and other in front of the photodetector. With this setup, the focal plane of the
specimen and the image plane at the retina of the eye or the surface of a camera
detector belong to a set of image-forming conjugate planes. By definition, an object
that is in focus at one conjugate plane also is in focus at the other conjugate planes
of that light path. That is,conjugate planesarepaired sets of in-focus planes. This
definition gives rise to the termconfocal microscope.
The feature of the conjugate planes allows only the light that is emitted from the
desired focal spot to pass through the microscope to the viewer. The screen at
the photodetector blocks all other diffracted or scattered light that is outside of the
desired focal plane. In Fig.8.10the blue lines represent the light cone that comes
from the pinhole at the laser, strikes the dichroic mirror, and illuminates a volume
within the sample. The solid green lines represent rays that come from spots on the
focal plane in the specimen, pass through the lens and dichroic mirror system, and
then pass through the pinhole aperture at the photodetector. The dashed green lines
originate from out-of-focus points in the sample and are blocked by the pinhole
aperture.
The confocal technique allows the specimen to be imaged through the use of a
rapid two-dimensional point-by-point serial scanning method in the xy-plane. To
obtain a three-dimensional image, the objective lens is focused to another depth and
the two-dimensional scan is repeated. Thereby a layered stack of virtual, confocal
image planes can be stored in a computer and used later to make three-dimensional
tomographic images of the specimen.
Slit scanningis an alternative method to the pinhole-based point-by-point
scanning for high-speed confocal imaging. This slit scanning method uses a 1-pixel
wide scan bar of excitation light to rapidly sweep across the sample. This procedure
allows video-rate image acquisition (for example, 120 images/s) by repeatedly
sweeping the bar at a high rate across the sample and then detecting thefluores-
cence with high-speed detectors through a slit aperture.
Because of its optical sectioning capability, confocal microscopy is a
well-established and widely used methodology in biophotonics. This feature
enables the analysis of morphologic changes in thick biologic tissue specimens with
sub-cellular resolution. Traditionally, confocal microscopy was limited to in vitro
studies of biopsy specimens and to in vivo analyses of easily accessible sites such
as the cornea, the skin, and lip and tongue areas, because it required large micro-
scope objectives and relatively long image acquisition times. These limitations can
be overcome through the use of opticalfiber-based techniques such as laser scan-
ning through a coherent-imagingfiber optic bundle or by means of a needle-based
imaging probe [ 15 – 18 ].
A confocal setup using a laser scanning mechanism with an opticalfiber bundle
is shown in Fig.8.11. First the light from a confocal pinhole aperture is collimated
and sent through a beam splitting mirror to a xy laser scanning mechanism. This
mechanism focuses the light sequentially onto each individualfiber in the coherent
bundle, so that the light gets transmitted to the sample. The light coming from the
248 8 Microscopy