3
High Resolution in Space and Time
Confocal laser scanning microscopes (LSMs) are
distinguished by their high spatial and temporal
resolving power. They clearly outperform classical
light microscopes especially by their axial resolu-
tion – a quality that enables users to acquire opti-
cal sections (slices) of a specimen. An object can
thus be imaged completely in three dimensions
and subsequently visualized as a 3D computer
image. In another group of applications, exactly
defined areas of a specimen can be selectively illu-
minated by laser light. This functionality is essen-
tial for quantitative investigations of dynamic proc-
esses in living cells using techniques such as FRAP
(fluorescence recovery after photobleaching),
FRET (fluorescence resonance energy transfer),
photoactivation and photoconversion.
New acquisition methods on the LSM permit the
detection of additional properties of the emitted
light including spectral signatures and fluores-
cence lifetimes. With such information it is possi-
ble to increase the number of fluorescent labels
used in an experiment, or to use fluorochrome
combinations unthinkable with conventional
detection methods. The advantages are obvious:
the more components in a cellular process that are
observed simultaneously, the greater the yield of
information.
Because of its versatility, laser scanning micros-
copy has become an established mainstream
method in biomedical research – a tool permitting
scientists to follow innovative experimental paths.
This article will show which basic functions and
applications of laser scanning microscopy can be
taught in academic tuition. The modern method of
confocal laser scanning microscopy can be taught
on the basis of classical light microscopy, an estab-
lished part of fundamental biomedical teaching.
In the non-confocal image, the interesting information of the
focal plane mixes with unwanted information from extrafocal
specimen planes; differently stained details result in a color mix.
In the confocal image, object details
blurred in the non-confocal image
are visible clearly and in greater contrast.
Triple-labeled tissue section
of mouse intestine
