Page 225 - Fundamentals of Light Microscopy and Electronic Imaging

208 CONFOCAL LASER SCANNING MICROSCOPY

In this chapter we discuss the optics and essential electronic features of the confo-
cal microscope, review briefly the parameters that are important in acquiring and evalu-
ating an electronic image, and then consider how these parameters are affected by the
operating controls of the confocal microscope.

THE OPTICAL PRINCIPLE OF CONFOCAL IMAGING

The confocal microscope is an integrated microscope system consisting of a fluores-
cence microscope, multiple laser light sources, a confocal box or scan head with optical
and electronic equipment, a computer and monitor for display, and software for acquir-
ing, processing, and analyzing images. The scan head generates the photon signals
required to reconstruct the confocal image and contains the following devices:

• Inputs from one or more external laser light sources
• Fluorescence filter sets
• A galvanometer-based raster scanning mechanism
• One or more variable pinhole apertures for generating the confocal image
• Photomultiplier tube (PMT) detectors for different fluorescent wavelengths

The general arrangement of components in the scan head is shown in Figure 12-3. A
computer converts the voltage fluctuations of the PMTs into digital signals for image
display on the computer monitor.
The optical principle of confocal microscopy is shown in Figure 12-4 and is
described as follows:

• Epi-illumination is used, where the light source and detector are both on the same
side of the specimen plane and separated from it by the objective lens, which func-
tions as both a condenser and objective. The components of fluorescence filter sets
(exciter filter, dichroic filter, emission filter) perform the same functions as they do
in wide-field fluorescence microscopy.
• A laser beam is expanded to fill the back aperture of the objective and forms an
intense diffraction-limited spot that is scanned from side to side and from top to
bottom over the specimen in a pattern called a raster. This procedure is called point
scanning.
• The heart of confocal optics is the pinhole aperture, which accepts fluorescent pho-
tons from the illuminated focused spot in the raster, but largely excludes fluores-
cence signals from objects above and below the focal plane, which, being out of
focus, are focused on the pinhole as disks of much larger diameter. Because the size
of the disk of an out-of-focus object is spread out over such a large area, only a frac-
tion of light from out-of-focus objects passes through the pinhole. The pinhole also
eliminates much of the stray light in the optical system. Examine Figure 12-4 care-
fully to see how the pinhole blocks out-of-focal-plane signals. The combination of
point scanning and the use of a pinhole as a spatial filter at the conjugate image
plane are essential for producing the confocal image.
• Fluorescent wavelengths emanating from an excited point in the specimen at any
time t are collected by the same objective and focused as a small diffraction spot