Confocal Microscopy - Biomedical Optical Imaging Technologies: Design and Applications - page 212

Fig. 6.1 Schematic diagram of a confocal microscope: ( a ) based on an upright microscope,
( b ) based on an inverted microscope
a scanning optical microscope by placing a pinhole in front of the detector, forming
effectively a point detector. The major advantage of the confocal microscope is its
optical sectioning, or depth discrimination, property. Light from different depths
of the object is brought to a focus at different planes, and only the light from the
in-focus region passes efficiently through the pinhole.
Figure 6.2 shows a confocal reflection microscope in which the sample is a
mirror. If the mirror is in the focal plane of the system, the reflected light is brought
to a focus on the pinhole, and a strong signal detected. When the mirror is moved
away from focus, the light is no longer focused on to the pinhole and the detected
signal drops. This optical sectioning property also occurs in confocal fluorescence
microscopy. It can be used in two distinct ways. If the sample is in the form of a
surface, it can be brought into the focal plane by maximizing the signal, and the
surface profile hence measured.
Or if the specimen is in the form of a thick object, a series of optical sections
at different focus positions can be recorded, resulting in a three-dimensional (3-D)
image stack. When surface profiling, usually the intensity is sampled at only a series
of discrete depth positions (Fig. 6.3 ). The brightest sample can be taken to give a
rough estimate of the surface height, or the position of maximum intensity can be
determined by curve fitting.
Figure 6.4 a shows the measured surface height from a semiconductor device
sample. As well as recording the axial position of the brightest intensity, the value
of this intensity maximum can also be recorded to give an autofocused image where
every point of the image is brought to its position of best focus Fig. 6.4 b. This is
sometimes called a maximum intensity projection. From the intensity and height
information, an isometric view of the sample can be reconstructed in the computer
(Fig. 6.4 c).
Figure 6.5 shows the theoretical axial intensity recorded from a mirror for
different numerical apertures (NAs) of dry objective lens. It is seen that the width