Page 235 - Fundamentals of Light Microscopy and Electronic Imaging

218 CONFOCAL LASER SCANNING MICROSCOPY

• Detector pinhole aperture
• Laser scan rate and image size
• Zoom factor
• Laser selection and laser intensity
• Gain and offset of the PMT detector
• Objective lens selection

Pinhole Adjustment

Parameters such as focal plane thickness, spatial resolution, image brightness, and sus-
ceptibility to photodamage are affected by the size of the detector pinhole. Since the pin-
hole is frequently adjusted to control these parameters, the confocal operator needs to
become familiar with its effects. The effect of the pinhole diameter on the thickness of
the optical section is shown in Figure 12-9. As already described, a small pinhole gives
the thinnest optical section. It also improves horizontal resolution and contrast in the
image. However, reducing the size of the pinhole also decreases the number of photons
reaching the detector. If we compare two images of comparable amplitude made at dif-
ferent pinhole settings, the image corresponding to the smaller pinhole diameter
requires a longer exposure for the same signal-to-noise ratio. However, increasing the
exposure time can cause significant photobleaching in immunofluorescence specimens
or compromise viability in live cell preparations. In contrast, a wide pinhole increases
photon flux and reduces photobleaching by requiring less exposure of the specimen to
the laser. If we compare images made at different pinhole settings but the same exposure
time, we see that the image acquired using a wider pinhole is brighter and contains less
noise.
The optimum average pinhole setting corresponds to an aperture giving 50% of
maximum intensity. At this position, 75% of the light of the Airy disk still passes the
pinhole, and resolution will be 20% better than obtained by a wide-field fluorescence
system. This corresponds to a pinhole diameter that is slightly smaller than the first dark
ring in the Airy disk pattern. Generally, higher magnification objectives require larger
pinhole settings because they produce larger Airy disks.

Scan rate, Image Size, and Dwell Time

The galvanometer-based scanning mechanism determines the size of the area in the spec-
imen that is covered by the raster and the rate at which the specimen is scanned. These
parameters determine, in part, the pixel size in the captured image, the spatial resolution,
and the laser dwell time—that is, the length of time the laser dwells on the specimen per
pixel element in the image. If the amplitude of deflection of the galvanometer mirrors is
small and the scan rate is high, a smaller image area can be presented on the monitor at
rates up to hundreds of frames per second. High-speed sampling (20–30 frames/s) gives
the appearance of a live image on the monitor and is convenient for focusing the speci-
men. High speed is also useful for monitoring rapid kinetic events in the specimen. In
contrast, a large-amplitude deflection of the galvanometer mirrors coupled with a slow
scan rate produces a large-size image at high resolution but at a frame rate of 1 frame/s or
less. For fixed, stained fluorescent specimens, the capture rate is typically 2 frames/s for