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

Abstract

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

A membrane‐bound FRET‐based caspase sensor for detection of apoptosis using fluorescence lifetime and total internal reflection microscopy

“…These values remain almost unchanged for the intensity I 5 I 0 T(Y) e 2D/d of the electromagnetic wave on the cell surface (15), if a transmission factor T 5 3.2 (for Y 5 67.58) and an average cell-substrate distance D 5 100 nm (17) are assumed. An additional unit for Köhler's illumination and phase contrast microscopy (consisting of an additional lens and a ring aperture) enabled adjustment of the samples before fluorometric measurements.…”

“…A penetration depth between about 60 and 200 nm (depending on the wavelength and the angle of incidence) is commonly achieved above the surface of the glass slide, so that the illuminated layer captures the plasma membrane of cells in close vicinity of the glass surface. For selective measurements of fluorescent molecules in close vicinity to the plasma membrane, TIRFM appears to be a well-suited method, since the low penetration depth of the evanescent wave results in an $100-fold lower light exposure of cells (15) compared with whole cell illumination in conventional and confocal laser scanning microscopy. This implies that light doses applied upon TIRFM are probably less toxic to living cells.…”

“…However, it should be taken into account that in comparison with conventional fluorescence microscopy, TIRFM signals are by about a factor 100 lower and need very sensitive optical detection systems in order to obtain good signal-to-noise ratios. TIRFM has been first described in 1981 (14), and since that time has been used increasingly for measuring cell-substrate contacts (15)(16)(17), membrane or protein dynamics (18), membrane proximal ion fluxes (19) as well as endocytosis or exocytosis (20)(21)(22).…”

Multi‐dimensional fluorescence microscopy of living cells

“…When a fluorescent dye is distributed rather homogeneously either in the cytoplasm or in the plasma membrane, and when fluorescence intensity I F is determined as a function of Q, cell-substrate distances can be calculated with nanometre precision, as described in Ref. [17]. Two custom-made illumination devices have recently been reported.…”

“…Two custom-made illumination devices have recently been reported. While in a first one the angle of incidence of a parallel laser beam can be varied in multiple steps with a precision of AE0.25 [17], this angle is fixed at 67.5 AE 1.3 in a second one permitting a penetration depth d ¼ 110 AE 35 nm [20]. This latter condenser unit contains a focusing lens for selective illumination of small fluorescent samples, e.g.…”