2
Following a description of the fundamental diffe-
rences between a conventional and a confocal
microscope, this monograph will set out the
special features of the confocal LSM and the capa-
bilities resulting from them.
The conditions in fluorescence applications will be
given priority treatment throughout.
Image generation
The complete generation of the two-dimensional
object information from the focal plane (object
plane) of a confocal LSM essentially comprises
three process steps:
1. Line-by-line scanning of the specimen with a
focused laser beam deflected in the X and Y
directions by means of two galvanometric
scanners.
2. Pixel-by-pixel detection of the fluorescence
emitted by the scanned specimen details, by
means of a photomultiplier tube (PMT).
3. Digitization of the object information contained
in the electrical signal provided by the PMT (for
presentation, the image data are displayed,
pixel by pixel, from a digital matrix memory to a
monitor screen).
Fig. 1 The quality of the image gener-
ated in a confocal LSM is not only
influenced by the optics (as in a con-
ventional microscope), but also, e.g.,
by the confocal aperture (pinhole) and
by the digitization of the object infor-
mation (pixel size). Another important
factor is noise (laser noise, or the shot
noise of the fluorescent light).
To minimize noise, signal-processing
as well as optoelectronic and electronic
devices need to be optimized.
Object
Resolution
Ideal optical theory
Noise
Detector, laser, electronics,
photons (light; quantum noise)
Digitization
Pixel size
Resudial optical
aberations
Pupil
Illumination
Confocal aperture
Image
337_Zeiss_Grundlagen_e 25.09.2003 16:16 Uhr Seite 5
