Chapter 12 Microscopy Applications 147
Adjusting the Parfocality of the Camera
To adjust the parfocality on an F-mount system, begin collecting images with a short
exposure time and focus the light on the camera by rotating the ring on the Diagnostic
Instruments relay lens without touching the main focusing knobs on the microscope.
While adjusting parfocality, you will need to acquire images rapidly to minimize the delay
between the time a setting is changed and the time when the effect of the change can be
observed. The specifics of how to proceed will vary according to the application software.
In the WinX application software, select Acquisition, Video Focus. Begin with an
exposure time of 0.1 sec. Then use Focus to begin data acquisition and Stop Acquisition to
end it when you are finished focusing. See your WinView/32 or WinSpec/32 manual for
additional information.
In IPLab, begin by selecting Set Camera from the Ext. menu. Set the dialog to
Asynchronous, 100 msec exposure, and 2 cleans. Establish Focus mode operation by
selecting Focusing from the Ext. menu. Set this dialog to 0.1 pixel seconds, 0-256 gray
scale, and 1×. Press Start Camera to begin focusing and Stop Camera when finished. See
your IPLab manual for additional information.
Many PI cameras, both F-mount and C-mount, also make provision for extending the
focus range by providing a focus adjustment on the camera lens mount. If necessary, this
focus can be changed to bring the image into range of the relay lens or other microscope
focus adjustment.
Imaging Hints
Determine the gray levels of the image by placing the cursor within the image and
monitoring the values shown. For optimal image quality of a 12-bit image, the highest
value in the field should be near 4000 counts but not at 4095 (which is saturating). You
may increase the number of counts by increasing your exposure, increasing the intensifier
gain, or by increasing the amount of light illuminating the specimen.
Note that adjusting the intensifier gain also affects the dark charge of the intensifier
(EBI). To properly perform background subtraction a background must therefore be
measured at each intensifier gain setting.
Fluorescence
Once you have acquired a suitable image in transmitted light mode, you may switch to
fluorescence mode.
In fluorescence mode you generally want to minimize the bleaching of your sample,
usually achieved by placing several neutral density filters in the excitation pathway to
minimize the illumination intensity. There will always be a trade-off here; when you
maximize signal quality by increasing the illumination intensity, you need to consider
whether your preparation can tolerate these conditions. In general, it is better to expose
longer with a lower intensity than to expose for a shorter time with a higher intensity;
nevertheless, your experimental conditions will dictate which path you take.
In fluorescence measurements you may not wish to maximize the gray levels in the
image, since this may cause bleaching of the dye or photodamage to the cell. Maintain
the minimum exposure required to get a sufficiently high quality image.
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