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 10, 2006.)