Page 240 - Fundamentals of Light Microscopy and Electronic Imaging

PHOTOBLEACHING 223

of the PMT is being utilized. Figure 13-10 shows the effect of gain and offset on image
contrast.

Objective Lens Selection

The quality of the confocal image is critically dependent on the optical performance of
the objective lens. The lens should be well corrected for the principal lens aberrations
discussed in Chapter 4. Different color wavelengths must be focused precisely in the
confocal pinhole, and spherical and off-axis aberrations such as coma and astigmatism
must also be corrected to a high degree. This is because the laser must be focused to a
well-defined diffraction spot at locations in the specimen that are off the optic axis, and
fluorescent wavelengths must be collected and focused to a spot in the pinhole. As we
now also appreciate, the NA of the objective is important, because it determines the spa-
tial resolution and brightness of the image and the depth of focus in the specimen. The
role of the NA in determining spatial resolution has already been discussed (Chapters 3
and 6). Image brightness (photon flux per area per unit time) is also determined by the
NA and is of great practical importance for confocal microscopy. Since the amount of
light reaching the photomultiplier tube is usually small, we are frequently forced to
operate the confocal system at high electronic gain, which tends to produce noisy
images. The gain can be reduced and the image quality improved by providing a brighter
diffraction spot at the pinhole with a high-NA objective. Since image brightness is pro-
2
4
portional to NA /Mag (Chapters 4 and 6), you should select the lowest magnification
commensurate with the resolution requirements, and within that constraint, choose the
highest available NA. For these reasons, a well-corrected plan-neofluorite or plan-
apochromatic objective of 60–100 magnification is ideal for the examination of cel-
lular details.
In addition to the confocal pinhole, the objective lens plays an equally important
role in determining the depth of field in the specimen and the depth of focus in the
image, and therefore the thickness of a confocal image. The thickness d of the focal
2
plane along the z-axis in the specimen is defined as λ/NA and is determined by the
wavelength λ and the numerical aperture NA of the objective lens (Chapter 6). Since in
epi-illumination mode the objective focuses the laser spot on the specimen, high-NA
lenses reduce the thickness dimension of the diffraction spot for laser excitation and
reduce the focal plane thickness at the exit pinhole. Reducing the size of the pinhole
diaphragm limits the thickness of the confocal optical section, but only to the limit set
by the microscope optics. Therefore, the combination of high-NA lenses and small pin-
hole apertures generates the thinnest optical sections. The effects of the mechanical
adjustments on image brightness and spatial resolution are summarized in Table 12-2.

PHOTOBLEACHING

Confocal specimens are subject to photobleaching from repetitive exposure to the
intense beam of the laser. The rate and extent of bleaching are generally greater than for
specimens examined with a wide-field fluorescence microscope and can make it diffi-
cult to obtain suitable images of weakly fluorescent specimens. If an immunolabeled
specimen appears dim and its image looks unacceptable due to improper staining, a new
specimen should be prepared. In other cases, dyes with lower rates of photobleaching