Chapter 2
Microscopy and Imaging Systems
ANDREAS PLESCH
Introduction
Any FISH technique remains useless without visualization of the probes.
In many routine FISH applications visual interpretation of the FISH
images by looking down the microscope may be sufficient. An imaging
system with its image enhancement capabilities will usually facilitate
the analysis and add the convenience of documentation and archiving
of the results. In a number of modern FISH techniques, however, quantitative digital image analysis is indispensable to obtain the relevant information. Each step in the imaging process (or each element in the chain of
imaging components) has the inherent risk of influencing (generally deteriorating) the image. The following chapter summarizes the fundamental aspects and limitations of the imaging process.
Basic concepts
Optical resolution
Any lens or optical instrument suffers from a physical phenomenon called
diffraction. Diffraction is due to the wave nature of light and will make an
infinitely small point-like object appear blurred (Fig. 1). The apparent size
of the blurred image depends on the wavelength of the light and of the
ratio of focal length and diameter of the lens and is given by the following
equation:
OVERVIEW
d = 2.44 A flO
(1)
~ Andreas Plesch, MetaSystems GmbH, Robert-Bosch-Str. 6, Altlussheim, 68804,
Germany (phone +49-6205-39610; fax +49-6205-32270;
e-mail aplesch@metasystems.de)
Microscopy and Imaging Systems
ANDREAS PLESCH
Introduction
Any FISH technique remains useless without visualization of the probes.
In many routine FISH applications visual interpretation of the FISH
images by looking down the microscope may be sufficient. An imaging
system with its image enhancement capabilities will usually facilitate
the analysis and add the convenience of documentation and archiving
of the results. In a number of modern FISH techniques, however, quantitative digital image analysis is indispensable to obtain the relevant information. Each step in the imaging process (or each element in the chain of
imaging components) has the inherent risk of influencing (generally deteriorating) the image. The following chapter summarizes the fundamental aspects and limitations of the imaging process.
Basic concepts
Optical resolution
Any lens or optical instrument suffers from a physical phenomenon called
diffraction. Diffraction is due to the wave nature of light and will make an
infinitely small point-like object appear blurred (Fig. 1). The apparent size
of the blurred image depends on the wavelength of the light and of the
ratio of focal length and diameter of the lens and is given by the following
equation:
OVERVIEW
d = 2.44 A flO
(1)
~ Andreas Plesch, MetaSystems GmbH, Robert-Bosch-Str. 6, Altlussheim, 68804,
Germany (phone +49-6205-39610; fax +49-6205-32270;
e-mail aplesch@metasystems.de)