EXPLORING LIFE.

Scanning Guide
How to produce
Optimal 2D Gel Images

DECODON
Scanning Guide

Introduction
2D gel electrophoresis is an extremely valuable but also demanding technique for performing
global proteomic analysis. Making your own gels takes time and needs hard work. After weeks of
adapting the sample preparation setup, optimizing the gel running protocol, and finding the
corresponding gel staining technique, the final step on the way to computerized analysis is the
generation of images from your 2D gels by using the adequate imaging technology.

This last step in the process is at least as important as all the other previous steps for the successful
analysis of your experiment. This guide will help you preserve the information that is contained in the
gel and produce the best possible input for computerized 2D gel image analysis.
 EXPLORING LIFE.

2D Gel Scanning Check List

Here is our check list for scanning 2D gels. We hope that it helps you to
achieve the best possible input for computerized image analysis. Please send
your questions and comments to support@decodon.com.

● Use grayscale instead of color images.

● Try to use the complete available grayscale range. Check this using the image
histogram.
● Choose the image resolution such that the smallest spots you want to analyze
have a diameter of at least 5 pixels. Use our image resolution table on the
next page as a guide.
● Scan all gel images using the same orientation.
● Place each gel at the same position on the scanner plate. Avoid scanning too
much of the area around the gel.
● Use presets in the scanner's software to make the scanning process easy and
reproducible. Save resolution and scanning area as presets and reuse them for
all gels in your experiment.
● Avoid Photoshop and similar general image processing software if you do not
know exactly what you are doing. Use the software that came with your
scanner / imager for image post-processing.
● Limit post-processing of images to crop, mirror, and rotation by 90, 180, 270
degrees.
● Avoid producing TIFF files if you can process calibrated image file formats
such as *.IMG/INF and *.GEL. Usually, the TIFF files are produced without
grayscale calibration. This means you loose precision or grayscales are
distorted nonlinearly, making quantitation questionable.
● Avoid using JPEG files for quantitative analysis.

DECODON
Scanning Guide

Resolution
The image resolution is determined during presetting the scan parameters in the scanner
software. During the scan the 2D gel is resolved into a mosaic of equally sized squares (pixels).
Smaller pixels mean higher image resolution, i.e. more details are visible on the image.

Resolution is measured in several units. Typical units used by scanners are micron (1 micron
corresponds to a pixel size of 1 micrometer), ppi / dpi (pixels per inch / dots per inch) or in the metric
system pixels per millimeter. For image analysis of protein spots, the smallest spots should have about
5 to 10 pixels in diameter. As an example, take a very small spot on a two-dimensional electrophoresis
gel with 1 mm in diameter. With a resolution of 200 dpi this spot will have a diameter of about 8
pixels.
 EXPLORING LIFE.

Image Resolution

Higher resolutions mean that you have more data available for the analysis.
However, the increase in precision that comes with higher resolution is marginal
once you have a certain minimum resolution. On the other hand, every doubling of
the resolution results in a fourfold increase of the image file size. Scanning takes
more time in higher resolutions, files take up more disc space, and image analysis
requires more time and memory.

The following table shows the relation between resolution and spot sizes for
some typical resolutions.
20cm gel image [pixel]
diameter [pixel] of a

diameter [pixel] of a
1mm-diameter-spot

1mm-diameter-spot

5mm-diameter-spot

5mm-diameter-spot
pixels in a circular

pixels in a circular
image resolution

image resolution

8 bit graylevels

16 bit graylevels
[dots per inch]

file size [MB]

file size [MB]

pixel width

width of a
[micron]
[inch]

100 0.0100 254 4 12 20 304 787 0.6 1.2

150 0.0067 169 6 27 30 685 1181 1.3 2.7
200 0.0050 127 8 49 39 1217 1575 2.4 4.7
300 0.0033 85 12 110 59 2739 2362 5.3 10.6
400 0.0025 64 16 195 79 4869 3150 9.5 18.9

Typical image resolutions and corresponding spot and file sizes

Increasing the resolution after the scan brings no additional information to the
image. Reducing the image resolution after the scan results in loss of data.
However, this can be acceptable provided that the spot diameter for small spots
does not drop below 5 pixels.

DECODON
Scanning Guide

P TI F JPG
IMG BM

PNG GEL TIFF

File Formats
Signal intensities are encoded as numbers in the image file, one number for each pixel. For
color images, things are a bit more complicated: for each color channel, one number has to be
stored, for example one intensity each for red, green, and blue. Make sure you use grayscale
instead of color images: the extra color information usually provides no benefit for the subsequent
analysis of 2D gels.

When your image file is, for example, a "16-bit TIFF" file this means that image intensities are
encoded with 16 bit numbers, giving 65,536 (2 to the power of 16) possible different values for each
pixel. In contrast, an 8-bit image file only stores 256 different values per pixel. The number of bits per
pixel is also called the color depth. The following table shows some examples.
 EXPLORING LIFE.

Image File Format

color depth intensity levels example

1 bit 2 black and white FAX image
8 bit 256 GIF image
10 bit 1,024 TIFF image
12 bit 4,096 TIFF image
16 bit 65,536 TIFF image

Typical color depths

TIFF images can have different color depths, this is one of the reasons why TIFF
is a widely used image file format.

Generally, having more possible intensity values per pixel (higher color depth) is
better for the analysis. The tradeoff is between higher accuracy and need for more
space to store the information: a 16 bit TIFF file is twice as large as the equivalent
8-bit file, but gives you 256 times more nuances in the image. Another
consideration is that many everyday image processing programs are not able to
deal with color depths greater than 8 bit.

Decreasing color depth in the scanned image file normally results in loss of
accuracy. It is not recommended to increase color depth after the scan is
completed: if your scanner produced only an 8-bit image you have at most 256
different intensity values in the image. Converting the file to a 16-bit image will
only give you at most 256 out of 65,536 possible gray values.

You can find more information about image file formats in the Wikipedia
1 2
Category: Graphics File Formats . At the DeltaStat Proteomics Image Analysis
Forum there is a discussion thread "Compatibility of Image formats across different
Image Analysis Softwares" that focuses on experiences from the proteomics
community with vendor specific formats.

1 - http://en.wikipedia.org/wiki/Category:Graphics_file_formats
2 - http://forums.deltastat.org/

DECODON
Scanning Guide

Data reduction
and calibration
Some imaging devices can measure more intensity values than what fits into the available
image formats. One way to deal with this is distributing the intensity values linearly over the
whole intensity range that is offered by the image file. An example: Say, the Analog to Digital
signal converter of the scanner can deliver 1024 intensity levels, but the image file is limited to 256
levels (8-bit color depth). Linear transform condenses 4 A/D converter levels to 1 intensity level in
the image. Of course, this process effectively wastes accuracy.

Especially if light scanners are used for image generation this technique can be improved by using
only the real dynamic range delivered by the A/D converter. For our example this could mean: The A/D
converter can deliver 1024 different intensity levels but in our experiment the gel only has intensity
values in the range from 128 to 920.
 EXPLORING LIFE.

Data reduction and image calibration

The 792 intensity levels have to be encoded in the image file - so only 3 A/D
converter levels have to be condensed to 1 image intensity level. Compared to the
former approach that wastes the lower and higher dynamic range this improvement
results in a 25% finer resolution.

There are instruments that can distinguish 100,000 and more intensity levels, far
more than can be encoded in a TIFF or similar file format. In order to save as much
information as possible in these files, intensity levels are encoded using a nonlinear
calibration curve. During scanning, measured intensities are converted to pixel gray
values according to this curve. During quantitation, the image analysis software
has to decode the pixel values to arrive at the originally measured intensities. The
curve is designed such that lower intensities will be encoded with higher accuracy
than higher intensities.

Example image calibration curve

During image analysis, it is important that the image analysis software

recognizes the calibration curve so it can do quantitation using the intensities
originally measured by the scanner. General image processing packages such as
Photoshop ignore grayscale calibration. It is even possible that calibration
information is lost during processing with these packages. Additionally, TIFF files
do not include calibration information so you will have to use vendor specific
formats.

DECODON
Scanning Guide

File compression
Compression of image data means the usage of algorithms to reduce the size of a gel image
file while retaining all or most of the image information. If you use calibrated image file formats
then image compression is not an option because compressed file formats do not store the
calibration information.

Image compression algorithms can be classified as lossy or lossless methods. Lossy compression
means that information can be lost in the image details, giving a higher compression ratio than
lossless methods. Uncalibrated files may be saved in file formats that use lossless data compression
e.g. subformats of *.tiff / *.tif or *.png. Often compression to 50 % of the original size is possible. The
commonly used JPEG format uses a lossy compression method. Large compression ratios may heavily
change or destroy your data. For low compression ratios, there is still an unknown influence of
compression on spot quantities. That is why we recommend to avoid using the JPEG file format for image
analysis purposes.
 EXPLORING LIFE.

Image Compression and Orientation

Image compression only saves disc space but does not affect the amount of
working memory (RAM) needed for image analysis because compressed files will
also be completely extracted before starting analysis.

The following table summarizes the properties for commonly used image file
formats.

use for
quantitative
format compression gray levels analysis calibration
gif lossless 256 no no
bmp no 2; 256 (yes) no
png lossless 256 yes no
jpg lossy 256 no no
tif(f) no (lossless) 2; 256; 1024; 4096; 65536 yes no
img inf no 1024; 4096; 65536 yes yes
gel no 65536 yes yes

Commonly used image formats

Scan all gel images using the same orientation. A common trick is to cut off one
corner of the gel. Cut off the low mW, low pI corner. Make sure that this corner
becomes the lower left corner in the image for every gel you scan. This has the
added advantage that your images will have the orientation that is usually required
by the proteomics journals (pI increasing from left to right, mW decreasing from top
to bottom).

If you need to rotate or flip the image it is recommended to use the software
that came with your scanner for this. General purpose image processing software
(such as Adobe Photoshop) may not preserve the vendor specific grayscale
calibration information in the image file.

DECODON
Scanning Guide

Scanner Settings
Scientific scanners and imagers are complex instruments. The following section should give
you some background information to make the right choices for scanning your gels.

The principle of operation of a scanner is quite simple: a pixel in the image file is created from
light intensity that is measured at a certain point in the gel. In more detail, light is emitted from a
light source, reflected or transmitted by the gel, and then measured by a detector. The measured
value is converted to a number by an A/D (analog to digital) converter. The numbers from the whole
gel are then processed to give the image file that is the output of the scanning process.

The Scan Mode describes how the light travels from the light source to the detector.
● In transmisson mode the light passes through the gel and is directly measured by the detector on
the opposite side of the gel (see left figure below).
● In reflection mode the gel is between the light source and a reflector (normally in the scanners lid).
Light travels through the gel, is reflected, goes again through the gel, and is then detected.
 EXPLORING LIFE.

Scanner Settings I

Three scanning principles: light transmission, light reflection, fluorescence

For high quality data we recommend using transmission mode because

saturation effects are less likely than with reflection mode. Saturation means that
differences in higher spot intensities are not properly resolved -- dark spot centers
become completely black. In reflection mode this happens more often because the
light travels twice through the gel, so it can be more easily absorbed completely.
Saturated regions appear as "plateau spots" in the 3D view of a spot (see the
following figure).

Saturated “plateau spot”

Consideration of the scan mode is important if you use staining techniques

absorbing the light, e.g. all types of Coomassie or silver nitrate staining. For
fluorescence scanning the scan mode is less relevant because the light is emitted
by the proteins within the gel.

DECODON
Scanning Guide

Light Intensity
The intensity of the light source directly influences differentiation of high density spots. It is
common to use office document scanners to detect absorbing dyes (coomassie, silver, x-ray film).
The manufacturers usually claim a differentiation of gray levels until an optical density (OD) of 2.0.
This is mostly sufficient for gel documentation purposes. However, it is insufficient for differentiation
of high density spots reaching optical densities near 3.7 to 4.0.

Document scanners resolving ODs from 3.7 to 4.0 can be found in the premium or professional
product lines of well known manufacturers (e.g. Quato, Umax). Warming up the lamp before starting
the scan process is highly important because the lamps become more powerful when they reach their
working temperature. Extensive usage of the scanner causes aging of the light source resulting in
weaker light intensity.
 EXPLORING LIFE.

Scanner Settings II

Especially for fluorescence imaging high performance light sources are absolutely
necessary. In this field of imaging normally lasers or UV lamps are used. Lasers
deliver highly concentrated light and can effectively excite fluorescent dyes.
Depending on the fluorescent dyes different colored lasers and excitation and
emission filters are needed. Lasers need a warm up phase too to reach their highest
performance.

The light harvesting detector and the A/D converter collect the light that has
passed through the gel and translate this information to computer readable signals.
The detector (CCD element or PMT) converts the light intensity to an electrical
signal that is subsequently digitized by the A/D (analog to digital) converter. These
data are stored in the image file.

In many high perfomance imaging devices for fluorescence detection also very
weak signals have to be detected. That is why these scanners allow to tune the
voltage of the light detector (PMT - photo multiplier tube). Higher voltages increase
the sensitivity for very weak signals but also increase image background.
Unfortunately increasing PMT voltage also results in amplifying noise. Because of
this fact PMT voltage should be modified with care. Another pitfall is simultanous
enhancement of strong signals bringing very intense spots to saturation.

In desktop scanners the A/D converter can very often differentiate a larger
dynamic range of intensities than what can be stored in the file format. This is the
reason, why the internal (A/D tranducer) color depth (e.g. 12 bit) may be larger
than the output (e.g. 8 bit). The advantage of such a constellation is the flexibility
for the selection of the right intensity range after a prescan for the final data
storage. An alternative approach is the data processing of the complete data
coming from the A/D converter by using gradation curves (see also image
calibration).

DECODON
Scanning Guide

Intensity Range
Enlarging the intensity range between signal and background is one of the major tasks in
image generation. The larger the distance between background and top intensity in an image the
larger the dynamic range that is available for coding intensity levels. The more intensity levels are
coded in the image the more accurate is the quantitative analysis. There are several ways to
increase the signal to background range:

Cutting off a high background by a scanner preset. In the prescan the user can traditionally specify
the lowest and highest intensity signals that will be used as lowest and highest intensity signal in the
resulting image file. The scanner will then distribute the measured intensity values evenly between the
given minimum and maximum. This makes sense if the A/D converter can distinguish more intensity
levels than what can be stored in the image file. Note that this approach only reaches highest
performance if you avoid scanning areas that are not covered by the gel.
 EXPLORING LIFE.

Scanner Settings III

Controlling the conversion from color to grayscale. Most office scanners create
grayscale images by converting a color scan to a grayscale image file. This is done by
averaging intensities over the different color channels. You can improve the resulting
image in some situations by controlling the color to grayscale conversion yourself. For
example:

A coomassie blue stained image can be better scanned by excluding the blue
color channel from the color image before converting it to a grayscale image.

A similar approach can be used for the brownish silver stains. As a rule,
extract those color channels with high absorption. Brownish silver stains absorb
the blue light most efficiently, so extracting the blue color channel will give the
best results.

Increasing the voltage of the light detector. If the intensity range is not completely
used by intensity signals in a fluorescence scan the voltage of the light detector may
be increased. This will increase the signals overall but also increases the absolute
distance between background signal and strongest intensity signal on the gel image.
Please be aware of saturation effects!

DECODON
Scanning Guide

Saturation
Effects
A perfect Spot without any saturation effect

Saturation effects heavily affect quantitative data because they virtually truncate the
peaks of high intensity spots. These truncations distort the quantities of spots. Many
scanner software packages highlight saturated image regions. Saturation effects may occur
for several reasons:

Intense staining can cause complete absorption of light passing through the spot. This
results in planar truncated spots in the 3D view of the gel image. Try to use a scanner with a
more intense light source (e.g. a laser scanner) or to warm up the scanner's lamp for a longer
time. Reducing staining or increasing destaining time may also solve this problem.

Dyes can appear in very high concentration. Some staining techniques accumulate pigment on
top of high intensity spots losing the amount / intensity correlation. This can be observed in all
techniques based on the photographic silver nitrate staining (silver staining, x-ray film).

While for classical silver staining techniques the typical doughnut spots may be observed the
saturations from x-ray films have a planar appearance. To avoid these problems use shorter staining
or developing times, or load less protein on the gel. Silver staining should not be used in quantitative
proteome analysis because of saturation problems, and, more importantly, because the relation
between silver stain intensity and protein amount is usually not linear.

A similar effect can be observed during phosphorimaging. If a gel is overexposed to a phosphorscreen

the screen’s capacity to accumulate energy from radioactive decay events may be exhausted. This also
results in planar truncated spots in the 3D view. Here reducing the exposure time will solve the problem.
 EXPLORING LIFE.

Scanner Settings IV

A saturated protein spot

Heavily fluorescing gels may overload the light detector or heavily excited light
detectors may overload the A/D converter. This results in a loss of differentiation
ability of strong signals. This also results in planar truncated spots. Here reduction
of the PMT voltage may help.

Subsequent manipulation of the image data by using image processing software

not designed for quantitative image analysis may ruin your images. Visually
enhanced images often show truncations in the high and low intensity regions. Gel
image files are not only pictures but also collections of quantitative data. Only
flipping, mirroring, rotating in 90 degrees steps and cropping leave the original
data intact. Free rotating, enlarging or reducing, any kind of gray level, contrast or
gamma adjustion distorts the quantitative data and may destroy your image files
(see also image calibration).

Silver stain: A "doughnut protein spot" that lost its central quantity.

DECODON
Scanning Guide

Artifacts,
Noise, Background
Avoiding background, artifacts and noise is essential. Such features may disturb the spot
detection process: Gel breaking separates spots, sprinkles may mislead the spot detection, noise
can cover low intensity spots etc. The weaker the signals the stronger is the effect of artifacts on
quantitative analysis. Very low signals may be observed due to faint spots, very low sample
amounts or bad incorporation of radioisotopes. Very often it is better and less frustrating to repeat
the experiment instead of doing analysis of suboptimal images.

Background may be caused by insufficient destaining, fluorescing glass plates, gel coverings and
backings. Using low fluorescence equipment will minimize such problems. Also misusing optical filters
for fluorescence imaging may cause background. Reevaluate which light sources and filters are useful for
your analysis.
 EXPLORING LIFE.

Avoiding artifacts

Noise can be produced by high PMT voltages enhancing also random signals.
Adapting the PMT voltage will solve this problem. Also phosphor screens that have
not been used for a longer time accumulate noise. Erasing the screen immediatly
before exposure solves this problem. Try to optimize the scanning procedure and
avoid post scan manipulation of your images.

Artifacts affect the quantitation process. However, they can often be avoided
very easily. The following list shows some problems and the corresponding solution.

● Artifacts on the gel surface

Try to focus the laser beam inside the gel if your imaging device supports this
feature.
● Fingerprints on the gel
Use (powderfree) gloves and/or clean the glass panels of your scanners
● Fluorescent sprinkles (s. figure)
Rinse the gel with destainer or water and avoid evaporation. Use powderfree
gloves, dust free experimental equipment in a dust free environment.

Fluorescent sprinkles can disturb the spot detection.

DECODON
Scanning Guide

More Artifacts ● Artifacts from phosphorscreens

Decontaminate or replace phosphorscreens; scan autoexposed
phosphorscreens before use to locate and remove contaminations.
● Artifacts and scratches on glass panels
Handle your imaging devices with care and only use a mild detergent and wood
free tissue for cleaning. Keep all glass panels clean and free of dust and fat.
Avoid regions containing artifacts during scanning.
● Ghost images or spot shadows
Do not move the gels during exposure or scan. Be
aware of scanner vibrations during the scan process.
Fix the gel on the scanner surface (use suction cups)
and erase phosphorscreens before using.
● Air bubbles and water droplets
Use enough water between glass panel and gel but be
aware of water running inside your scanner. Composition
rubber may help to seal problematic grooves. Ghost images are problematic
because they are hard to
recognize.

Bubbles may interfere with quantitation.

● Newtonian rings
Try to avoid using glass plates. If this is not possible focus the laser beam inside the gel.
 EXPLORING LIFE.

More Artifacts

● Horizontal or vertical lines

Sometimes single CCD elements in CCD bars of a desktop scanner fail to
work. The corresponding line of the image remains empty. The only way to
solve this problem is replacing the CCD element or the whole scanner.
● Gel breakings and gel pieces

Breaks in the spot cause problems with spot detection. This may be corrected
manually.

Work carefully and use gel strengthener to give the gel more stability. If
necessary try to puzzle the gel pieces to close the breakings before scanning.
● Labels on the gel
Using permanent markers for gel labeling sometimes interferes with the scan.
To simplify the subsequent analysis please make sure that the label does not
overlay parts of the gel or spot pattern.

DECODON
Delta2D
DECODON produces Delta2D, a leading 2D gel image
analysis software. With advanced image processing,
robust quantitative analysis, proteome maps, and web
scouts, Delta2D lets you extract more information from
your gels, in a fraction of the time needed by
conventional packages.

Learn more at http://www.decodon.com.

Contact
web: www.decodon.com
email: info@decodon.com
phone: +49 (0)3834 51 52 30
fax: +49 (0)3834 51 52 39

DECODON GmbH
Walther-Rathenau-Str. 49a
D-17489 Greifswald
Copyright and Trademarks
Germany
All material in this brochure is Copyright © 2005 DECODON GmbH.
All Rights Reserved. DECODON, DECODON logo, Delta2D, and
Protecs are trademarks or registered trademarks of DECODON
GmbH in Germany and in several other countries all over the world.
All other products mentioned are trademarks or registered
trademarks of their respective companies.
DECODON