Non-Linear Microscopy - Biophotonics - page 51

point-like source. This can be achieved by using the condenser lens to project
a pinhole aperture (a small aperture in an opaque screen) on the focal plane
or by using a laser source focused on the focal plane. In this way the amount
of light delivered on the sample is reduced by order of magnitudes without
affecting the focal brightness. However, even in this way, all the points along
the focal axis will be able to scatter light (or to produce fluorescence signal)
that will contribute to the image. The solution to this problem resides in
placing a second pinhole aperture in the image plane that lies beyond the
back aperture of the objective lens, so that all the light coming from out of
focus scattering sources (or fluorescence sources) will be rejected. As shown in
Fig. 4.2, the final result is a symmetrical set-up, made up of two lenses at the
two sides of the specimen with two point-like apertures beyond them: in this
way the role played by the lens on the excitation side is identical to the one on
the detection side and their combined effect results in a relevant improvement
of the axial response of the imaging system [49]. The strong symmetry of
the system results also in the possibility (adopted by modern raster scanning
confocal microscopes) of using the same lens both for the excitation and for
the acquisition, in an epi-fluorescence scheme.
Because of the point-like nature of the focused beam, in order to acquire
microscopic images of an extended portion of the sample, it is necessary to
perform a scan. This is usually obtained with one of these methods.
The first one is based on raster scanning (point by point in a line and then
line by line) of the image field. This operation requires a finite time in order

objective lens
(L2)

condenser lens
(L1)

detector

light source

excitation pinhole

detection pinhole

Fig. 4.2. Simplified view of a confocal microscope. A point-like source (a lamp with
an excitation pinhole mask or a laser) is projected on the specimen by a condenser
lens L 1 . All the fluorescent molecules illuminated by the cone of light will emit.
This emission is brought to the detector by the objective lens L 2 . Right before the
detector the collection pinhole allow to exclude all the light emitted by out of focus
planes