Free Flow Cytometry Software Detailed Description Form (form revised 2/2/95) ---------------------------------------------------------------------- **SECTIONS** The sections of this form are: **SHORT OVERVIEW** **BASICS** **DOCUMENTATION** **INPUT/OUTPUT** **DOT PLOTS** **CONTOUR & DENSITY PLOTS** **HISTOGRAMS** **KINETICS** **CELL CYCLE ANALYSIS** **TECHNICAL MATTERS** **COMMENTS/SPECIAL FEATURES** ---------------------------------------------------------------------- **SHORT OVERVIEW** (Paragraph of not more than 10 lines) Fast batching including graphics and overlays, fully corrected intensities, automatic peak detection, kinetics, gate ratios. Labels listmode files, converts to ASCII for spreadsheets. Tutorial with sample data. ---------------------------------------------------------------------- **BASICS** Name of program: MFI (Median Fluorescence Intensity) Date this form was completed/revised: 2/95, version 3.4I Computer operating systems supported (if MS-DOS, does the program require Windows?): MS-DOS (Windows is NOT required but program will run in a DOS window under Windows). Author(s) of program, contact addresses and phone numbers: Eric Martz, Dept. Microbiology, Univ. Mass., Amherst MA 01003-5720. FAX 413-545-1578. emartz@microbio.umass.edu. Where/how to get the program: ftp://marlin.bio.umass.edu/pub/flowcyt/mfi http://www.bio.umass.edu/mcbfacs/flowhome.html ---------------------------------------------------------------------- **DOCUMENTATION** On-line help: extensive. Is there a tutorial? If so, length? Major topics covered? Extensive tutorial with chapters on titration, kinetics, and subpopulations. Are sample data files provided with the tutorial? Yes. Manual, other documentation or published reference: Manual is included in the on-line help. ---------------------------------------------------------------------- **INPUT/OUTPUT** Input data file formats accepted: Broad range of FCS1.0/2.0 including B-D, Coulter, Winlist, Pro2FCS, Cytomation. Video screen types supported: CGA, EGA, VGA (640 x 480), Hercules. Printers and other output devices supported: Dot matrix (9 or 24 pin), HP Laserjet/Deskjet/Paintjet/Thinkjet/Pen Plotters, Postscript. Output disk file types created (if any): Text file of results (medians, percentages in gate, etc.). Listmode or histograms, optionally gated or smoothed, as ASCII data file for input to spreadsheets or plotting software. Mouse control: None. ---------------------------------------------------------------------- **DOT PLOTS** Can dot plots be displayed? Yes. Can multiple dot plots be displayed on the screen simultaneously? Yes: one, four, or nine. Dot plot regions (polygonal, rectangular, elliptical, etc.): Rectangular only. Gating logic options for regions: Events inside region only, region can involve two or three parameters. Are dots in different regions colored differently? If yes, do these colors carry over into dot plots of different parameters? No, no. 3D dotplots? If yes, can they be rotated and tilted to any desired angle? No. Is quadrant analysis supported? If yes, can the results be shown on the dotplot itself? No. ---------------------------------------------------------------------- **CONTOUR & DENSITY PLOTS** 2D contour plots? If yes, with colored contour lines? Smoothable? No. 3D contour plots? If yes, with rotation and tilting? Smoothable? No. Can density plots be displayed? If yes, in color? Smoothable? No. ---------------------------------------------------------------------- **HISTOGRAMS** Are medians/means for log acquired data converted to a linear intensity scale? Yes. Can arbitrary markers be placed on histograms to give percentages and medians/means? No (but see automatic peak detection under comments). What statistics are available for marked segments? (see comments) Can statistics for a marked segment be shown on the histogram itself? (yes, see comments) Can histograms be filled solid with a user-selected color? No. Is smoothing available? If yes, which algorithms, and can user specify number of cycles? Yes, by pooling of adjacent channels (e.g. 1024 to 256 or 128 total channels); Savitzky-Golay with level and cycles user specified. Can histograms be overlayed (maximum number per panel): Yes, 3. Histogram/overlay vertical scaling options: Scaling is automatic but can be common, independent, and off-scale pileups can be clipped. Overlayed histogram lines: can different colors be used? If yes, can user select colors? No. Overlayed histogram lines: can thickness, dotted/broken be selected? Dotted/broken is automatic. Can histograms be displayed as a 3D arrangement? If yes, can the display be rotated and tilted? No. ---------------------------------------------------------------------- **KINETICS** Are dots on dotplots sampled evenly throughout the listmode file? Always. Can time be used as a region/gating parameter? Yes, also event number. Can the median Y line be drawn vs. time? Yes. Can event number be used as a dotplot X parameter if time is not a recorded parameter? Yes. Can medians be obtained for a user-specified number of slices of each listmode file? Yes. ---------------------------------------------------------------------- **CELL CYCLE ANALYSIS** Is cell cycle analysis of DNA fluorescence histograms supported? No. Models/algorithms available: N/A. ---------------------------------------------------------------------- **TECHNICAL MATTERS** Program configuration information saved between sessions: Everything (which would not lead to later inadvertant misinterpretation) including all gates, run descriptions and subsets, output device/path, printer type, and over a dozen options. Configuration is saved separately for each subdirectory where program is run; defaults can be changed by putting configuration file on DOS path. Computer requirements (memory, video display, floating point hardware, etc.): 286 or better, floating point hardware required for acceptable performance. Source code programming language used, interpreters required (if any), approx. total lines of source code: About 30,000 lines of C. Compiler manufacturer and version: Borland 3.1, program uses dynamic overlay manager included with compiler. Commercial libraries or development tools required to produce the executable program from the free source code? Quinn-Curtis Scientific and Engineering Library, about $100. Seconds required to read/process a 10,000 event, 5-parameter file (specify computer system[s] used for timing): 3 sec on an 80486DX at 33 MHz. ---------------------------------------------------------------------- **COMMENTS/SPECIAL FEATURES** MFI is designed for batch analysis of large numbers of files. It can show preconfigured histograms/dotplots, stepping from file to file simply by pressing the Enter key, or produce a table of medians etc. in an unattended batch run. Different subsets of the files present can be tagged for different runs. Control files can be marked, causing their intensities to be subtracted from those of subsequent files. Parameters can be omitted from the tabular output, and the included parameters can be in any order desired. Listmode files can be labeled with one line of text. The label shows for the highlighted file on the run organization screen. Labels are inserted into the listmode file itself so cannot become separated from the file. Peaks in histograms are detected automatically with criteria of adjustable sensitivity. Percentages and medians are given for each peak both in the tabular output and shown directly on the histograms. Calculates ratios of events in any specified pair of gates. Warns the user when potential problems with the data are detected, such as off-scale pileups of events, shifting of the event cloud within the scatter gate. Factors can be entered for each fluorescence parameter for conversion to MESF. ---------------------------------------------------------------------- [END OF FORM] ----------------------------------------------------------------------