for Fluorescence Correlation Spectroscopy (FCS) data
from Zeiss ConfoCor Systems
Earl F Glynn
Stowers Institute for Medical Research
4 May 2007
The purpose of the FCSViewer program is to decompress a file with Zeiss Fluorescence Correlation Spectroscopy (FCS) data (in either ConfoCor 2 or ConfoCor 3 formats) and display the two channels. In addition, time "calipers" can be used to select and enlarge any time interval, and this can be repeated until the full resolution of the data is displayed.
Experimental Principles of Fluorescence Correlation Spectroscopy, DrBio, Cornell Biophysics
Fluorescence Correlation Spectroscopy: An Introduction to its Concepts and Applications, Petra Schwille and Elke Haustein, Max-Planck-Institute.
ConfoCor 2 System
Klaus Weisshart, Volker Jungel and Stephen J. Briddon, "The LSM 510 META - ConfoCor 2 System: An Integrated Imaging and Spectroscopic Platform for Single-Molecule Detection," Current Pharmaceutical Biotechnology, 2004, 5 135-154. Publisher Abstract.
pp. 141 of this paper describes "The FCS Data Format." Table 4, Structure of Recorded Word, and Table 5, Example of Recorded Word, give details of the compression technique used in the FCS file. The information from Table 4 and 5 is also on the web page, ConfoCor 2 -- Description of the Raw Data Format.
Carl Zeiss Advanced Imaging Microscopy claims a patent on this process, including the FCS data file format.
The FCS data stream represents two channels of discrete events. Because these events do not occur often, interleaved run-length-encoding is used as a form of lossless data compression. Two-byte words are used to contain a run-length byte and a data byte. This diagram gives an overview of the bit manipulations needed to extract and reorder the separate channels of information. This diagram shows the mapping of bits, bytes, time, and bins.
ConfoCor 3 System
The ConfoCor 3 data is actually much easier to work with the ConfoCor 2 data, however, the FCSViewer program does not take advantage of this. The ConfoCor 3 data were read and "converted" to the same in-memory bit stream as used with ConfoCor 2. The analysis examples (in R and IDL) do take advantage of the new format.
Tested only using Windows 2000 and XP, but should work on other versions of Windows.
You will want a 1024-by-768 screen (even larger is preferred) to work with this software.
1. Download executable and put in any convenient directory. Download and unzip the test data and put it in the same or another directory.
2. Double click on executable to start the program.
3. Press the Read button. For ConfoCor 2, select a single input file, such as Sample488.011.
For ConfoCor 3, first select the Channel 2 file, and then use Ctrl-Click to select the Channel 1 file:
4. If the Auto box is checked, the data will automatically be displayed. If the Auto box is not checked, after the data are read you can specify the T1, T2 time interval boundaries first, and then press the Show button manually. This manual procedure can also be used to change the default number of bins to be used in each of the subsequent plots.
5. The time "calipers" initially are at the far left (green) and far right (magenta) of the chart area. To move either of the time calipers, move the mouse over one of these vertical lines. When the cursor changes from the normal arrow to an index finger, press down and drag the caliper left or right to the desired time and release. The color of the caliper vertical line is coordinated with the T1, T2 values in the box in the middle at the top.
Note that when the Max Count for a Channel is more than 10, the Line format is automatically used to display the data, but the Bar or Point options can be manually chosen.
6. After picking a new time interval, press the Zoom button to see this interval enlarged.
Note that when the Max Count for a Channel is10 or less (see Channel 1 above in red), the Bar format is automatically used to display the discrete data.
7. If desired, yet another new time interval can be chosen and enlarged.
8. Close any of the enlargement windows by pressing the red "X" at the upper right of the window.
9. Close everything by exiting the base window by pressing the red "X" at the upper right of that window.
Here's a ConfoCor 3 example:
The "About" screen shows version and license information
Fluorescence Correlation Spectroscopy (FCS) Viewer
Version 2.0, 4 May 2007
Winfried Wiegraebe, PhD
Advanced Instrumentation and Physics
Earl F. Glynn
Copyright © 2007 Stowers Institute for Medical Research.
All Rights Reserved.
Distributed under GNU General Public License
4 May 2007