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环境病理学,毒理学和肿瘤学期刊
影响因子: 1.241 5年影响因子: 1.349 SJR: 0.519 SNIP: 0.613 CiteScore™: 1.61

ISSN 打印: 0731-8898
ISSN 在线: 2162-6537

环境病理学,毒理学和肿瘤学期刊

DOI: 10.1615/JEnvironPatholToxicolOncol.v26.i2.30
pages 83-88

Cell-Substrate Topology upon ALA-PDT Using Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

Henri-Pierre Lassalle
Hochschule Aalen, Institut für Angewandte Forschung, Anton-Huber-Str. 21, 73430 Aalen, Germany. Permanent address: CRAN UMR 7039, Nancy University, CNRS, CAV, Avenue de Bourgogne, 54511 Vandoeuvre les Nancy, France
Harald Baumann
Hochschule Aalen, Institut für Angewandte Forschung, Anton-Huber-Str. 21, 73430 Aalen, Germany
Wolfgang S. L. Strauss
Institut für Lasertechnologien in der Medizin und Messtechnik, Helmholtzstr. 12, 89081 Ulm, Germany
Herbert Schneckenburger
Hochschule Aalen, Institut für Angewandte Forschung, Anton-Huber-Str. 21, 73430 Aalen, Germany; and Institut für Lasertechnologien in der Medizin und Messtechnik, Helmholtzstr. 12, 89081 Ulm, Germany

ABSTRACT

Because of the low penetration depth of an evanescent electromagnetic field, total internal reflection fluorescence microscopy (TIRFM) proved to be a powerful technique to examine fluorescent dyes or photosensitizers in close vicinity to the plasma membrane of living cells. In addition, on variation of the angle of incidence of exciting laser light, the penetration depth is varied, so that cell-substrate topology can be examined with nanometer resolution. Using a specific illumination device for TIRFM and a highly sensitive electron multiplying (EM) CCD camera, fluorescence of the photosensitizer protoporphyrin IX (PPIX) was studied in human cancer cells after application of 5-aminolevulinic acid (5-ALA) prior to and after irradiation with sublethal light doses (635 nm, 4 J/cm2). For cells growing on microscope cover slides, cell-substrate distances varied between approximately 20 and 250 nm with a mean distance of approximately 120 nm. On light exposure, these distances generally decreased, and a mean value below 100 nm was attained. Moreover, focal contacts visualized with a fusion protein of yellow fluorescent protein and focal adhesion kinase were maintained on light exposure, i.e., light-induced detachment of cells from their substrate was not likely to occur.


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