Zeiss LSM 880, Operating Manual

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CHAPTER 1 - SYSTEM OPERATION
ZEISS Hardware Aspects LSM 880
10 000000-2071-464 10/2014 V_01
2 Hardware Aspects
2.1 Principle of Laser Scanning Microscopy
To yield information on their inner structure by conventional transmitted-light microscopy, specimens
have to be very thin and translucent; otherwise image definition will be poor. In many cases it is a
problem to satisfy these requirements.
The essential considerations have led to trailblazing changes in conventional microscopy and supplied a
successful solution of the problem above.
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Unlike the practice of even illumination in conventional microscopy, the LSM technique projects the
light of a point light source (a laser) through a high-NA objective onto a certain object plane of
interest as a nearly diffraction-limited focus. However, if not for another "trick", the stray light
produced outside the object plane, or the fluorescence of fluorescent specimens, would disturb the
in-focus image of object point of interest, resulting in a blurred image of poor contrast. The
problem therefore is how to capture only the light coming immediately from the object point in
focus, while obstructing the light coming from out-of-focus areas of the specimen.
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The light reflected, or the fluorescence light
produced, at the focus of the high
NA-objective is projected onto a variable
pinhole diaphragm by the same objective
and a tube lens. The focus inside the
specimen and the pinhole are situated at
optically conjugate points (
Confocal
imaging ). The decisive advantage of this
arrangement is the fact that essentially no
other light than that coming from the object
plane of interest can pass the narrow pinhole
and be registered by a detector. Unwanted
light coming from other specimen areas is
focused outside the pinhole, which passes
only a small fraction of it. The smaller the
pinhole, the less stray light or fluorescence
from out-of-focus areas will get on the
detector. The image point thus generated is
largely free from blur caused by unwanted
light.
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In order to obtain an image of the selected
object plane as a whole, it is necessary to
scan the object plane in a point-by-point,
line-by-line raster by means of an XY light
deflection system. The detectors - as a rule, photomultipliers - convert the optical information into
electric signals. This allows the image of any object plane to be generated and stored within less
than a second. By a defined focusing (Z axis) movement it is possible to look at any object plane of
interest. By scanning a succession of object planes in a specimen, a stack of slice images can be
produced.
This way, the LSM technique in conjunction with ICS optics (Infinity Color-Corrected System) has brought
decisive improvements over conventional microscopy in terms of resolving power and confocal depth contrast:
Object features in the order of 0.2 µm can be resolved, and height differences of less than 0.1 µm made
visible, without the use of interference methods.
Fig. 2 Principle of confocal imaging