Analysis of histology specimens using lifetime multiphoton microscopy

University of Wisconsin-Madison, Laboratory for Optical and Computational Instrumentation and Laboratory of Molecular Biology, Madison, Wisconsin 53706, USA.
Journal of Biomedical Optics (Impact Factor: 2.86). 08/2003; 8(3):376-80. DOI: 10.1117/1.1584053
Source: PubMed


Observations of cells or tissues with fluorescence microscopy can provide unique insights into cellular physiology and structure. Such information may reveal the pathological state of a tissue to the physician or information on cytoskeletal dynamics to the research scientist. However, problems of overlapping spectra, low signal, and light scatter impose serious limitations on what can be achieved in practice with fluorescence microscopy. These problems can be addressed in part by the development of new imaging modalities that make maximum use of the information present in the fluorescence signal. We describe the application of a new technology to the study of standard histological pathology specimens: a multiphoton excitation fluorescence microscope that incorporates a novel, photon-counting detector that measures the excited-state lifetimes of fluorescent probes. In initial investigations, we have applied this system to the observation of C. elegans embryos and primate histology specimens, with the objective of identifying potentially diagnostic signatures. Our findings demonstrate that lifetime multiphoton microscopy has considerable potential as a diagnostic tool for pathological investigations.

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    • "As a consequence, numerous studies have been reported in recent years that have utilized FLIM to monitor diverse parameters including pH, molecular association, oxygen concentration, Ca 2+ concentration and proteolysis processing (Lakowicz, 1999; Periasamy, 2001). FLIM has also proven to be particularly useful as a diagnostic tool, finding scope in a diverse range of studies including oncological applications (Wagnieres et al., 1998), histopathology (Eliceiri et al., 2003), imaging of tissue constituents (Dowling et al., 1998), studies of human skin (Cubeddu et al., 1999; Becker et al., 2002), cell cultures (Bastiaens and Squire, 1999), metabolic mapping of human breast cells (Bird et al., 2005) and surveying inorganic contaminants present on the marble surface of the Statue of David (Toniolo et al., 2004). It is interesting to note, however, that although the unique advantages FLIM has to offer are proving to be particularly useful in the biosciences, to date they have been largely unexploited by other research disciplines. "
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