Biology Reference
In-Depth Information
3.2. Microscope Setup and Data Acquisition
In a confocal-FRAP experiment, a prebleach image is acquired, the sample
is bleached, and a recovery image series is collected and analyzed. Scanning
confocal microscopes excite fluorescence with a laser and detect emitted light
with a photomultiplier (PMT). The final image intensity is a complex function
of the sample concentration, laser intensity, scan rate, optical path, confocal
pinhole size, and PMT gain. These parameters must be set so that imaging of
the sample is within the linear region of PMT response for imaging prebleach
and during the recovery. The essential elements of a protocol, based on a square
bleach and a moments recovery analysis will be given. This protocol, though
general, does require that the switching between microscope configurations is
directed by software, a facility which is available on all recent generations of
confocal scanners. The analysis is not dependent on the shape of the bleach as
long as it is symmetrical about the bleach center, and whereas a circular bleach
may give the optimum geometry, it is much easier and faster to generate a
square bleach because of the scanning action of a confocal microscope.
1.
Determine instrument settings for bleach and prebleach/recovery and save as
separate set-up configurations to be accessed by the bleaching software (
see
Note 5
).
Typically, for prebleach/recovery the scan time = 2 s, laser intensity = 1% of
maximum, PMT gain = 1000 V, iris size = 75% of maximum. Typical bleach
settings are scan time = 2 s, laser intensity = 100%, PMT gain = 0, iris size =
minimum (
see
Note 6
).
2.
At the start of the bleaching and data acquisition program, a square region for
bleaching is defined at the center of the field of view. In general, the more mobile
species the larger the bleach and the faster the image scan rate required.
3.
The microscope is set and one or more prebleach images are scanned, centered
on the proposed bleach. Scan dimensions are
4 the bleach width, optical zoom = 1.
Several prebleach images may be averaged to reduce noise.
×
4.
The microscope switches to bleach configuration and sample bleaching is per-
formed at 100% laser power (
see
Note 7
). The bleach scan area is
16 smaller in
area than the prebleach area, although the scan time is the same. The total energy
delivered to the bleach scan area is therefore
×
1600 that conveyed to the same
area during a single prebleach or recovery scan.
×
5.
The microscope resets to the prebleach setup, and a series of recovery images (up
to 50) are collected. For slowly moving species, two or more images should be
taken and averaged in real time at each scan time-point (Kalman type averaging
is suitable). More mobile species require faster data acquisition, the rate of image
acquisition can be increased by saving images direct to the computer's random
access memory (use a software utility such as MS RAM-disk) rather than the
hard drive (
see
Note 8
).
6.
Final images should be taken at two long time points and compared to check that
the recovery is complete. This is important as it enables the mobile fraction to
be calculated.
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