Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 34)

Imaging Systems
Bio-Rad’s GS-900 calibrated imaging densitometer
has transmittance and true reflectance capabilities
that allow accurate scans of samples that are
either transparent (gels and film) or opaque (blots).
It provides high-quality imaging to resolve close
spots and a variable resolution feature to preview
and crop images.
Bio-Rad’s ChemiDoc MP supercooled CCD
system provides maximum flexibility. It offers
transillumination of both UV light (for imaging UV
fluorescent stains) and white light (for imaging visible
stains). It also offers optional LED epi-illumination
in red, green, and blue for single fluorescent stains
or fluorescent multiplexing. In addition, it can also
image stain-free gels, which require no staining or
destaining and are ready for imaging in a matter of
minutes after completing the SDS-PAGE run.
System Type and Application
GS-900 ™ PharosFX ™ and
Densitometer ChemiDoc ™ MP Gel Doc ™ EZ PharosFX Plus
Type of imager Densitometer CCD camera-based CCD camera-based Laser-based
Light source options Epi- and Transillumination of UV Transillumination of UV 488 nm external laser
transillumination of and white light* and white light 532 nm internal laser
white light 635 nm external laser
Epi-illumination by LEDs
(red, green, blue, and white)
Optimized applications
Visible stains
• • • —
UV light–excited — • • —
fluorescent stains
Visible light–excited
— • — •
fluorescent stains and labels
Fluorescent multiplexing
— • — •
Chemiluminescence — • — —
Stain-Free ™ — • • —
* White light conversion screen is required.
The PharosFX systems use multiple lasers, which
enhance application flexibility and allow optimum
excitation of single- or multicolor fluorescent
samples to enable detection of most fluorescent
dyes and labels. Computer-controlled, user-
accessible filter wheels have eight filter slots,
supporting multiplex or multicolor fluorescence
imaging applications in gels and blots, such as
Qdot multiplex blotting, DIGE, and gel staining with
Pro-Q stains. The Molecular Imager
® PharosFX
system has all the features of the PharosFX Plus
imager for fluorescence and visible detection,
but it lacks the storage phosphor option for
imaging radioisotopes.
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2-D Electrophoresis Guide Theory and Product Selection Chapter 6: Image Acquisition, Analysis, and Spot Cutting
■ ■
Charge-coupled device (CCD) camera systems can
feature different light sources for greater application
flexibility. They can be used for visualization
of visible and fluorescent stains and of
chemiluminescence in some cases. Systems
offer transillumination (visible or UV light source
underneath the gel or blot) or epi-illumination
(colored or white light positioned above the sample).
Heat in the camera system can manifest as noise,
and this noise can prevent detection of faint
chemiluminescent signals above the background.
Supercooled CCD cameras reduce image noise,
allowing detection of faint signals
■ ■
Laser-based scanners offer the highest sensitivity,
resolution, and linear dynamic range. They are
powerful image acquisition tools for electrophoresis
gels and blots stained with fluorescent dyes.
Lasers can be matched to the excitation wavelengths
of a multitude of fluorophores
Finding Protein Spots of Interest
After 2-D gels are stained, the protein patterns can
be digitized and analyzed with an image evaluation
system comprising an imaging device and analysis
software. Following analysis, spots of interest can
be excised from gels for further analysis, by mass
spectrometry for example (see Chapter 7).
Image Acquisition
In proteomic applications, selecting the image
acquisition device depends on the staining technique
used. A number of imaging systems are capable of
multiple detection modes and can be used with a
variety of applications.
■ ■
Densitometers enable the visualization of gels
stained with visible light–absorbing stains such as
Coomassie, negative, or silver stains
Image Analysis
Following image acquisition, a robust software
package is required to analyze and present the
data and to draw conclusions from 2-D gel images.
The software should provide a variety of tools to
enhance the user’s ability to evaluate the acquired
data. For example, the software should be able to
adjust contrast and brightness and magnify, rotate,
resize, and crop gel images. It should measure total
and average quantities and determine relative amounts
of protein. It should also be capable of determining
the presence/absence and up- or downregulation of
proteins, their molecular weight, pI, and other values.
Following this initial analysis, computer-assisted
image analysis software should allow:
■ ■
Storage and structuring of large amounts
of collected experimental image data
■ ■
Rapid and sophisticated analysis of
experimental information
■ ■
Supplementation and distribution of data among labs
■ ■
Establishment of 2-D-protein data banks