Page 222 - Fundamentals of Light Microscopy and Electronic Imaging

CHAPTER
12

CONFOCAL LASER SCANNING
MICROSCOPY

OVERVIEW

Thick fluorescent specimens such as rounded cells and tissue sections can pose prob-
lems for conventional wide-field fluorescence optics, because bright fluorescent signals
from objects outside the focal plane increase the background and give low-contrast
images. Confocal and deconvolution microscopy solve the problem by rejecting signals
from nearby sources above and below the focal plane. In confocal microscopes this is
accomplished optically by illuminating the specimen with a focused scanning laser
beam (point scanning) and by placing a pinhole aperture in the image plane in front of
an electronic photon detector. Both fluorescent specimens and reflective surfaces can be
examined using this technique (Fig. 12-1). Confocal images can also be produced using
a spinning Nipkow disk that gives tandem scanning with literally thousands of scanning
beams. In deconvolution microscopy a standard wide-field fluorescence microscope is
used, and the image of an optical section is obtained computationally with a computer
that removes out-of-focus light from the image. (Deconvolution microscopy is not cov-
ered in this book. For discussion of this topic, the reader is directed to an excellent per-
spective by Chen et al., as well as early pioneers and developers of the method, David
Agard and John Sedat (Pawley, 1995).) The high-contrast images provided by confocal
and deconvolution methods can provide clear answers to commonly asked questions
about fluorescent microscope specimens: Is a fluorescent signal distributed on a mem-
brane surface or contained throughout the cytoplasm as a soluble factor? Within the lim-
its of resolution of the light microscope, are different fluorescence signals colocalized
on the same structure? What is the three-dimensional structure of the specimen?
By using a stepper motor that changes the microscope focus in 100 nm steps along
the z-axis, confocal and deconvolution microscopes make it possible to acquire a stack
of images or z-series at different focal planes and generate a three-dimensional view of
the specimen using computer software. Microscope savants and manufacturers foresee
a time when it will become routine for microscopists to acquire z-section stacks of live
cells in multiple color channels, with stacks acquired at regular time intervals, so an
entire color movie can be constructed showing dynamic events in a cell in three dimen-
sions—a truly valuable and exciting experience! Such sequences are called five-
dimensional, because intensity information for every point in x, y, and z dimensions in 205