4
The
Confocal Principle
In this chapter, the mode of operation of an LSM
will be explained using a fluorescence-labeled spe-
cimen as an example. Fluorescent dyes, also known
as fluorochromes, are used as markers in most bio-
medical applications to make the structures of inter-
est visible. But laser scanning microscopes can just
as well be combined with other microscopic contrast
techniques such as reflected light or polarization.
An LSM can be easily understood as a modified
light microscope supplemented by a laser module
that serves as a light source, and a scanning head
(attached to the microscope stand) that is used to
detect the signal. Signal processing is effected by
an electronic system contained in a box. The
whole system is controlled by a computer.
To generate a confocal LSM image, let us first
excite the fluorescence marker in a defined speci-
men area with a laser. For this purpose, mono-
chromatic light from the laser module is coupled
into the scanning head via a fiber optic. In the
scanning head, the beam is made parallel by
means of a collimator, and reflected into the
microscope’s light path by the principal dichroic
beam splitter. The objective focuses the excitation
beam onto a small three-dimensional specimen
region called the excitation volume. The spatial
extension of this volume is directly related to the
system’s resolving power. The greater the numeri-
cal aperture of the objective, the smaller the focal
volume, and the higher the resolution. The posi-
tion of the excitation volume can be shifted later-
ally (in X and Y) by means of two scanning mir-
rors, and vertically (in Z) with the microscope’s
focusing knob. The current Z position marks the
system’s focusing plane.
Laser light
source
Collimator
Principal dichroic
beam splitter
Pinhole in the
confocal plane
Detector
Scanning mirrors
Objective
Specimen
Focal plane
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