Use of the approximations in cell studies by total internal reflection fluorescence microscopy (TIRF)

In determining cell parameters by the use of total internal reflectance fluorescence microscopy it is necessary to evaluate the electric field strength in the neighbourhood of the cell. It has been suggested that the true field distribution be assumed to be of exponential form. In some circumstances, this approximation gives rise to errors and seriously incorrect results are obtained. The true field distribution is easily obtained numerically so the use of an exponential approximation is unnecessary and errors are avoided.     

A ‘pocket guide’ to total internal reflection fluorescence

The phenomenon of total internal reflection fluorescence (TIRF) was placed in the context of optical microscopy by Daniel Axelrod over three decades ago. TIRF microscopy exploits the properties of an evanescent electromagnetic field to optically section sample regions in the close vicinity of the substrate where the field is induced. The first applications in cell biology targeted investigation of phenomena at the basolateral plasma membrane. The most notable application of TIRF is single‐molecule experiments, which can provide information on fluctuation distributions and rare events, yielding novel insights on the mechanisms governing the molecular interactions that underpin many fundamental processes within the cell. This short review intends to provide a ‘one stop shop’ explanation of the electromagnetic theory behind the remarkable properties of the evanescent field, guide the reader through the principles behind building or choosing your own TIRF system and consider how the most popular applications of the method exploit the evanescent field properties.     

Total internal reflection fluorescence imaging using an upconverting cover slip for multicolour evanescent excitation

Total internal reflection fluorescence microscopy is well known as a means of studying surface‐bound structures in cell biology. It is usually measured either by coupling a light source to the sample using a prism or with a special objective where light passing through the periphery of the lens illuminates the contact region beyond the critical angle. In this study we present a new and simple approach to total internal reflection fluorescence microscopy where the sample is mounted on a cover slip prepared from a high‐index upconverting glass‐ceramic. Excitation of the cover slip with a low‐cost near‐infrared laser diode generates intense narrow‐band visible emission within the cover slip, some of which is totally internally reflected. This emission gives rise to an evanescent wave at the interface and hence can excite surface‐bound fluorescent species. Depending on the excitation conditions the cover slip can generate violet, green and red emission and hence can excite a wide range of fluorescent labels. Fluorescence emission from the sample can be detected in spectral regions where the direct emission from the cover slip is very weak. The advantages and limitations of the technique are discussed in comparison with conventional total internal reflection fluorescence microscopy measurements and prospects for novel total internal reflection fluorescence microscopy geometries are considered.     

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

A 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 prism combination for near isotropic fluorescence excitation by total internal reflection

Total internal reflection fluorescence (TIRF) microscopy is finding increasing application for selectively detecting molecules at or near a glass–water surface. As with all fluorescence methods, the efficiency of excitation of a fluorophore is potentially sensitive to the polarization state of the source. In TIRF, s‐polarized excitation produces an evanescent field that is perpendicular to the incident plane (y direction), whereas p‐polarized light generates a more complex pattern but one dominated by a field that is vertical to the surface (z direction). Thus, fluorophores whose absorption dipoles are fixed in the x direction are not favourably aligned for excitation. Here we describe a beam‐splitting prism arrangement that allows excitation by two orthogonal beams, thus giving isotropic excitation in the x–y plane with s‐polarized light. With linearly polarized light at the magic angle, near isotropic excitation in three dimensions should be achieved. This prism design should find application in polarized fluorescence microscopy to investigate the rotational motions of macromolecules or to minimize flickering of fluorescence emission arising from molecular rotations in single molecule studies.     

Even illumination in total internal reflection fluorescence microscopy using laser light

In modern fluorescence microscopy, lasers are a widely used source of light, both for imaging in total internal reflection and epi-illumination modes. In wide-field imaging, scattering of highly coherent laser light due to imperfections in the light path typically leads to nonuniform illumination of the specimen, compromising image analysis. We report the design and construction of an objective-launch total internal reflection fluorescence microscopy system with excellent evenness of specimen illumination achieved by azimuthal rotation of the incoming illuminating laser beam. The system allows quick and precise changes of the incidence angle of the laser beam and thus can also be used in an epifluorescence mode.     

Tracking of secretory vesicles of PC12 cells by total internal reflection fluorescence microscopy

Total internal reflection fluorescence microscopy is used to detect cellular events near the plasma membrane. Behaviours of secretory vesicles near the cell surface of living PC12 cells, a neuroendocrine cell line, are studied. The secretory vesicles are labelled by over‐expression of enhanced green fluorescent protein‐tagged Rab3A, one of the small G proteins involved in the fusion of secretory vesicles to plasma membrane in PC12 cells. Images acquired by a fast cooled charge‐coupled device camera using conventional fluorescence microscopy and total internal reflection fluorescence microscopy are compared and analysed. Within the small evanescent range (< 200 nm), the movements of the secretory vesicles of PC12 cells before and after stimulation by high K⁺ are examined. The movements of one vesicle relative to another already docked on the membrane are detected. Total internal reflection fluorescence microscopy provides a novel optical method to trace and analyse the exocytotic events and vesicle specifically near a cell membrane without interference of signals from other parts of the cell.     

Elimination of the effects of stray light in measurements by total internal reflection aqueous fluorescence (TIRAF)

Total internal reflection aqueous fluorescence has been shown to be capable of achieving spatial resolution in surface contours of about 1 nm. When used with highly structured objects, errors in measurements can arise from light scattered either by the object or within the body of the microscope. We describe how these errors can be eliminated when studying surface contours of human platelets.     

A method to simulate the optical image from far-field scattering numerical data and its application to the total internal reflection microscopy of metallic nanowires

Computational electrodynamics modelling plays an important role in understanding and designing new photonic devices. The results offered by these simulations are usually close-range field distributions or angular power emission plots. We describe a procedure to compute the optical microscopy image from simulated far-field scattering data using three-dimensional discrete Fourier transforms that can be used when the simulation software package do not include proper far-field to optical imaging projection routines. The method is demonstrated comparing simulated images with real images of nanowires obtained with a total internal reflection microscope.     

Quantitative analysis of variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) of cell/substrate contacts

Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) allows controlled variation of the illumination depth with the potential of measuring both membrane/substrate separation distances and sizes of focal contacts. VA-TIRFM images are collected from well-spread bovine aortic endothelial cells (BAEC) stained with a membrane-bound carbocyanine dye. Quantitative determination of absolute membrane/substrate separation distances and individual focal contact area are attempted using a simplified model of TIRFM optics. For angles slightly greater than the critical angle of 64°, both the dorsal and ventral membranes were illuminated, while images excited above 66° illuminated only focal contacts. Above 74° the fluorescence of focal contacts was dominated by background noise. Direct application of the simplified optical model without accounting for background intensity was unsatisfactory. However, correction for background fluorescence and nonlinear regression of the untransformed data over the working range yielded focal contact separation distances of 24 ± 13 nm. Focal contact areas estimated by TIRFM (1·3 ± 0·7 μm²) agreed closely with areas observed by immunofluorescence staining of vinculin (1·5 ± 0·3 μm²).     

Signal analysis of total internal reflection fluorescent speckle microscopy (TIR-FSM) and wide-field epi-fluorescence FSM of the actin cytoskeleton and focal adhesions in living cells

Fluorescent speckle microscopy (FSM) uses low levels of fluorescent proteins to create fluorescent speckles on cytoskeletal polymers in high‐resolution fluorescence images of living cells. The dynamics of speckles over time encode subunit turnover and motion of the cytoskeletal polymers. We sought to improve on current FSM technology by first expanding it to study the dynamics of a non‐polymeric macromolecular assembly, using focal adhesions as a test case, and second, to exploit for FSM the high contrast afforded by total internal reflection fluorescence microscopy (TIR‐FM). Here, we first demonstrate that low levels of expression of a green fluorescent protein (GFP) conjugate of the focal adhesion protein, vinculin, results in clusters of fluorescent vinculin speckles on the ventral cell surface, which by immunofluorescence labelling of total vinculin correspond to sparse labelling of dense focal adhesion structures. This demonstrates that the FSM principle can be applied to study focal adhesions. We then use both GFP‐vinculin expression and microinjected fluorescently labelled purified actin to compare quantitatively the speckle signal in FSM images of focal adhesions and the actin cytoskeleton in living cells by TIR‐FM and wide‐field epifluorescence microscopy. We use quantitative FSM image analysis software to define two new parameters for analysing FSM signal features that we can extract automatically: speckle modulation and speckle detectability. Our analysis shows that TIR‐FSM affords major improvements in these parameters compared with wide‐field epifluorescence FSM. Finally, we find that use of a crippled eukaryotic expression promoter for driving low‐level GFP‐fusion protein expression is a useful tool for FSM imaging. When used in time‐lapse mode, TIR‐FSM of actin and GFP‐conjugated focal adhesion proteins will allow quantification of molecular dynamics within interesting macromolecular assemblies at the ventral surface of living cells.     

The effects of total internal reflection on the spread function along the axis in confocal microscopy

A broadening and splitting of the axial spread function is observed when high-numerical-aperture (NA) oil-immersion objectives are used on a confocal microscope to examine dielectric interfaces when the refractive index below the boundary is lower than the NA of the objective. The phenomena is due to total internal reflection probably as a consequence of the Goos-Hänchen shift. If total internal reflection occurs when undertaking confocal microscopy, this shift creates obvious problems when the optical sectioning capabilities must be optimal in reflectance mode and more subtle difficulties can arise when examining fluorescent emission. Alternatively, deliberately inducing total internal reflection can be used to estimate the refractive index in component parts within foams, emulsions and aerated specimens where such measurements can be relatively difficult to make by other means. Furthermore, the examination of total internal reflection with a confocal microscope permits the phenomena of total internal reflection itself to be probed with very high illumination intensities without disturbing the boundary conditions with an external probe. Finally, other changes in the apparent position of the focus were noted to occur when high-NA oil-immersion objectives are used to examine specimens such as metal mirrors.     

Epifluorescence, confocal and total internal reflection microscopy for single-molecule experiments: a quantitative comparison

Epifluorescence, confocal and total internal reflection microscopy are the most widely used techniques for optical single‐molecule experiments. Employing these methods, we recorded the emission intensity of the same single molecule as a function of the excitation rate under otherwise identical experimental conditions. Evaluation of these data provides a quantitative comparison of the signal‐to‐background ratios that can be achieved for the three microscopic techniques.     

Near‐field microscopy and fluorescence spectroscopy: application to chromosomes labelled with different fluorophores

We have coupled a spectrophotometer with a scanning near‐field optical microscope to obtain, with a single scan, simultaneously scanning near‐field optical microscope fluorescence images at different wavelengths as well as topography and transmission images. Extraction of the fluorescence spectra enabled us to decompose the different wavelengths of the fluorescence signals which normally overlap. We thus obtained images of the different fluorescence emissions of acridine orange bound to single or double stranded nucleic acids in human metaphase chromosomes before and after DNAse I or RNAse A treatment. The analysis of these images allowed us to visualize some specific chromatin areas where RNA is associated with DNA showing that such a technique could be used to identify multiple components within a cell.     

Correlative fluorescence and transmission electron microscopy: an elegant tool to study the actin cytoskeleton of whole-mount (breast) cancer cells

Elucidating the structure and dynamics of lamellipodia and filopodia in response to different stimuli is a topic of continuing interest in cancer cells as these structures may be attractive targets for therapeutic purposes. Interestingly, a close functional relationship between these actin-rich protrusions and specialized membrane domains has been recently demonstrated. The aim of this study was therefore to investigate the fine organization of these actin-rich structures and examine how they structurally may relate to detergent-resistant membrane (DRM) domains in the MTLn3 EGF/serum starvation model. For this reason, we designed a straightforward and alternative method to study cytoskeleton arrays and their associated structures by means of correlative fluorescence (/laser)- and electron microscopy (CFEM).
  CFEM on whole mounted breast cancer cells revealed that a lamellipodium is composed of an intricate filamentous actin web organized in various patterns after different treatments. Both actin dots and DRM's were resolved, and were closely interconnected with the surrounding cytoskeleton. Long actin filaments were repeatedly observed extending beyond the leading edge and their density and length varied after different treatments. Furthermore, CFEM also allowed us to demonstrate the close structural association of DRMs with the cytoskeleton in general and the filamentous/dot-like structural complexes in particular, suggesting that they are all functionally linked and consequently may regulate the cell's fingertip dynamics. Finally, electron tomographic modelling on the same CFEM samples confirmed that these extensions are clearly embedded within the cytoskeletal matrix of the lamellipodium.