. Alternatively, ready prepared histogram templates with all selections can be used as well.
When switching to a lower number of histogram plots, the first plots of the page will be displayed. E.g. 8 initially plots:
[1] [2] [3] [4] [5] [6] [7] [8] 3 plots: [2] [1] [3] 1 plot: [1]
Selecting the page layout
The page layouts allow a quick change or setup of the histogram page. Here, the number of histograms on the page can be selected.
The page layout also allows limitation of the analysis and display to a given range of particles, e.g. the first 10000 lymphocytes.
Selecting a predefined page layouts
Click View in the menu bar - the view menu
appears.
Click Page Layout... in the view menu - the Page
Layout dialog box appears.
To select the number of plots (histograms or dotplots) on the page, click on one of the page layout buttons.
Selecting a range for data analysis and display
To limit the data display and analysis to a range of events (cells), click on one of the Event Range options:
All Events: all events of the file used.
Events in Counting Volume: Only events in the
counting volume are used (requires volumetric absolute counting from a Partec flow cytometer).
First 10000/20000 Events: The first 10000/20000
events of the file are used.
Preselected Events: Events between the two
limits below (from-to) are used.
Event Count Gate: A previously defined gate can
be specified to define the event range for a gate. Cannot be used together with Events in Counting Volume.
Select a gate to use a given number of subpopulations of cells.
Example: Exactly 10000 lymphocytes can be analysed and displayed in cases the file contains data from more lymphocytes.
Selecting Histogram Properties
You may create a histogram template once with all the histogram settings optimized for your application. To create a histogram template, select type and properties of the histograms and save it with File-Save As..., as a regular FCS file without data.
To use a histogram template for a new acquisition, open this template (FCS file without data) before starting an acquisition.
You may also load a usual FCS file with data and use the layout stored within the file as a template.
For each of the histograms on the page, its type and properties can be individually changed.
Open the histogram property box
Right click on a histogram
or
doubleclick on a histogram
- the property box for that histogram is opened.
Select the histogram type
To select a 1P histogram or a 2P dotplot, click on one of the radio buttons:
• 1P Histogram
• 2P Dotplot
Change the histogram properties
See the following pages about the properties and settings.
To apply the settings, click Apply - new settings are applied to the histogram and the histogram property box stays open.
Click Close - new settings are applied to the histogram and the histogram property box is closed.
You can simply switch to the property box of another histogram by right clicking onto that histogram while the property box remains open.
Black plot selection marks show which plot is actually selected.
Histogram Properties
In the parameter selection list, only parameters which were (or are) stored in the previously loaded file or enabled for acquisition can be selected.
Location of parameter names and labels
In case of a linear scale, the numbers on the histogram axis correspond to channel numbers. In case of logarithmic scaling, the axis always cover a range up to 1000.
Parameter
Click on the little arrow at the parameter-box - a list of the parameters appears.
Click one of the parameters.
Label
Click on the little arrow at the label-box - a list of labels appears.
Click one of the labels.
or
Enter a label for the parameter by keyboard.
Color (1P histograms only)
Click on the little arrow at the color-box - a list of available colours appears.
Click one of the colors.
Channels
Click on the little arrow at axis channels-box - a list of available channel resolutions appears. Click on the desired number of channels.
Automatic Rescale
Click on this to enable/disable an automatic vertical rescaling during acquisition or rerun from file.
y-Axis Maximum (1P histograms only)
Enter a number to define which y-scale will be used for the histogram.
z-Axis Maximum (2P histograms only)
Enter a number to define which channel content will be used for the last color of the color scale.
Optimize
To optimize the channel content scale according to the histogram or dotplot, click this button.
Optimize All
All histograms are scaled optimally at once by one click to this button.
Parameter Label Parameter
Name Gate
Histogram Properties
Scatter dotplot: z-level = 0
Scatter dotplot: z-level = 6
z-level (2P dotplots only)
Enter a number to define which is the minimum channel content to be displayed. You may use this to separate subpopulation clusters visually.
Gate
To activate a gate for the histogram or dotplot, click on the little arrow at the gating-box - a list of available regions and gates appears.
Click on the desired region or gate.
Color Gating
Switches between color gating display and z-level-color display.
Analysing Channel Contents
Channel Analysis in one parameter histogram.
Channel Analysis in two parameter dotplot.
Channel contents are not displayed for dotplots which show color gated data. Make sure color gating is switched off for the plot before analysing channels.
In case of a linear scale, the numbers on the histogram axis correspond to channel numbers. In case of logarithmic scaling, the axis always cover a range up to 1000.
Analysing Channel Positions and Content
Move mouse cursor across a one parameter histogram or two parameter dotplot.
- the mouse cursor changes to a crosshair and the status line displays information on the channel number, position, and content.
Meaning of Channel Information
a) One parameter histograms Example: x=166 (39.2) count = 198 channel number: 166 relative intensity value: 39.2 count: 198 b) Two parameter dotplots
Example: x=171, y=64 (47.0, 1.0) count = 2 x-channel number: 171 y-channel number: 64 x-relative intensity value: 47.0 y-relative intensity value: 1.0
count: 2
Channel Number
The range of channel numbers depends on the axis resolution set for the histogram. Typical values are 0…255 or 0…1023. For a log scale channel values do not correspond directly to intensity values.
Relative Intensity Value
The range of relative intensity values is [0…1000] independent of the scale or histogram resolution used. These values takes into account the scale used (linear, 3 or 4 decade logarithmic). For log scale histograms, relative intensity values correspond to the axis values.
Count
The count is the channel content or number of particles of the channel currently addressed. crosshair cursor
Absolute Cell Counting / Analysing the Particle Concentration
The Partec True Volumetric Absolute Counting system allows to analyse the concentration of cell subpopulations during or any time after acquisition, e.g. by defining or adjusting regions and gates, provided a count phase has been completed.
FloMax displays the particle concentrations in the analysed tube. Remember to take into account possible dilution factors.
CD34+ volumetric absolute counting without reference beads.
For data for which a volumetric absolute count has been performed, concentration results of all or a subset of cells are displayed.
The concentration, i.e. the number of all analysed
particles per ml is shown in the result line on top of
the histogram page.
The result line also displays the total number of analysed particles.
The concentration of particle subpopulations is displayed in the region statistics field for each region or gate.
For quadrants, the concentration of particle-subpopulations is additionally directly displayed in the quadrants.
Two methods can be used for absolute cell counting
i) a reference method,
ii) Partec’s unique volumetric absolute counting method.
Direct display of the concentration of cell subsets is supported for method (ii). For (i), the ratio of counts from the subpopulation and the reference beads has to be multiplied with the specified bead concentration.
Rerun From File Function
Rerun button.
For huge files with several hundred thousand events and complex displays, the rerun function can take some tens of seconds, depending on the computer speed. FloMax displays a progression percentage in the status line.
For a simpe “replay” animation, data from the active file can be rerun in the sequence of acquisition (but faster). This can be a simple means to check the stability of the acquisition, e.g. if peaks shifted over time. It might also help to demonstrate how an acquisition proceeds.
Click Rerun button in the toolbar or
Select Analysis – Rerun From File in the menu or
Simultaneously press Ctrl and R on the keyboard - data is rerun and reclassified into the histograms as during acquisition.
Zoom Function
Zoom button.
Zoomed histogram page for precise region entry.
The Zoom function can also be used to generate high quality copy & paste results, e.g. for publications.
For more zooming, select View-Zoom+ and
Zoom- in the menu.
Using the Zoom Function
For a closer look to the data plots, a zoom function is provided. The Zoom function magnifies an area of interest. It e.g. helps to precisely define regions when using histogram pages with 6 or 8 plots.
1. Click the Zoom-button in the toolbar
or
Press F2 on the keyboard
or
Select View-Quick Zoom in the menu
- the screen zooms to a portion of the histogram page.
2. Click on the scroll bars to move to the plot of interest.
3. Perform the analysis which requires a closer look.
4. Click the Zoom-button in the toolbar again - the screen reverts to normal size.
Gating - Defining Regions
Entry of a polygon to define the lymphocyte cluster.
Quadrant to define lymphocyte subpopulations.
Range in one-parameter histogram.
See next page on how to apply gates.
Why Gating?
By gating, subpopulations of particles can be analyzed. The display and statistical evaluation can be reduced to a subpopulation defined by a gate. Typically, a gate is a simple range in a 1P histogram or a polygon region in a 2P dotplot. Gates can also consist of a logical combination of ranges, polygon regions, and quadrants.
To perform gating, two steps are required: 1. Defining gates (or regions).
2. Applying gates to histograms.
Defining regions
Click on one of the gating region buttons in the toolbar or click on the G-button - the gating dialog appears.
Polygon entry in a 2P dotplot 1. Click the polygon button.
2. If desired, specify a name for the polygon in the Region entry of the gating dialog. If not specified, default names as R1, R2 etc. are used.
3. Click the first point of a polygon in a 2P dotplot. 4. Draw the polygon,
a) click once for every corner of the polygon
or
b) draw a region while keeping the mouse key pressed.
5. To close the polygon,
a) click in the box around the first point
or
b) press the right mouse button - the polygon is closed and defined.
Quadrant entry in a 2P dotplot 1. Click the quadrant button.
2. If desired, specify a basic name for the quadrants in the Region entry of the gating dialog. If not specified, standard names as Q1-Q4, QA1-QA4 etc. are used.
3. Move quadrant cross to desired point in a 2P dotplot and enter by mouse click
- four quadrant regions are defined.
Range entry in a 1P histogram 1. Click range button.
2. If desired, specify a name for the ranges in the Region entry of the gating dialog. If not specified, standard names as RN1, RN2 etc. are used. 3. Move cursor to left border of desired channel range in a 1P histogram. Enter left border by mouse click.
4. Move to right border of range. Enter right border by mouse click.
Gating - Applying Gates
If a gate is applied to a histogram, this is displayed at the top of the histogram.
2P dotplot of FL1 - FL2, gated by a polygon region "Lympho" defined in the FSC - SSC dotplot.
Gating can be applied during or after an acquisition: online and offline gating.
In any case, all events are saved. This allows to completely reanalyse the measurement, starting from the original data.
Applying gates or regions to histograms
1. Right click the histogram for which a gate is to be applyed - the histogram property box is displayed.
2. Click the little arrow in the gate drop down list and select the gate or region.
3. Press OK - the gate is applied and the histogram property box is closed.
Removing gates or regions from a histogram
1. Right click the histogram for which a gate is to be applyed - the histogram property box is displayed.
2. Click the little arrow in the gate drop down list and select the <None>.
3. Press OK - the gate is removed from the histogram and the histogram property box is closed.
Gating - Logical Gates
Logical Gates dialog
It is not neccessary to define gates for simple regions, e.g. G1=R1. Simple regions can be directly applied as a gate.
Defining logical gates
If not already opened, open the gating dialog by pressing the gating button in the toolbar.
Click Logical Gates…
- the Logical Gates dialog appears.
In the Logical Gates dialog, select a name for the gate and assign to it a logical gate expression. As an example to define a gate for particles belonging to R1 and R2 as well, enter
G1=R1 AND R2
By using the operands AND, OR and NOT
combined with the brackets ( ), any logical combination of regions is possible.
You may select regions from the regions list and use the buttons to enter operands and brackets. Press the [ = ] button to validate the gate definition.
Note: CLR clears the entry for a gate.
Press OK – the logical gates appear in the left gates list and can now be used for gating.
Gating - Saving and Loading Gates and Regions
Regions, ranges and quadrants are stored in a mathematical space spanned by the parameters. E.g. when quadrants are defined for a FL1-FL2 dotplot, they will allways appear in a FL1-FL2 plot, no matter where this plot is displayed on the screen. The quadrants will even stay defined if no FL1-FL2 plot is on the screen at all.
Saving gates and regions
A complete set of gates and regions may be saved in a file. These gates can later be reload and applied to other measurements.
1. Press G= to open the gating dialog.
2. Click on Save... in the gating dialog - the Save As dialog box appears.
3. In the Save in field, select the folder where the gating file is to be saved.
4. In the File name field, enter a file name. It is not neccessary to enter the extension - the file automatically will get the extension .gat used for gating files.
5. Click Save button - the gating file is saved.
Loading gates and regions
1. Press G= to open the gating dialog.
2. Click on Load... in the gating dialog - the Open
dialog box appears.
3. In the Look in field, select the folder where the gating file has been saved.
4. Click on the gating file to be opened. The extension is .gat.
5. Click Open button the gating file is opened -gates and regions are shown in the histograms.
Saving a gating strategy protocol
A complete gating strategy, consisting of gates, regions and the histograms where they are applied, can be stored within a histogram template, which is nothing else than a FCS file without data. To use this,
1. Prepare a histogram template with suited histogram types and parameters on the axis. Tip: Reload a measurement.
2. Define regions and gates and apply these to the histograms.
3. Save this file by the File - Save As... function as a FCS file.
Loading a gating strategy protocol
Load a histogram template or measurement which contains a gating strategy. This can be used for new acquisitions.
Gating - Moving, Resizing, and Deleting Regions
To delete all gates and regions, press Clear All
in the gating dialog.
Moving regions
1. Select the polygon or quadrant region to move: click with the mouse near the region or quadrant center - a selection frame appears.
2. While the mouse is above the selection frame, a double cross is displayed. While holding down the left mouse button, move the region.
3. Release the mouse button - the region is moved and region statistics are updated.
Resizing polygon regions
1. To select the polygon or quadrant region to resize, click with the mouse near the region or quadrant center - a selection frame appears. 2. While the mouse is above one of the square trackers, a double arrow is displayed. While holding down the left mouse button, move the tracker.
3. Release the mouse button - the region is resized and region statistics are updated.
Deleting regions
1. To select the polygon or quadrant region to delete, click with the mouse near the region or quadrant center - a selection frame appears. 2. Press the Del key on the keyboard - the region or quadrants are deleted.
or
1. Open the gating dialog with the gating button. 2. In the Gates drop down list, select the region, quadrant, or range to delete.
3. Press the Delet button in the gating dialog - the region is deleted.
Deleting logical gates
1. Open the gating dialog with the gating button. 2. In the Gates drop down list, select logical gate to delete.
3. Press the Delet button in the gating dialog - the logical gate is deleted.
Gating - Obtaining Statistics
For log-scale data, geometrical mean, mean and CV-calculations are performed after transforming all data to the linear scale first. See mathematical appendix for details of calculations.
For each of the polygon regions, quadrants or 1P ranges defined, various statistical information can be displayed.
Select display of region statistics
Make sure View - Region Statistics is checked in the menu
- results are displayed on the lower part of the histogram page.
Use the scroll bar on the right side or reduce the size of the histogram window to display results at the lower part of the histogram page.
Select statistics information
To specify which results are to be displayed 1. Select Analysis - Region Statistics... in the menu
- the Region Statistics box appears, containing the regions defined so far.
2. Select the results to show/to hide. 3. Click Close
- the Region Statistics box is closed and the results are displayed on the lower part of the histogram page.
Region Statistics Results
Region name of the region
Gate name of the gate in effect. <None> for ungated results.
Ungated total number of cells or particles
belonging to the region.
Count number of particles belonging to the
region, gated by the given gate.
Count/ml number of particles in the region per
ml. Only available if absolute counting has been performed. Gated, if a gate is specified.
% Gated percentage of particles related to the
number of particles in the given gate, if a gate is active.
% Total percentage of particles related to all
particles.
GMean geometrical mean of the region in x-
or y- direction.
Mean mean of the region in x- or y-direction ("center of gravity").
CV% relative coefficient of variation (standard deviation divided by mean value) of the region in x- or y-direction.
Gating – Saving Region Statistics to a File
The Save to File function generates an ASCII text file in “tab-text” format. This format can be imported as a data table by almost any other software. FloMax adds the extension “.txt” to the exported file.
To import the region statistics from the ASCII text file, select the appropriate import function in the other software. Be sure to specify the correct extension (.txt). Select a “tab-text” format.
Saving Region Statistics to a File
The region statistics results can be saved to a text file (ASCII) for export and simple further evaluation by other software, e.g. Word or Excel. 1. Select Analysis - Region Statistics... in the menu
- the Region Statistics box appears, containing the statistical results.
2. Select the results of interest. 3. Press Save To File... - a Save As dialog appears.
4. Specify filename and folder for the export file and press OK
- Region Statistics is saved in an tab-text ASCII file.
Gating – Exporting a Gated File
To import gated data by other software, select FCS as data type and “.A” (for Analysis) as file extension.
The generation of a second gated or compensated file as specified in Save Options will be active unless explicitely deactivated. This facilitates rountine operation when e.g. always having to save gated files.
When no more required, be sure to deactivate the generation of a gated/compensated file by clearing the save options.
Exporting a Gated File
For other evaluation software it might be necessary to work on data which is already analysed to some degree, e.g. gated or compensated. E.g. an external cell cycle analysis software could be used to evaluate subpopulations contained in the FCS data set, but might itself not offer gating options.
For this reason, FloMax offers the export of gated or compensated files.
1. Define the gates, as decribed further above. 2. Select FIle-Save Options…
- the Save Options dialog appears.
3. In the gate drop list, select the gate to use for the export.
4. Enable the “Gated by” checkmark.
5. Press OK – the Save Options dialog is closed. 6. Select File-Save As… - the Save As dialog appears.
7. Select a folder and a filename and press OK - Together with a normal FCS file with all data, a second file with the same name plus an extension “.A” is saved. The “.A”-file is a regular FCS file but only contains the gated data.
Example
Filename selected in Save As is “EAT” – two FCS files are generated:
1. EAT.FCS, containing all data.
Gating - Color Gating
MultiColor Gating. Circle overlaps are associated with an independant color each.
The color gating setting is stored within FCS files or, together with the regions and gates, in separate gating files, which can be applied easily.
Color Gating
Color gating is used for simple visual association of cell-clusters in different dotplots.
Generally, by color gating, a unique color is associated to regions or gates, called “color gates”. Cells belonging to a color gate, are then displayed with the associated color.
However, things may become more complicated when a cell belongs to more than one color gate. FloMax offers two different color gating modes for these cases:
• Priority Color Gating
• MultiColor Gating
Priority Color Gating
• Events in color gates are displayed on top of each other, according to the priority: color gate 1 > color gate 2 > color gate 3.
• The color for each of the three color gates can be freely selected.
• Any range, region, quadrant, or logical gate (logical combination of ranges, regions, quadrants) can be associated with color gate 1, 2, or 3.
MultiColor Gating
• All combinations of color gates are displayed by an independant color.
• All colors are freely selectable.
Gating Procedure
1. Define polygon-regions, quadrants, and one-parameter ranges in dotplots and histograms, e.g. R1 = “Lymphs”, R2, Q1...Q4, QA1...QA4, RN1.
2. Define logical gates, as required, e.g. “G1 = Lymphs AND Q2”.
3. From the Gating dialog, click “Multi Color Gating...”
- the Color Gates dialog appears.
4. Select the color gating mode: MultiColor. 5. Select the regions/gates to be used as color gates.
6. Change color of the color gates, if required. 7. Click OK - the Color Gates dialog is closed. 8. Make sure “Color Gating” is enabled for each dotplot you want to use color gating.
- the events are displayed with the corresponding gate color.
Gating - Color Gating
Selecting/deselecting Color Gating for all plots.
Selecting/deselecting Color Gating for individual plots.
Selecting/Deselecting Color Gating for all plots
Select View-Color Gating – all plots on the page are displayed according to the new setting.
Selecting Color Gating for individual histograms
1. Right click onto dotplot for which to change color gating display – the histogram property box is opened.
2. Select/deselect the Color Gating flag .
3. Press Close – the dotplot is displayed according to the selection.
Crosstalk Compensation - Overview
You can compensate any parameters, e.g. FL2 to FL1, FL3 to FL1, FL1 to FL3, and SSC to FL4.
You can correct and repeat the compensation anytime, even after reloading a measurement file from disk.
Graphical entry of crosstalk entries.
FloMax N-Color software compensation requires the FCS files to be stored without previous compensation. If you are not using a Partec flow cytometer, switch off your instrument compensation in order to use FloMax’ compensation.
FloMax provides stepwise compensation, e.g. starting with FL1 – FL2, then FL2 – FL3, and so on in the order of strength of crosstalk.
Compensating one parameter against another can influence the distributions of all parameters (all histogram plots) included in the compensation matrix.
Why Crosstalk Compensation?
Crosstalk compensation is a means to compensate for spectral overlap of fluorescent dyes, when two or more dyes are used.
FloMax provides a software based mathematical compensation. This allows to take into account the crosstalk between any (of N) parameters, i.e. not only the neighbored colors. The algorithm used is correspondingly called N-color
compensation. Unique advantages of a software
based algorithm are:
a) Compensation settings can be corrected any time during or after the acquisition without need to rerun samples.
b) Compensation is reproducible and does not depend on possibly variing amplifier noise and offsets in the millivolt region which are usually difficult to control for the first decade on a 4 decade log scale.
FloMax offers graphical entry of crosstalk values, thereby facilitating the compensation setup.
Crosstalk Compensation - Setup
or
The traceline corresponds to the values, which will be compensated to zero (or onto the axis). All events below the traceline will also be compensated to zero.
Different compensation modes can be specified. Log is the preferred mode taking into account if and what log scales are used.
Lin always deals with data as if it was acquired with linear amplification. This mode does not change the general cluster shape. However, it this mode is not strictly correct. Only use in special cases.
Random Bias can be selected to redistribute data
otherwhiles falling onto the histogram axis graphically (see notes for details).
When starting a FloMax controlled instrument, the previously set compensation values are active.
Warning
Be sure not to overcompensate too much. The result could be an underestimation of dimly
Set up the Compensation
1. Select a display with dotplots showing the parameters to compensate against each other, e.g. FL1-FL2, FL2-FL3.
2. Open the compensation box:
a) Click the compensation button on the toolbar or
b) Select Analysis - Compensate Crosstalk...
- the Compensation toolbox appears.
3. To work with new settings, press Clear All... in the compensation toolbox.
4. Click in the 2P dotplot showing the largest crosstalk, e.g. FL1-FL2. If data was already compensated by FloMax, press Undo to show the compensation tracelines.
5. Enter slope (compensation percentage) and intercept (autofluorescence values):
a) Method 1: Graphically by compensation trace lines:
Move the mouse cursor above the square line trackers - the cursor is changed to a double arrow. With the left mouse button pressed, move the the trace line to set the slope and intercept. The corresponding values are displayed in the compensation box.
b) Method 2: Numerical entry of values.
In the boxes “Crosstalk of” and “to” select the parameters from and to which crosstalk to compensate. Enter slope and intercept for this compensation.
6. Click Compensate Crosstalk. 7. If desired, select Random Bias.
8. Click the + and – buttons left of the slope entry box to increase/decrease the compensation value of the actual parameters – the effect is directly visualized in the plots.
9. Repeat steps 4-8 with the dotplots remaining for compensation.
10. To close the compensation toolbox, click
Close.
Revert to Uncompensated Data for the Actual Dotplot
Click Undo - the uncompensated of the actual dotplot data is shown.
To correct the compensation, repeat the steps above.
Revert to Uncompensated Data for All Parameters
Click Clear All – the original uncompensated data is shown.
To correct the compensation, repeat the steps above.
Crosstalk Compensation – Saving and Loading
Saving and Loading Compensation Settings
Click Save... / Load... in the compensation dialog and specify a filename and folder in the Save As / Open dialog box:
- Compensation settings will be saved / loaded from disk.
Crosstalk Compensation – Exporting a Compensated File
To import compensated data by other software, select FCS as data type and “.A” (for Analysis) as file extension.
Files written this way contain compensated data in FCS format. These files can be read by FloMax, but compensation cannot be corrected or undone. If you do not need to export data, it is recommended to save files using FloMax standard FCS format. Opening standard format files with FloMax will show compensated data but allow to revert to the original data, since FloMax saves original data plus the compensation matrix.
The generation of a second gated or compensated file as specified in Save Options will be active unless explicitely deactivated. This facilitates routine operation when e.g. always having to save compensated files.
When no more required, be sure to deactivate the generation of a gated/compensated file by clearing the save options.
Why Exporting Compensated Files?
For other evaluation software it might be necessary to work on data which is already compensated. E.g. an external software could be used for special 3D data display, but might not offer a compensation function.
For this reason, FloMax offers the possibility of exporting compensated files.
Exporting Compensated Files
1. Compensate the data, as decribed above. 2. Select File - Save Options…
- the Save Options dialog appears. 3. Enable the Compensated checkmark. 5. Click OK – the Save Options dialog is closed. 6. Select File - Save As… - the Save As dialog appears.
7. Select a folder and a filename and press OK - Together with a normal FCS file with all data, a second file with the same name plus an extension “.A” is saved. The “.A”-file is a regular FCS file but contains compensated data.
Example
Filename selected in Save As is “Apoptosis” – two FCS files are generated:
1. Apoptosis.FCS, containing original data plus compensation settings.
2. Apoptosis.FCS.A, containing compensated data.
Crosstalk Compensation - Notes
FL1 -> FL2 = 20% FL1 -> FL2 = 10% mit Autofluoreszenz ohne AutofluoreszenzOriginal data example before compensation with appropriate compensation line (schematical).
Compensated data showing increase of cluster size.
The effect of cluster size increasing has nothing to do with the method of compensation, e.g. by analogue means or by software.
Why is the Cluster Size Increased by Compensation?
Correct compensation tends to increase the cluster size graphically on a logarithmic scale (see example on the left).
Even if correct in a strict mathematical sense, the increase of cluster size due to compensation is often undesired especially when operators are familiar with less precise electronic compensation techniques.
Example
Assume a fluorescence plot on a 4 decade logarithmic scale as on the left.
Your uncompensated FL1 negative and FL2 positive cluster extends from fluorescence values 3 to 10 in FL1 and 30 to 100 in FL2. Its mean value is at (FL1, FL2) = (7.3, 70).
A correct compensation would be 10% (slope). Accordingly, the FL1 mean value is compensated to:
FL1 - 10% x FL2 = 7.3 - 10% x 70 = 0.3. The right border of the cluster is compensated to
10 - 10% x 70 = 3.
The left border of the cluster is compensated to 3 - 10% x 70 = - 4.
How would this compensated cluster appear on a 4 decade log scale?
The cluster mean value will be presented about in the middle of the first decade (value 0.3).
The right border (value 3) will appear about in the middle of the second decade.
The left border (value –4) cannot be displayed on the log scale since it is non-positive. However, negative values will be limited to the minimum displayed value on the axis, which is 0.1 for a 4 decade scale.
The cluster will consequently range from the axis to the middle of the second decade. It's size is increased significantly.
Generally, this effect will grow with increasing compensation values.
without autofluorescence with
Crosstalk Compensation - Notes
Original uncompensated data with suggested setting of compensation traces.
Q1 Q2
Q4 Q3
Slightly overcompensated data without random bias.
Q1 Q2
Q4 Q3
Data from above with with random bias. Data is redistributed in the first decade of the log scale. Quadrant results will be similar with or without use of random bias.
Warning
Especially when using random bias, be sure not to overcompensate too much. The result could be an underestimation of dimly stained positive cells, which will appear in the negative distribution.
What is “Random Bias”?
Random Bias is a means to simulate electronic noise and offsets present in traditional analog compensation circuits.
How to use Random Bias?
In order to graphically present data as more narrow clusters, data may slightly be overcompensated by setting the trace lines slightly above the "negative-positive" clusters. By compensation, mathematically, this cluster will be completely moved to the axis. By adding a
Random Bias to the data, a small artificial offset
with Gaussian-like distribution, this cluster will be moved back from the axis to a narrow cluster near the axis. The effect of all this on region and quadrant statistics usually is small. However, if you prefer a mathematically correct compensation, it is recommended to turn off the Random Bias.
Should I Use Random Bias?
If you want to work with a mathematically correct compensation, no.
If you need to display data of negative clusters with a small distribution in the first decade, yes. - The influence onto the quadrant statistics usually is small.
How is Data Analysed on the Axis?
Data on the axis will be included in quadrant analysis.
What is the Influence of Gain Values onto Compensation Values?
Required compensation slopes heavily depend on the gain values used for the optical detectors. E.g. when using doubled amplification for FL1 (usually by increasing gain by 50) in the left example, compensation would have been set to 20% instead of 10%.
Can I Analyse without Compensation?
If or if not spectral crosstalk compensation is necessary, depends on the characteristics of the used fluorochromes.
Generally, for a two color analysis, the cluster percentages and concentrations can always be analysed without compensation as well. In cases where quadrants do not separate the clusters, polygon regions can be used.
For multiparametric data, analysis without compensation can be difficult if not impossible.
Cell Cycle Analysis
or
Cell Cycle Analysis should be performed on a single histogram layout. Linear amplification must be used for the DNA parameter.
Press Clear All to revert to the original display without analysis.
See mathematical appendix for details about cell cycle algorithms.
Performing a Cell Cycle Analysis
1. Make sure the DNA distribution showing the cell cycle is shown on a single one parameter histogram page. A linear scale is required for Cell Cycle Analysis.
2. Click the Cell Cycle Analysis button in the toolbar or
Select Analysis - Cell Cycle Analysis…
- the Cell Cycle Analysis box is opened. 3. Select a fit method.
a) For a complete numerical fit of a single cell cycle, click Fit One Cycle.
or
b) To perform only a Gaussian fit to G1 and G2/M data and analyse S-phase data "as-is" (without fit), click Fit G1/G2M only.
- a numerical fit to the experimental data is performed, according to a mathematical model (see mathematical appendix for details).
The numerical result is shown in the Cell Cycle Analysis Box . A graphical representation of G1, S, and G2/M phase is displayed together with the experimental data. Channel contents are now displayed as dots.
4. Click Close to close the Cell Cycle Analysis box
- Results stay displayed in the histogram.
Quantitative Cell Cycle Results
G1% percentage of G1 phase cells related to all cells in the cell cycle
S% percentage of S phase cells related to all all cells in the cell cycle
G2M% percentage of G2/M phase cells related to all cells in the cell cycle
CV%G1 relative coefficient of variation of G1
peak (relative width of Gaussian fit peak).
CV%G2M relative coefficient of variation of
G2M peak (relative width of Gaussian fit peak).
MnG2M mean channel value of G2/M peak
(mean of Gaussian fit peak).
MnG1 mean channel value of G1 peak (mean of Gaussian fit peak).
G2M/G1 ratio of MnG2M and MnG1. The ratio
is 2 for normal cell cycles.
ChiSqu. measure of the variation between
experimental data and the fitted mathematical model. The smaller ChiSqu., the better the fit.
drawing curve: numerical fit (sum of G1, S and G2/M fits).
dottet points: experimental
blue: G1 phase fit red: S phase fit green: G2/M phase fit
Peak Analysis – Numerical Fit Method
or
Numerical Peak Analysis should be performed on a single histogram layout. For the calculations, a linear amplification is assumed.
Press Clear All to revert to the original display without analysis.
See mathematical appendix for details about peak analysis algorithms.
By the numerical fit method a mathematical model (sum of bell-shaped Gaussian distributions) is fitted to the histogram data. In the model, each peak is represented by a Gaussian distribution with given position, width, and height.
Performing a Peak Analysis
1. Make sure the data is displayed on a single one parameter histogram page. A linear scale is assumed for numerical Peak Analysis.
2. Click the histogram – selection marks are displayed in the corners.
3. Click the Peak Analysis button in the toolbar or
Select Analysis - Peak Analysis...
- the Peak Analysis box is opened. 4. Click Fit Gauss Peaks:
- a numerical fit to the experimental data is performed, according to a mathematical model (see mathematical appendix for details).
The numerical result is shown in the Peak Analysis Box . A graphical representation of the fit is displayed together with the experimental data. Channel contents are now displayed as dots. 5. Press Close to close the Peak Analysis box - Results stay displayed in the histogram.
Quantitative Peak Results
Peak peak number (from left to right)
Index relative peak position related to the
first peak (or a reference channel)
Mean mean channel position of the peak
Area peak area (of Gaussian peak). Corresponds to the number of particles belonging to that peak.
Area% percentage of peak area related to all
sum of all peak areas
CV% relative coefficient of variation of peak (relative width of Gaussian fit peak).
ChiSqu. measure of the variation between
experimental data and the fitted mathematical model. The smaller ChiSqu., the better the fit.
drawing curve: numerical fit (sum of peak fits).
solid area: peak fits dottet points: experimental measurement data
Peak Analysis – Find Peaks Range Method & Batch Analysis
Press Clear All to clear the Find Peak analysis.
Ranges automatically set by “Find Peaks”.
Selection of output file for batch analysis.
For data files with several histograms, batch mode analyses the first histogram in a file. Make sure this is the histogram of interest.
By the find peaks range method, the position of the peaks in the histograms is analysed. For