Biology Reference
In-Depth Information
diffraction of light by the anticlinal walls (parallel to the inci-
dent light), however, the image quality in this part of the cell
is not as high as closer to the cover slip.
16. FPs have both broad absorption and emission peaks. As a result,
we often observe bleed-through of one fl uorophore into the
fi lter set of another fl uorophore. For example, GFP will be vis-
ible with both CFP and YFP fi lters. Similarly, it is possible to
excite RFP with GFP or YFP fi lters. This problem becomes
more noticeable with multiwavelength fi lters, but it can also
occur with dedicated fi lter sets when one fl uorophore is very
bright and the other is very dim. In this case, the long exposure
times necessary to detect the weak second signal may make it
possible to pick up the small amount of bleed-through of the
brighter fl uorophore. To test whether this problem may occur,
perform a transient expression with a single marker only and
collect an image with optimal settings for this fl uorophore.
Then switch to another fi lter set and take an image with identi-
cal settings. Repeat taking images with increasing exposure
times (twice as long, four times as long, etc.) until the signal
becomes visible with this “wrong” fi lter. Depending on the fi l-
ters and the fl uorophores, a two- to fourfold difference in expo-
sure time may result in negligible bleed-through, but this has to
be determined for every microscope setup independently. Also
note that contrast enhancement may bring out weak signals
that may go unnoticed at fi rst. Once these “bleed-through lim-
its” have been established, it is possible to detect this problem
simply by comparing exposure settings for marker and unknown.
Should this be a problem, it may be possible to reduce the
amount of DNA for the marker construct to reduce its signal.
17. The maximal signal intensities per image pixel should be about
10 % below the maximum that can be handled by the camera.
This would be an intensity of about 3,700 for 12-bit cameras,
15,000 for 14-bit cameras, and 59,000 for 16-bit cameras.
Bright signals ensure highest signal-to-noise ratios and hence
best image quality.
18. Organelles can often move with speeds exceeding 2
μ
m/s and
m/s. In this case, it is necessary
to reduce exposure times to 250 ms or shorter. This may
require increasing the electronic gain setting of the camera,
even though this tends to increase the noise more than longer
exposure times.
19. If possible, computer automation should be used to capture
the two images in close succession. Typically, the exposure set-
tings are fi rst determined and stored in the computer which
then controls the microscope to expose the camera with the
appropriate fi lter sets. With this approach, the delay between
the images is only limited by the exposure times and the speed
with which the fi lter sets can be switched. For this reason, a
reaching as high as 8 or 10
μ
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