Page 241 - Fundamentals of Light Microscopy and Electronic Imaging

224 CONFOCAL LASER SCANNING MICROSCOPY

TABLE 12-2 Effect of Increasing Confocal Parameters on Image Intensity and
Spatial Resolution

Parameter Effect
↑ Pinhole diameter ↑ Intensity
↓ Spatial resolution

↑ Zoom ↑ Intensity
↑ Spatial resolution

↑ Scan rate ↓ Intensity
↑ Temporal resolution

↑ Objective NA ↑ Intensity
↑ Spatial resolution

and antifade reagents (mounting medium with components that reduce the rate of pho-
tobleaching) can be used. Cyanine dyes (Amersham International, marketed by Jackson
ImmunoResearch Laboratories, West Grove, Pennsylvania) or Alexa dyes and new
antifade reagents such as SlowFade and ProLong (Molecular Probes, Eugene, Oregon)
are suitable reagents.
The microscope operator can reduce the rate of photobleaching through one or
more of the following methods:

• Reduce the laser power.
• Insert neutral density filters into the excitation beam.
• Scan at a faster rate.
• Lower the zoom factor (scan over a larger area).
• Widen the pinhole (allows shorter exposure time).
• Use a long-pass emission filter to capture as much fluorescent light as possible.
• Reduce the number of frames used for frame averaging.

For z-series intended for three-dimensional viewing, the operator can sometimes collect
the stack of images from front to back so that more distant details, made dimmer by pho-
tobleaching from repetitive scanning, match the viewer’s expectations that fluorescent
signals from more distant locations will appear fainter in the three-dimensional view.

GENERAL PROCEDURE FOR ACQUIRING A CONFOCAL IMAGE

The sequence of steps used to acquire an image with a confocal laser scanning micro-
scope usually proceeds as follows:

• Prepare for acquisition.
1. Focus the specimen in visual mode with the appropriate filter set.