Flicker Comparison of 2-Dimensional Electrophoretic Gels
Introduction
Flicker is an open-source stand-alone computer program for visually
comparing 2D gel images. Two-dimensional polyacrylamide gel
electrophoresis (2D-PAGE) gels are often difficult to compare because
of rubber-sheet distortions. Flicker allows you to visually compare your gel
images against each other or against those found in Internet
databases. Many published Internet gels have a subset of spots
identified which may make them useful to compare with your gels. Some
of these Internet gels are active maps that you can click on a spot to
inquire of its identity. You may be able to draw putative conclusions
as to the identification of some spots in your gels that visually
appear to be the same spots as in reference gels. The Flicker program
integrates these various needs to help you try to make putative spot
identifications. See the Quick start
examples below for a short list of some of these methods.
Of course Flicker might be useful for comparing other types of images
such as 2D-blots, 1D gels, serial microscope sections, time-lapse,
microarrays, etc. - any type of image where there are distortions or
intensity variation of the images, differing numbers of spots, and
other systematic and procedural experimental differences. It can read
black and white or color JPEG and GIF images, and black and white TIFF
images (some un-banded color TIFF images are available).
The Flicker application is Java program that runs under the MS
Windows, MacOS-X, Linux and Solaris operating systems. It is downloaded to and runs on your
computer. Flicker has been made open source and was contributed to the
Open2Dprot project. Both the executable binary as
well as the source code is available.
The original version of Flicker was a Java applet that ran in your Web
browser ([Lemkin97a]-[Lemkin97c],
[Lemkin99], [Lemkin02]) and is available at
http://www.ccrnp.ncifcrf.gov/flicker. However, being a Java applet,
it had many limitations in its capabilities. To resolve these
limitations it was converted in 2003 to a Java application by Peter
Lemkin and Greg Thornwall with help from Jai Evans. Code was added
from the open-source MicroArray Explorer program,
http://maexplorer.sourceforge.net/, to implement some of the new
features in Flicker. The new version of Flicker is much more flexible
and makes it easier for you to compare your gels with each other or
with reference gels on Internet databases. A limited measurement
functionality is available to estimate spot or region quantification.
The new version is described in a book chapter Comparing 2-D Electrophoretic Gels Across Internet
Databases, PF Lemkin, GC Thornwall, J Evans (2005) in "The
Proteomics Handbook", JM Walker (Ed), Humana Press Inc, Totowa, NJ, pp
279-305.
Notice: some individuals have problems looking at flickering images
Some individuals have problems with flickering images. The Flicker
program allows you to flicker-compare two images at flicker rates of 0.1 second/frame
up to 3 seconds/frame (individually selected on a per-image basis).
If you are bothered by flickering images, do not download
this program or do not use the flicker image comparison option.
However, the program can be used without flicker and may still be useful
to zoom/dezoom images, adjust brightness/contrast,
do image transforms, identify and label spots via comparison with SWISS-2DPROT
images, etc. In this case, don't enable the flicker option.
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Quick start examples
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The following are brief sketches of several ways that Flicker could be
used. There are many other ways of using Flicker, and these are detailed in
the Reference Manual.
A. Compare two gels to find spot differences.
- Open 2 gels to compare (your own or gels from the Internet).
- Flicker align
similar regions for the spot(s) of interest.
- When they are aligned, you can see local spot differences
between the gels.
B. Compare your gel against an Internet reference gel to try to
putatively identify the spot.
- Open 2 gels to compare (let one of them be an active reference gel).
- Flicker align similar regions for the spot(s) of interest.
- If one of the gels is an active reference gel, then you can
click on it to putatively identify
the protein.
C. Quantitate a list of spots. Flicker has a rudimentary spot list definition and
quantification capability that might be used as follows:
- Open 2 gels to compare.
- Flicker align similar regions for the each of the spot(s) of
interest.
- Add spots of interest
to spot lists (a separate list for each gel).
- Click on corresponding spots in each gel and pair them using
a common annotation id.
- List the spots in the paired spot list (this can be generated
as tab-delimited data for export to Excel).
D. Putatively identify a
list of spots in your gel that are identified in an active
reference gel by first identifying spots in the reference gel and then
using them to identify corresponding spots in your gel.
- Open 2 gels to compare (let one of them be an active reference gel).
- Flicker align similar regions for each of the spot(s) of
interest.
- Add spots of interest to spot lists (a separate list for each gel).
- Request Flicker to visit the active reference gel Web server
and try to lookup the protein id (e.g, Swiss-PROT) for the spots you
have defined in the active gel.
- Then click on corresponding spots in your gel and then
pair them using
a common annotation id from the reference gel.
- List the spots in the paired spot list (this can be generated
as tab-delimited data for export to Excel).
This approach may be useful for comparing similar protein samples
created in different laboratories to help putatively identify or
suggest possible protein spot identifications. The gels should be run
under similar pH and molecular weight ranges if possible. Although
available for over three decades, 2D polyacrylamide gel
electrophoresis (2D-PAGE) is still routinely used [1] even considering the now common
use of mass spectrometry [2-7]
and recently protein arrays [8]
for protein identification. If you have defined a list of spots in an
active gel (such as one of the Swiss-2DPAGE gels) and you are
connected to the Internet, you can request Flicker to lookup the
annotation information (Swiss-Prot id and name) for each spot in the
list. Then you can define a list of spots in your gel that correspond
to spots in the annotated active reference gel, and then assign these
protein identifications to your gel.
Recent advances, such as IEF "zoom" fractionation gels [9] that divide the protein sample
by pH range or immunoaffinity subtraction with LC [5], greatly increase the
resolution and numbers of spots able to be discriminated by subsequent
2D-gel electrophoresis. Another increasingly common image comparison
technique uses 2 to 6 cyanine dyes using dye multiplexing to label
multiple control and experimental samples run in the same gel such as
DIGE [10] and scanned with very
high resolution systems [11]. Multiple scans of the same
gel using different color filters can then be color mapped to see the
contributions of the different samples. This is useful if one has
control over the experimental design when determining the reference
gel, set of control gels, and experimental gels. However, it does not
solve the problem of trying to putatively compare one's own sample
against an Internet reference gel where they have identified protein
spots.
A number of 2D-gel image databases are available on the Web where some
of the proteins are identified for various types of samples. Both WORLD-2DPAGE and
2D-HUNT on
the SWISS-2DPAGE [12-15] server can be used to find
Web URL addresses for a number of 2D protein gel databases. The Web
site a large number of tissues with databases that include a wide
range of human tissues, mouse tissues, E. coli,
aribidopsis, dictyostelium, and yeast. You might also
try a Google
2D-gel search.
Some 2D-gel image Web databases have active maps (e.g., SWISS-2DPAGE)
where you can click on a
spot in an active gel image to identify the protein if it is in
their database (see WORLD-2DPAGE). Clicking
on a spot queries the associated Web server database to determine if
the spot you pointed to is in that database. If it is, it then
reports the protein identity of the spot with links to SWISS-PROT
etc. Alternatively, you can have it report the ID and protein name in
the reporting window or assign it to the selected spots annotation.
Using active maps with Flicker to putatively identify protein spots
We have integrated this capability in Flicker so that if you are
viewing an active map (say from SWISS-2DPAGE), you can switch from
Flicker mode to Clickable image database (DB) mode and
then just click on the spot to
query the database which then will bring up the specific protein annotation Web
page from the associated active DB server (e.g., SWISS-2DPAGE) in
a Web browser. This is most useful after you have flicker-aligned your
gel with the reference gel map image. Comparing one's own
experimental 2D gel image data with gel images of similar biological
material from such Internet reference databases opens up the
possibility of using the spots in these reference gels to suggest the
putative identification of apparently corresponding spots in your
gels. The image analysis method described here allows scientists to
more easily collaborate and compare their gel image data over the Web.
Methods for 2D gel comparison
When two 2D gels are to be compared, simple techniques may not
suffice. There are a several methods for comparing two gel images: 1)
put the images side by side and visually compare them; or 2) slide one
gel (autoradiograph or stained gel) over the other while back lighted;
or 3) build a 2D gel quantitative computer database from both gels
after scanning and quantitatively analyzing these gels using a 2D gel
database system; 4) more recently dye multiplexing has been used to
label different samples in the same gel. A variant if this is to warp
two gels so they are the same geometry, then generate pseudo color
images and look for differences by color differences. These methods
may be impractical for many investigators since in the first case the
physical gel or autoradiograph from another lab may not be locally
available. The first method may work for very similar gels with only
a few differences. The second method will work better for gels that
are not so similar but that have local regions that are similar. The
third method may be excessive if only a single visual comparison is
needed because of the costs (labor and equipment) of building a
multi-gel database solely to answer the question of whether one spot
is probably the same spot in the two gels. The fourth method may have
some problems if spot sizes vary for similar spots.
Flicker was developed to fill the need for a quick comparison of a
researcher's gel image against one of these internet reference
2D-gels. We have also provided a limited quantification facility
for manually measuring, annotating, a limited number of spots.
The Flicker Reference Manual describes
the operation of the various commands. The current status of the program and the revision history is updated as
changes occur in the software. You download
the software to install it on your computer. Flicker is downloaded as
a Zip file from the
Files Mirror.
Please contact us with
suggestions and comments. If you make interesting changes in the
source code, please send us a copy and describe your changes so we can
merge them in the released version.
