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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    • "Working with mouse models they compared fixed to unfixed live specimens and showed that the fixation process did not significantly impact the ability to obtain useful information from NADH and FAD measurements. Other reports also demonstrated that the chemical environment around the fluorophores is somehow preserved after the sample processing [22]. Our fixation process is similar to theirs which means, therefore, that we can compare metabolic states in different samples even after the fixing procedure. "
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    ABSTRACT: Nonlinear optical (NLO) microscopy techniques have potential to improve the early detection of epithelial ovarian cancer. In this study we showed that multimodal NLO microscopies, including two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third-harmonic generation (THG) and fluorescence lifetime imaging microscopy (FLIM) can detect morphological and metabolic changes associated with ovarian cancer progression. We obtained strong TPEF + SHG + THG signals from fixed samples stained with Hematoxylin & Eosin (H&E) and robust FLIM signal from fixed unstained samples. Particularly, we imaged 34 ovarian biopsies from different patients (median age, 49 years) including 5 normal ovarian tissue, 18 serous tumors and 11 mucinous tumors with the multimodal NLO platform developed in our laboratory. We have been able to distinguish adenomas, borderline, and adenocarcinomas specimens. Using a complete set of scoring methods we found significant differences in the content, distribution and organization of collagen fibrils in the stroma as well as in the morphology and fluorescence lifetime from epithelial ovarian cells. NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for serous and mucinous ovarian tumors. The results provide a basis to interpret future NLO images of ovarian tissue and lay the foundation for future in vivo optical evaluation of premature ovarian lesions.
    PLoS ONE 10/2012; 7(10):e47007. DOI:10.1371/journal.pone.0047007 · 3.23 Impact Factor
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    • "This has allowed a range of studies to utilize FLIM for monitoring and quantification of miscellaneous parameters including the pH gradient of the stratum corneum (Hanson et al., 2002), molecular association, oxygen concentration, proteolysis processing and Ca 2+ concentration (Lakowicz, 1999; Periasami, 2001). FLIM has also proven to be useful as a diagnostic tool in oncological applications (Wagnieres et al., 1998), histopathology (Eliceiri et al., 2003), imaging of tissue constituents (Dowling et al., 1998) and mapping metabolic activity in human breast cells (Bird et al., 2005). Although imaging studies based on the fluorescence lifetime of the intrinsic autofluorescence from skin have been reported (König et al., 2002), few (if any) have been specifically tailored for transdermal delivery. "
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    ABSTRACT: We demonstrate the potential of fluorescence lifetime imaging by time-correlated single-photon counting as a method for monitoring the transdermal diffusion pathway and diffusion rate of pharmaceuticals in human skin. The current application relies on observing subtle changes in the fluorescence lifetime of the intrinsic fluorophores present in the intracellular region between corneocytes of the stratum corneum. We have comprehensively characterized the measured fluorescence lifetimes from intracorneocyte junctions in three skin section types (dermatomed skin, epidermal membranes and stratum corneum) revealing statistically significant differences of the short lifetime component between each of the types, which we attribute to the sample preparation and imaging method. We show using epidermal membrane sections that application of a drug/solvent formulation consisting of ethinyl estradiol and spectroscopic grade ethanol to the surface gives rise to a slight but statistically significant shortening of the fluorescence lifetime of the long-lived emitting species present in the sample, from approximately 2.8 ns to 2.5 ns. The method may be useful for future studies where the kinetics and pathways of a variety of applied formulations could be investigated.
    Journal of Microscopy 03/2008; 230(1):61 - 69. DOI:10.1111/j.1365-2818.2008.01955.x · 2.33 Impact Factor
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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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