Biology Reference
In-Depth Information
To ensure that always one defi ned section of all gels within an
MFA experiment is scanned, it is useful to pre-defi ne a scan area
using the scanner control software. It might be helpful to use special
hardware extensions of the scanning devices, e.g. gel holding trays,
that may help to adjust gels or gel cassettes. In addition, the orien-
tation of the gel might be redefi ned by software set-up and should
be well documented. When scanning gels inside their cassettes, the
confocal optics of the laser scanning device has to be adjusted
focussing 3 mm above the scanner glass platen.
For multifl uorescence 2D gel imaging, the imaging device has to
be equipped with a defi ned combination of emission fi lters and
excitation fi lters (CCD scanning systems) or lasers (laser fl uorescence
scanners). For the three CyDye™ fl uorescence labels, the ideal
combinations of excitation and emission wavelengths for multifl u-
orescence 2D gel imaging are given below:
Laser, Filter and
Photomultiplier Settings
Cy2: Blue (480 nm) excitation fi lter/laser, 520 nm emission fi lter.
Cy3: Green (532 nm) excitation fi lter/laser, 580 nm emission fi lter.
Cy5: Red (633 nm) excitation fi lter/laser, 670 nm emission fi lter.
The specifi cations and the quality of the applied fi lters and lasers
are crucial for a reliable separation of the three signal channels. Best
possible results can be accomplished by a combination of excitation
lasers with high-quality band-pass emission fi lters. The scanner
control software provides the opportunity to select (only) the
appropriate combinations of excitation and emission wavelengths.
Each multifl uorescence 2D gel has to be scanned in all fl uores-
cence channels. The scans have to be carried out as individual scans
resulting in a set of individual gel images.
The intensity of the fl uorescence signals emitted by labelled
proteins in a gel can vary considerably depending on the total
amount of protein applied to the gel and the amount of fl uores-
cence dyes used for protein labelling. To maximize the dynamic
range of the imaging device, the signal amplifi cation of the scan-
ning CCD camera or the PMT has to be adapted to the strength of
the fl uorescence signals in order to prevent signal saturation. In
case of using fast CCD cameras, the adaptation can be performed
“on the fl y”. In case of using a laser fl uorescence scanner, the adap-
tation routine is a little bit more time consuming. As mentioned
above, the control of the signal amplifi cation is carried out by vary-
ing the PMT voltage. For voltage estimation, a pre-scan of the gel
has to be performed and the generated corresponding image fi les
have to be analysed, whether the pixel intensity values cover the
full range between 1 and 100,000. In particular, the maximum
pixel intensity value should be within this range, exceeding it would
mean signal saturation and would prevent an accurate quantifi cation
of spot volumes by image analysis software. An example for signal
Search WWH ::




Custom Search