Zeiss LSM 880, Operating Manual

Page: 655 / 690

Scanningprocess
In a conventional light microscope, object-to-image trans-
formation takes place simultaneously and parallel for all
object points. By contrast, the specimen in a confocal LSM is
irradiated in a pointwise fashion, i.e. serially, and the physi-
cal inter action between the laser light and the specimen
detail irradiated (e.g. fluorescence) is measured point by
point. To obtain information about the entire specimen, it
is necessary to guide the laser beam across the specimen, or
to move the specimen relative to the laser beam, a process
known as scanning. Accordingly, confocal systems are also
known as point-probing scanners.
To obtain images of microscopic resolution from a confocal
LSM, a computer and dedicated software are indispensable.
The descriptions below exclusively cover the point scanner
principle as implemented, for example, in laser scanning
microscopes from Carl Zeiss. Configurations in which several ob-
ject points are irradiated simultaneously are not considered.
Confocalbeampath
The decisive design feature of a confocal LSM compared
with a conventional microscope is the confocal aperture
(usually called pinhole) arranged in a plane conjugate to
the intermediate image plane and, thus, to the object plane
of the microscope. As a result, the detector (PMT) can only
detect light that has passed the pinhole. The pinhole di-
ameter is variable; ideally, it is infinitely small, and thus the
detector looks at a point (point detection).
As the laser beam is focused to a diffraction-limited spot,
which illuminates only a point of the object at a time, the
point illuminated and the point observed (i.e. image and
object points) are situated in conjugate planes, i.e. they
are focused onto each other. The result is what is called a
confocal beam path (see figure 2).
5
Fig. 2 Beam path in a confocal LSM. A microscope objective lens is used
to focus a laser beam onto the specimen, where it excites fluorescence, for
example. The fluorescent radiation is collected by the objective and efficiently
directed onto the detector via a dichroic beamsplitter. The interesting wave-
length range of the fluorescence spectrum is selected by an emission filter,
which also acts as a barrier, blocking the excitation laser line. The pinhole is
arranged in front of the detector, on a plane conjugate to the focal plane of
the objective lens. Light coming from planes above or below the focal plane is
out of focus when it hits the pinhole, so most of it cannot pass the pinhole and
therefore does not contribute to forming the image.
Pinhole
Depending on the diameter of the pinhole, light com-
ing from object points outside the focal plane is more or
less obstructed and thus excluded from detection. As the
corresponding object areas are invisible in the image, the
confocal microscope can be understood as an inherently
depth-discriminating optical system.
By varying the pinhole diameter, the degree of confocality
can be adapted to practical requirements. With the aper-
ture fully open, the image is nonconfocal. As an added
advantage, the pinhole suppresses stray light, which further
improves image contrast.
X
Z
Detector (PMT)
Pinhole
Emission filter
Dichroic mirror
Focal plane
Objective lens
Detection volume
Background
Beam expander
Laser