4
Optical slices
With a confocal LSM 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 condi-
tions, the thickness (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 fluorescence 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.
Unless this condition is satisfied, the in-focus
image information from the object plane of inter-
est 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).
A confocal LSM can therefore be used to advan-
tage especially where thick specimens (such as
biological cells in tissue) have to be examined by
fluorescence. The possibility of optical sectioning
eliminates the drawbacks attached to the obser-
vation of such specimens by conventional fluores-
cence microscopy. With multicolor fluorescence,
the various channels are satisfactorily separated
and can be recorded simultaneously.
With regard to reflective specimens, the main
application is the investigation of the topography
of 3D surface textures.
Figure 3 demonstrates the capability of a confocal
Laser Scanning Microscope.
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.
337_Zeiss_Grundlagen_e 25.09.2003 16:16 Uhr Seite 7
