Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM): realization and application of a compact illumination device

摘要: A novel compact illumination device in variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) is described. This replaces the standard condensor of an upright microscope. Light from different laser sources delivered via a monomode fibre and focused onto identical parts sample under variable angles reflection. Thus, fluorophores close proximity to cell-substrate interface are excited by evanescent wave with penetration depth, localized high (nanometre) axial resolution. In addition quantitative measurements solution, markers cytoplasm plasma membrane, i.e. calcein laurdan, were examined using cultivated endothelial cells. Distances between glass substrate membrane determined mathematical algorithm four-layer model, as well Gaussian-shaped intensity profile spot on samples. 0 30 nm focal contacts 100 300 other cell thus determined. topology, appears appropriate for numerous applications which resolution required, e.g. experiments endocytosis or exocytosis, ion concentrations proximal membrane. The also suitable combining TIRFM further innovative techniques, time-resolved spectroscopy, lifetime imaging (FLIM) resonance energy transfer (FRET).