Page 247 - Fundamentals of Light Microscopy and Electronic Imaging

230 CONFOCAL LASER SCANNING MICROSCOPY

Direct viewing of the confocal image through an eyepiece increases the speed, ease,
and efficiency of specimen positioning, focus, and examination.
• With a high-dynamic-range CCD camera, the gray scale range is extended to thou-
sands of levels, and light intensity can be quantitated with higher resolution and
accuracy. S/N is increased and images look considerably smoother and less noisy.
• Time resolution is greatly improved, because the rotating disk provides 360
frames/s (3 ms/frame) as opposed to 12–36 frames/s for single-point scanning con-
focals.
• The reduction in sampling time reduces the rate of photobleaching and the amount
of phototoxicity.
• The very rapid scan rate (12.5 s) results in very efficient fluorescence emission.
Among the benefits is improved detection of fine structural details represented by
clusters of only a few fluorescent molecules.
• Since there are no adjustments for the pinhole, scan speed, frame size, or zoom fac-
tor, the Nipkow confocal is considerably easier to use from the point of view of
microscope-operating software.

The pinholes in the Nipkow disk perform the same function as the pinhole in a single-
point scanning system in generating a confocal image of an optical section. Incorpora-
tion of a z-axis stepper motor and three-dimensional imaging software also permits
volume viewing and single-plane composite or projected views. Early Nipkow disk
imaging systems had the disadvantage of very low light transmission, but a recently
introduced double-disk design by Yokogawa Electronics, Inc., (Tokyo) remedies the
problem by including microlenses that greatly boost light-gathering efficiency and
transmission. The Yokogawa-Nipkow system has been thoroughly reviewed in a recent
publication by Inoué and Inoué (2000). The popularity of modified Nipkow disk confo-
cal systems is certain to increase in the future. The disadvantages of this system are that
the optical section is somewhat thicker than with a conventional confocal with a
stopped-down pinhole and that the microlenses of the disk do not transmit UV light,
which precludes the use of UV-excitable dyes.

Exercise: Effect of Confocal Variables on Image Quality

This exercise should be performed during or after a training session on the use of
a confocal microscope by an experienced user.

• The following adjustments can be used to brighten a confocal image. Exam-
ine the effects of each manipulation on image quality beyond the effect on
brightness:
1. Remove a neutral density filter or increase the laser power.
2. Open the pinhole diaphragm.
3. Increase the PMT gain.
4. Decrease the PMT offset.