Zeiss LSM 880, Operating Manual

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Fig. 3 Non-confocal (left) and confocal (right) image of a triple-labeled cell
aggregate (mouse intestine section). In the non-confocal image, specimen planes
outside the focal plane degrade the information of interest from the focal plane,
and differently stained specimen details appear in mixed color. In the confocal
image (right), specimen details blurred in non-confocal imaging become distinctly
visible, and the image throughout is greatly improved in contrast.
Opticalslices
With a confocal LSM and it's variable pinhole it is therefore
possible to exclusively image a thin optical slice out of a
thick specimen (typically, up to 100 µm), a method known
as optical sectioning. Under suitable conditions, the thick-
ness (Z dimension) of such a slice may be less than 500 nm.
The fundamental advantage of the confocal LSM over a
conventional microscope is obvious: In conventional fluores-
cence microscopy, the image of a thick biological specimen
will only be in focus if its Z dimension is not greater than
the wave-optical depth of focus specified for the respective
objective lens.
Unless this condition is satisfied, the in-focus image infor-
mation from the object plane of interest is mixed with out-
of focus image information from planes outside the focal
plane. This reduces image contrast and increases the share of
stray light detected. If multiple fluorescences are observed,
there will in addition be a color mix of the image information
obtained from the channels involved (figure 3, left).
Hence it follows that a confocal LSM can be used to advan-
tage especially where thick fluorescent specimens (such as
biological cells in tissue) have to be examined. The possibil-
ity of optical sectioning eliminates the drawbacks attached
to the obser vation of such specimens by conventional fluo-
rescence microscopy. With multicolor fluorescence, the vari-
ous channels are furthermore satisfactorily separated and
can be recorded simultaneously.
Figure 3 (right) demonstrates this capability of a confocal
Laser Scanning Microscope.