Chemical microscopy is
a development that could enable the study of chemistries within a cell in real time.
New techniques are necessary to execute sensitive, selective, fast and in situ chemical
measurements of micron-scale complex cellular systems and their functions. Edgar
A. Arriaga and his colleagues at the University of Minnesota in Minneapolis have
reviewed novel methods and applications of fluorescence microscopy that relate to
chemical microscopy techniques.
They report advancement in multiphoton
fluorescence microscopy, a nondestructive technique that is useful for in vivo measurements
of intracellular pH, detection of reactive oxygen species and examination of protein
interactions in living organisms. Other nondestructive techniques include Raman
and infrared microscopy, both of which provide information on molecular vibrational
modes. An ongoing development is coherent anti-Stokes Raman scattering (CARS), which
offers sensitivity, three-dimensional spatial resolution and high speed. Applications
for the technique include investigation of the chemical environments of different
regions of the retina and of single microbial cells attached to a surface.
Combining FRET and fluorescence lifetime
imaging microscopy enables investigation of interaction among proteins and allows
for better distinction between overlapping emission spectra. Similarly, combining
FRET with total internal reflection fluorescence microscopy enables the study of
molecular interactions in cell membranes. Other methods also are reviewed. When
choosing a technique, one must consider resolution, penetration depth, complexity
of the chemical information and the level of destruction imparted on the sample.
Because of vast improvements, the authors suspect that CARS will become the preferred
optical technique for many applications. (Analytical Chemistry, ASAP, May
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