Page 236 - Fundamentals of Light Microscopy and Electronic Imaging

ELECTRONIC ADJUSTMENTS AND CONSIDERATIONS 219

an area covered by 512 512 pixels. The scan rate options indicated in the confocal soft-
ware accommodate a range of scanning speeds and image sizes.
It should be remembered that the scan rate directly affects the rate of photobleach-
ing of the specimen (slower scan rate gives greater bleaching rate) and the signal-to-
noise ratio in the image (faster scanning reduces S/N). The scan rate also affects the
laser dwell time on a unit imaging area (pixel) in the image. With rapid scanning, the
dwell time can be reduced to about 1.0–0.1 s/pixel. The significance for live fluores-
cent specimens is that shorter dwell times reduce the rate of accumulated photodamage.

Zoom Factor

Electronic zoom is used for two reasons: (1) to adjust the sampling period of the laser
scanning mechanism to maintain spatial resolution, and (2) to magnify the image for
display and printing. Implementing the zoom command while maintaining the same
pixel format in the image causes the galvanometer scanners to cover a smaller area on
the specimen and reduce the scan rate. Since the zoomed image contains the same num-
ber of pixel elements, the image appears magnified on the monitor (Fig. 12-10). How-
ever, if significant changes in magnification are required, it is always preferable to use a
higher-power objective lens, rather than increase magnification electronically using the
zoom, because the higher-magnification, higher-NA objective gives better image reso-
lution and definition. In most cases, zoom control is used to make minor adjustments in

No zoom 2x zoom

3-dot specimen
and sampling
intervals

pixel display
on monitor

Figure 12-10
Effect of electronic zoom on electronic sampling and image resolution. Increasing the zoom
factor reduces the area scanned on the specimen and slows the rate of scanning. The
increased number of sampling operations along a comparable length in the specimen
increases the spatial resolution and magnifies the display on the monitor. Zoom is typically
employed when it is necessary or desirable to preserve the diffraction-limited optics of the
objective lens and optical system.