In Situ Characterizations of Porous Media for Applications in Biofuel Cells: Issues and Challenges - Porous Media: Applications in Biological Systems and Biotechnology - page 518

13.5.4 Other Spectroscopic Techniques
Confocal microscopy is an optical imaging technique employing in-focus pro-
cessing through a pinhole to eliminate out-of-focus light in a specimen that
is outside the focal plane. The ability to increase micrograph contrast and
to reconstruct three-dimensional images makes this technique popular in the
studies of life sciences and semiconductor industry. In a conventional wide-
field fluorescence microscope, a light source illuminates the entire specimen.
The resulting fluorescence from the specimen is detected by the photodetec-
tor or camera to construct the image. In a confocal microscope, a laser light
source provides a sharp small-area illumination and a pinhole in an optically
conjugate plane in front of the detector is used to eliminate out-of-focus light
signals. Therefore, in a confocal configuration, only light produced by fluores-
cence very close to the focal plane is detected for image resolution, particularly
in the sample depth direction. The image resolution of the confocal system
is much better than that of a wide-field microscope. The tradeoff is a longer
exposure time required to collect the light signals from the physical scan-
ning process. There are three major types of confocal microscopes available
commercially. LSCM usually provide better imaging quality than spinning-
(Nipkow) disk confocal microscopes (SDCM) or programmable array micro-
scopes (PAM) at a lower frame rate. SDCM can achieve higher frame rate
for video, thus it is preferred for dynamic observations in live cells. To apply
the fluorescence technique, a specimen is usually labeled with a fluorescent
molecule called a fluorophore (e.g., green fluorescent protein or fluorescein) to
enable light detection. A typical fluorescence microscope will comprise a light
source, an excitation filter, a dichroic mirror (dichromatic beamsplitter), an
emission filter, and a photodetector. The labeled specimen is illuminated with
light of a specific wavelength (e.g., a laser source) or a range of wavelengths
(e.g., a xenon arc lamp or mercury-vapor lamp). The fluorophore absorbs the
light and then emits longer wavelengths of light (fluorescence). An emission
filter will screen the spectrum for photo detection. Through proper control of
the filters and dichroic mirror, different wavelengths of light can be detected
and imaged with a distinct color. Different color images can be overlaid and
reconstructed into a two-dimensional or three-dimensional image to display
the specimen's unique property through fluorophore labeling. The best reso-
lution below 10 nm can be achieved to date with a confocal system combining
localization microscopy with spatially modulated illumination. Most of the
fluorescence microscopes are epifluorescence types, in which excitation and
observation of the fluorescence is from above (epi-) the specimen.
Figure 13.17 illustrates an experiment using an epifluorescence LSCM to
understand the interaction of enzyme and porous polymer backbone during
immobilization (Konash et al. 2006). In Figure 13.17a the distribution of the
alcohol dehydrogenase labeled with Alexa 488 near the surface of an East-
man AQ 55 polymer film was shown. Using the advantage of depth profiling,
the images of the distribution along the thickness of the film can be stacked
to reconstruct the three-dimensional image for visualization (Figure 13.17b).