- Listmode data (for dotplots), optionally gated
- Histogram data, optionally smoothed/gated
- Spreadsheet-ready median fluorescence intensity results
MFI can optionally produce plain text ASCII output that can be used as input to generic plotting software, mathematics or statistics software, or spreadsheets.
Flow cytometers
generally store data in a specialized
Flow Cytometry Standard (FCS) format,
in which the intensities of each scatter or fluorescence parameter
are encoded in a specialized, compact binary format that cannot be displayed
in a word processor, or imported easily into other software.
Conversion to
plain text ASCII
puts the intensity values in plain text that can be viewed and edited
in a word processor or used in a spreadsheet or generic data plotting
software. In addition, histograms or
results derived from listmode data by MFI can be output in plain text ASCII.
For examples, see below.
Generating plain text ASCII output with MFI.
MFI can produce plain text ASCII output files at three levels:
I. Plain Text ASCII Listmode Data (optionally gated)
The term "listmode" describes flow cytometry data files that list the values of the scatter and fluorescence parameters for each event, in the order in which the events passed through the cytometer's interrogation point (usually a laser beam). Listmode files are raw cytometer data files. Most cytometers create listmode files in the Flow Cytometry Standard (FCS) format, which is not understood by general purpose scientific software. Converting a listmode file to a plain text ASCII format enables it to be read by such software, or viewed and edited with a word processor.
MFI will optionally create one new plain text ASCII list mode data file for each listmode file tagged on the input list (or for each time slice!). MFI names these output files *.ALS (Ascii LiSt), [or, for time slices, *.L01-*.Lnn]. The output files include only in-gate events for the parameters selected, in the order selected. To produce an ungated file, select gate 0. These plain text ASCII list mode files are suitable for making dot-plots with general purpose plotting software.
Let's look at a concrete example. The first set of data files provided as an example for MFI's tutorial represent the titration of an antibody -- fluorescence intensities obtained with a series of dilutions of the antibody. (See the Tutorial for details.) One of the the files in this set, TITRA062 (normally located in c:\flowcytm\mfitutor\1titrat), is from unstained control cells.
A gated plain text ASCII file resulting from listmode file TITRA062
might contain this:
MFI-generated ASCII list mode data from FCS file TITRA062:
Active Gate: #1 "JY Cells [FSC vs. SSC]",
FSC = 174-359, SSC = 134-339
(See end of file for report of events in-gate.)
(If columns don't line up, increase display tabulation modulus to 8.)
Event FSC SSC FL1 FL2 FL3
1 246 273 141 3 4
2 301 301 159 3 4
3 275 309 142 3 4
4 232 246 124 3 4
5 349 313 153 3 4
6 235 165 93 3 4
7 313 321 133 3 4
8 276 252 98 3 4
... [lines 9 - 2559 omitted] ...
2560 262 176 107 3 4
2561 275 314 189 3 4
2562 303 318 121 3 4
2563 280 314 163 3 4
Total events in gate are 2563 out of 5000 in file (51.3%)
Each line represents a single event.
The numbers in the columns labeled FSC, SSC, and FL1 represent the
scatter and fluorescence intensities for each event. (In this
instance, FL2 and FL3 were not in use and the gains were set very low.)
If imported into generic scientific plotting or spreadsheet software, the above plain text file would most easily be plotted as dot plots.
In each run, MFI processes listmode files in a given folder that are tagged in its Run Organization screen. (See the Tutorial for details.) Whether MFI puts out plain text ASCII files is controlled by settings in MFI's Configuration Menu. There, item 9. Spreadsheet/histogram/listmode ASCII files opens a submenu that includes 2. Create ASCII gated list mode data files. When this option is set to YES, MFI puts out one plain text ASCII listmode file for each FCS listmode file it processes.
Gating. MFI allows you to define scatter gates (rectangular only; see MFI's Tutorial.) In MFI's Run Organization, you may apply gates to listmode files or not. If a gate is applied to a file, the plain text ASCII output will contain only in-gate events. To process a file without gating, select gate 0.
Parameter omission, re-ordering. MFI allows you to omit parameters that are not in use. Omitted parameters will not be included in the plain text ASCII output. Had FL2 and FL3 been omitted in the run below, they would not appear in the listing. Parameters can also be included in any order, and the plain text ASCII output will follow the order designated in MFI.
Event sampling. Spreadsheet programs may not have the capacity to process all of the events recorded in a list mode file. Therefore, MFI provides an option to export to plain text ASCII only the 'dot plot' events, a number that can be configured between 1 and 5,000. This capability is independent from the scatter gating capability. The 'dot plot' events are sampled uniformly from throughout the file; that is, if 2,500 events are specified, they are not the first 2,500 events in the file, but are taken evenly spaced from the entire file. This even sampling is crucial for kinetic studies.
In MFI's Configuration Menu, item 9. Spreadsheet/histogram/listmode ASCII files opens a submenu that includes 3. Limit events in list mode ASCII files to number in dot plots. Change this to YES to limit the events in plain text listmode output files to the number specified for dot plots.
The number of dots per dot plot is controlled
from MFI's Configuration Menu. Item
5. Graphics output options opens a GRAPHICS submenu that
includes
5. Dot plot options menu, which opens a DOT PLOTS submenu, including
2. Number of dots to show on dot plot.
II. Plain Text ASCII Histogram Data, Optionally Smoothed and/or Gated
MFI will optionally create one new plain text ASCII histogram data file for each input list mode data file (or for each time slice!). These histogram files are named *.HIS [or *.H01-*.Hnn for time slices]. They include only IN-GATE events for the parameters selected for the run in question. The degree of smoothing is controlled by the number of channels designated for histograms, and/or by invocation of Savitzky-Golay smoothing. These histogram files are suitable as convenient input for most general purpose scientific plotting software and for spreadsheets.
In MFI's Configuration Menu, item 9. Spreadsheet/histogram/listmode ASCII files opens a submenu that includes 5. Create ASCII data files of gated, smoothed histograms. Change this to YES to generate plain text ASCII histogram output files.
Histogram options such as smoothing are controlled from MFI's Configuration Menu. Item 5. Graphics output options opens a GRAPHICS submenu that includes 4. Histogram options menu.
Here is an example, a histogram output for file TITRA062:
MFI-generated 128 channel histograms for data from FCS file TITRA062:
Active Gate: #1 "JY Cells [FSC vs. SSC]",
FSC = 174-359, SSC = 134-339
No smoothing applied.
Total events in gate are 2563 out of 5000 (51.3%)
(If columns don't line up, increase display tabulation modulus to 8.)
Channel FSC SSC FL1 FL2 FL3
0 0 0 9 2563 2563
1 0 0 4 0 0
2 0 0 1 0 0
3 0 0 7 0 0
4 0 0 12 0 0
5 0 0 13 0 0
6 0 0 28 0 0
7 0 0 60 0 0
8 0 0 74 0 0
9 0 0 97 0 0
10 0 0 125 0 0
11 0 0 197 0 0
12 0 0 219 0 0
13 0 0 253 0 0
14 0 0 275 0 0
15 0 0 241 0 0
16 0 1 222 0 0
17 0 22 183 0 0
18 0 40 152 0 0
19 0 44 123 0 0
20 0 58 97 0 0
21 1 74 62 0 0
22 13 97 42 0 0
23 30 97 22 0 0
24 36 98 21 0 0
25 75 126 11 0 0
26 90 132 7 0 0
27 124 123 1 0 0
28 157 140 1 0 0
29 195 139 1 0 0
30 201 121 0 0 0
... [lines 31-125 omitted] ...
126 0 0 0 0 0
127 0 0 0 0 0
TOTALS 2563 2563 2563 2563 2563
III. Plain Text ASCII Results for Spreadsheets
MFI's primary results are corrected linear median fluorescence intensities for scatter-gated events. MFI is efficient at extracting results from large numbers of list-mode files. By default, the results are displayed on the screen, or saved into a plain text file designed for human readers. Here is an example where colors were added to help see the structure of the results output.
Alternatively, MFI can summarize its median fluorescence intensity results in file designed as input for a spreadsheet program. These files are named MFIRUNnn.SPR, where "nn" is the Run Number.
Here is an
example of the spreadsheet output for the same gate and files as shown
in the primary results example. You will have
to scroll to the right to see the very wide table below.
Columns are delimited by tab characters.
28 columns by 7 rows. MFI'S RUN INFO BEGINS AT "##" AFTER DATA. Tab modulus 8.
ROW FILENAME DATE WARNINGS THR_P THR_V EVENTS SECS EV/SEC GATE INGATE G1FSC G1SSC G1FL1 G1FL2 G1FL3 G2FSC G2SSC G2FL1 G2FL2 G2FL3 MdFSC MdSSC MdFL1 MdFL2 MdFL3 LABEL
1 TITRA062 14-JAN-93 C FSC 52 5000 18 277 1 61.94 E-1 307 476 150 150 682 100 LOG 100 100 263 267 0.0000 0.0000 0.0000 JY cells alone
2 TITRA063 14-JAN-93 E4 FSC 52 5000 17 294 1 62.10 E-1 307 476 150 150 682 100 LOG 100 100 265 267 0.140 0.0000 -1.00 JY cells + 2nd
3 TITRA011 14-JAN-93 FSC 52 5000 17 294 1 56.14 E-1 307 476 150 150 682 100 LOG 100 100 267 264 128 -1.00 -1.00 JY cells + TS1/
4 TITRA012 14-JAN-93 FSC 52 5000 17 294 1 57.06 E-1 307 476 150 150 682 100 LOG 100 100 267 264 119 -1.00 -1.00 JY cells + TS1/
5 TITRA013 14-JAN-93 E4 FSC 52 5000 17 294 1 57.96 E-1 307 476 150 150 682 100 LOG 100 100 267 264 89.2 -1.00 -2.00 JY cells + TS1/
6 TITRA014 14-JAN-93 FSC 52 5000 18 277 1 58.98 E-1 307 476 150 150 682 100 LOG 100 100 267 262 49.8 -1.00 -2.00 JY cells + TS1/
7 TITRA015 14-JAN-93 FSC 52 5000 15 333 1 59.22 E-1 307 476 150 150 682 100 LOG 100 100 267 265 22.0 -1.00 -2.00 JY cells + TS1/
##MFI RUN INFORMATION BLOCK:
Run 1:
These log-acquired parameters were converted to a linear scale:
FL1
Peaks detected when excursion >5% using 32-channel histograms.
Gate: #1 "JY Cells [FSC vs. SSC]",
FSC = 169-374, SSC = 154-409
Warning codes: (n = parameter number)
(Details for each listmode file given in MFI's non-spreadsheet primary results.)
C: Control, FL medians subtracted from those of subsequent files.
[: previous control no longer applies.
En: End pile-up, param. n. >10% of events in first/last occupied channel.
I: Instrument settings changed (gains, threshold, etc.).
L?: can't tell which parameters if any use Log scale.
P-: Peak detection disabled.
P<: Peak detection unreliable, too few events in gate.
Pn: multiple Peaks in histogram for parameter n.
!: warnings truncated because longer than 15 character column width limit.
Despite the width of the above table, spreadsheet-ready results are not as complete as the primary results. Spreadsheet data lack medians for individual peaks in multimodal histograms, do not give details on end-pileups, and do not show results prior to control subtraction or KMESF calculation when these options are in effect. The spreadsheet column WARNINGS should be examined carefully; if warnings are present, consult the primary results for further information.
In the above example, the G1xxx columns are the cytometer Gain-1 settings; G2xxx are the Gain-2 settings. At the far right are Mdxxx columns giving linear median intensities. The MdFLx fluorescence medians are corrected for the control designated in MFI's Run Organization. The labels in the last column are truncated, a bit unfortunate in this case since the dilutions are lost.
Controlling Spreadsheet Output. On MFI's Configuration Menu, pressing T toggles spreadsheeT output, and item 9. Spreadsheet/histogram/listmode ASCII files controls the details of spreadsheet output, under the submenu 6. Spreadsheet data file option menu. Under this submenu SPREADSHEET RESULTS FILE, the run information block was set to "short". Here also is 10. Spreadsheet data file column omission menu. For the example output below, this submenu was used to omit row numbers, date, threshold, time, and cytometery settings.
In the example data, FL2 and FL3 were not used, so they can be omitted. (This is done from Configuration Menu, item 3. Parameters. You must blank out the last parameter first, then work backwards.)
Here is MFI's spreadsheet output for the same run as above, but with
settings to produce a more succinct and less detailed output (11 columns
instead of 28!). Nevertheless, the key results for the titration
are in the column MdFL1. The LABELs were shortened so the dilutions
are included in the last column.
Columns are delimited by tab characters.
11 columns by 7 rows. MFI'S RUN INFO BEGINS AT "##" AFTER DATA. Tab modulus 8.
ROW FILENAME WARNINGS EVENTS GATE INGATE MdFSC MdSSC MdFL1 LABEL
1 TITRA062 C 5000 1 61.94 263 267 0.000 JY cells alone
2 TITRA063 5000 1 62.10 265 267 0.140 JY + 2nd Ab
3 TITRA011 5000 1 56.14 267 264 128 JY, 1/300
4 TITRA012 5000 1 57.06 267 264 119 JY, 1/900
5 TITRA013 5000 1 57.96 267 264 89.2 JY, 1/2,700
6 TITRA014 5000 1 58.98 267 262 49.8 JY, 1/8,100
7 TITRA015 5000 1 59.22 267 265 22.0 JY, 1/24,300
##MFI RUN INFORMATION BLOCK:
Run 1:
These log-acquired parameters were converted to a linear scale:
FL1
The following parameters, present in the listmode data files, are not shown:
FL2, FL3
Peaks detected when excursion >5% using 32-channel histograms.
Gate: #1 "JY Cells [FSC vs. SSC]",
FSC = 169-374, SSC = 154-409
In Microsoft Excel, the file MFIRUN01.SCR can be imported by using
Excel's File, Open, Files of Type: All files.
In MFI's output file, columns are delimited by tab characters.
Here is a snapshot of the above output imported into a Microsoft Excel spreadsheet:
In addition to using MFI's spreadsheet-ready output as input to spreadsheet software, spreadsheet-ready output provides an alternative format for printing results, more compact (but less complete) than the primary results.
Definitions
Flow Cytometry Standard Data Format
Although there is a published FC standard data format (see references below), cytometer and software manufacturers usually adhere only loosely to this standard. (This required that MFI have special case handling code for nearly every combination of software and cytometer that it supports!) Since MFI development stopped in 1996, data files from newer cytometers may not be handled properly by MFI. The best way to find out is to examine MFI's output carefully, comparing it with that of trusted software (see the MFI Validation Suite). A list of cytometers and software supported is given in MFI's internal help (available by pressing F1 when running MFI, especially item 4 "Cytometers and listmode acquisition software supported", and item 19 "Compliance with FCS data file standard").
MFI was designed to obey the FCS 2.0 data format. Note that the FCS 3.0 standard was published after MFI development ceased in 1996.
Flow Cytometry Standards:
"ASCII" designates a standard method of coding plain text or numbers on computers. Most textual information on computers is stored in ASCII, including this document. ("Plain text" files, conventionally named ending with .txt, contain only the text in ASCII. Word processors add proprietary non-ASCII codes to specify how the text is formatted.) "ASCII" stands for American Standard Code for Information Interchange. ASCII was recommended in 1968 by the American National Standards Institute (ANSI) in their recommendation X3.4 (International Standards Organization ISO 646).