INTRODUCTION TO LASER SCANNING MICROSCOPY
LSM 510
DuoScan
Carl Zeiss
Principle of Laser Scanning Microscopy
LSM 510 META
DuoScan
3-2
B 45-0021 e
03/06
3
INTRODUCTION TO LASER SCANNING MICROSCOPY
3.1
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 to the above problem.
•
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.
•
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.
•
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
μ
m can be resolved, and height differences of less than
0.1
μ
m made visible, without the use of interference methods.
Fig 3-1
Principle of confocal imaging
