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: 2.75). 08/2003; 8(3):376-80. DOI: 10.1117/1.1584053
Source: PubMed

ABSTRACT 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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    ABSTRACT: Just as medical research has advanced, diseases have also adapted to many treatments and continue to plague society. Disease may never be completely eradicated, but that has not discouraged the advancement of scientific technology. In order to effectively treat a disease it must first be identified and characterized. Fluorescence Lifetime Microscopy (FLIM) is an emerging optical method that shows great promise for pathology detection. FLIM measures the lifetime of a fluorophore which is the measure of how the photons stay in the excited state. As fluorescence lifetime is affected by changes in the microenvironment, FLIM can potentially be used to non-invasively monitor changes key to pathology. These measured fluorophores may be intrinsic, a natural component of the tissue, or extrinsic, added during tissue preparation. By observing the intrinsic and/or extrinsic fluorescence researchers are able to obtain information about the cellular environment of malignant and normal tissues. In a previous study FLIM was used to distinguish between structures in histology slides, and demonstrated the potential to distinguish between normal and malignant tissue. FLIM is a component of the Spectral Lifetime Imaging Microscopy (SLIM) system at LOCI which uses multi-photon excitation for fluorescence imaging and advanced photon counting electronics for lifetime collection. The SLIM system incorporates several novel detectors to obtain the spectra, lifetime, and intensity data, which are all properties of a fluorophore. We plan to evaluate the potential of FLIM for histopathology identification and to do so we must compare malignant and normal histology samples of the same tissue type. FLIM can be used to observe live and fixed tissue, but we have solely focused on H&E fixed tissue samples as these are the standards for histopathology. While we did not conclusively determine whether FLIM can be used for malignancy in the short time frame available we did get the following promising results. As a result of experimentation we were able to identify eosin as the lifetime altering constituent of the H & E staining process. By pin-pointing eosin as the unusually short lifetime component that we observed, we were able to eliminate some of the technical issues that have hindered experimental progression. We also composed a detailed protocol explaining the methodology of image and data acquisition. The protocol will aid future researchers as the project is continued. The collection and analysis of data this summer has reinforced the belief that Fluorescence Lifetime Microscopy has the potential to be an effective histopathology diagnostic tool.
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    ABSTRACT: We demonstrate the applicability of time-correlated single photon counting multiphoton microscopy to the spatio-temporal localisation of protein-protein interactions in situ. An Example of a new fluorescent protein variant with enhanced properties are given and the development of a FRET biosensor for simultaneous measurement of multiple intra- and inter-molecular interactions is illustrated by experimental evidence of an energy transfer cascade via multiple acceptors. The juxtaposition of interacting population and FRET efficiency is elucidated, with a priori knowledge, by multi-exponential analysis.
    Proc SPIE 01/2003; 5139.
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    ABSTRACT: Previously, a confocal 'Forster' resonance energy transfer (FRET)-based assay has been used to establish a clustered organization for receptor-ligand complexes containing polymeric IgA-receptor or transferrin-receptor (TFR) during endocytic trafficking in polarized epithelial MDCK cells. Here, the experimental system has been extended to internalizing transferrin (Tfn) labeled with donor fluorophore (Alexa Fluor-488) and/or acceptor fluorophore (Alexa Fluor-555) and applying two-photon fluorescence lifetime imaging microscopy (FLIM)-FRET. The fluorescence lifetime distribution should provide insights, not available with confocal FRET, due to FLIM's ability to reflect the diverse micro-environments of the polarized endocytic pathway. This pilot study confirms that a range of fluorescence lifetime values are detected both in cells containing donor-labeled Tfn (single-label specimens) and cells containing both donor and acceptor-labeled Tfn (double-label specimens) at the level of the basolateral and peri-nuclear common endosomes. Furthermore, significant reduction is detected in the fluorescence lifetime in the presence of donor and acceptor -labeled TFR-Tfn receptor-ligand complexes, when compared with that of donor-labeled, confirming the existence of FRET among these complexes.
    Proc SPIE 01/2006;


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