Characterization techniques - Micromixers Fundamentals Design and Fabrication - page 300

sets are selected by a multiposition filter wheel. Because the sample only works with two wavelengths,
a fluorescence microscope needs to ensure that only shorter excitation wavelength is directed onto the
sample and only longer emitted wavelength is used to form the image on the sensor.
A dichroic mirror can fulfill these two requirements. The mirror works as a high-pass filter, which
reflects the shorter excitation wavelength and directs it to the sample, while letting the longer emitted
wavelength go through ( Fig. 8.4 (b) ). The intensity of the emitted signal is extremely weak compared
to the excitation intensity. Thus, the excitation light and the emitted light must be completely separated
by using narrow-band filters or fluorescent dyes with a large Stokes shift.
8.1.3 Confocal laser scanning microscopy
Confocal laser scanning microscope allows the three-dimensional reconstruction of semitransparent
microstructures using two-dimensional sections of the object. The two-dimensional image is formed
by scanning a focused point across the imaged section. This imaging concept requires both the
condenser lens of the illumination and the objective lens to have the same point of focus, or are
confocal. Strictly speaking, standard optical microscopes also use high-NA lenses to both illuminate
and image the sample and are therefore also confocal. However, the terminology of confocal
microscope as a synonym for confocal laser scanning microscope is well established in the liter-
ature. A confocal microscope allows imaging a particular plane without any information of the out-
of-focus planes. To avoid the accurate alignment needed for the condenser and objective lenses,
a single lens can be used for both illumination and imaging. Modern confocal microscopes use
a laser beam to generate a focused spot on the imaged section. The laser beam is scanned through
the objective in a raster pattern. The reflected light is collected on the image sensor through the same
optical system and a pinhole. The speed of the confocal microscope depends on the scanning speed
of the light spot.
Figure 8.5 shows the schematic concept of an epi-fluorescence confocal laser scanning microscope
with two pinholes for illumination and imaging. The illumination system consists of a laser source,
a pinhole, and a beam expander. Because the laser source provides a single wavelength, the excitation
filter may not be needed. The laser should have a stable intensity during the scanning period, because
a change in source intensity will lead to error in the measured image. In a confocal microscope, the
objective lens is used for both illumination and receiving the reflected light. Thus, the effect of
aberration is very crucial for the objective lens. The light passes the objective lens and is focused at the
sampling point. The stage holding the device is scanned in a raster pattern to form the two-dimensional
image point by point. The scanning stage should have high speed, positioning accuracy, and stability.
The scanning level in z -direction is usually controlled by a piezoelectric transducer.
The pinholes determine both axial and transverse resolution of the confocal measurement. A large
pinhole allows more light to pass through, leading to a stronger signal but lower resolution. A small
pinhole results in a higher resolution but weaker signals with lower signal-to-noise ratio.
Light from the sampling point passes through the detector pinhole and impinges on the detector.
The optical detector can be a photodiode or a photomultiplier tube. A relay lens between the
pinhole and the detector can be used for imaging the pinhole on the detector surface. Furthermore,
a narrow-band filter can be used to reduce the noise level caused by stray light. The detected signal
is recorded together with the x , y position of the sampling point to form the two-dimensional
image.