Standing-wave Fluorescence Microscopy Research Articles

An evaluation of multi-excitation-wavelength standing-wave fluorescence microscopy (TartanSW) to improve sampling density in studies of the cell membrane and cytoskeleton

Conventional standing-wave (SW) fluorescence microscopy uses a single wavelength to excite fluorescence from the specimen, which is normally placed in contact with a first surface reflector. The resulting excitation SW creates a pattern of illumination with anti-nodal maxima at multiple evenly-spaced planes perpendicular to the optical axis of the microscope. These maxima are approximately 90 nm thick and spaced 180 nm apart. Where the planes intersect fluorescent structures, emission occurs, but between the planes are non-illuminated regions which are not sampled for fluorescence. We evaluate a multi-excitation-wavelength SW fluorescence microscopy (which we call TartanSW) as a method for increasing the density of sampling by using SWs with different axial periodicities, to resolve more of the overall cell structure. The TartanSW method increased the sampling density from 50 to 98% over seven anti-nodal planes, with no notable change in axial or lateral resolution compared to single-excitation-wavelength SW microscopy. We demonstrate the method with images of the membrane and cytoskeleton of living and fixed cells.

Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells

We present the first application of standing wave fluorescence microscopy (SWFM) to determine the size of biological nanostructures in living cells. The improved lateral resolution of less than 100 nm enables superior quantification of the size of subcellular structures. We demonstrate the ability of SWFM by measuring the diameter of biological nanotubes (membrane tethers formed between cells). The combination of SWFM with total internal reflection (TIR), referred to as SW-TIRFM, allows additional improvement of axial resolution by selective excitation of fluorescence in a layer of about 100 nm.

Cell reactions with biomaterials: the microscopies.

The methods and results of optical microscopy that can be used to observe cell reactions to biomaterials are Interference Reflection Microscopy (IRM), Total Internal Reflection Fluorescence Microscopy (TIRFM), Surface Plasmon Resonance Microscopy (SPRM) and Förster Resonance Energy Transfer Microscopy (FRETM) and Standing Wave Fluorescence Microscopy. The last three are new developments, which have not yet been fully perfected. TIRFM and SPRM are evanescent wave methods. The physics of these methods depend upon optical phenomena at interfaces. All these methods give information on the dimensions of the gap between cell and the substratum to which it is adhering and thus are especially suited to work with biomaterials. IRM and FRETM can be used on opaque surfaces though image interpretation is especially difficult for IRM on a reflecting opaque surface. These methods are compared with several electron microscopical methods for studying cell adhesion to substrata. These methods all yield fairly consistent results and show that the cell to substratum distance on many materials is in the range 5 to 30 nm. The area of contact relative to the total projected area of the cell may vary from a few per cent to close to 100% depending on the cell type and substratum. These methods show that those discrete contact areas well known as focal contacts are frequently present. The results of FRETM suggest that the separation from the substratum even in a focal contact is about 5 nm.

The Actin-Based Nanomachine at the Leading Edge of Migrating Cells

Two fundamental parameters of the highly dynamic, ultrathin lamellipodia of migrating fibroblasts have been determined—its thickness in living cells (176 ± 14 nm), by standing-wave fluorescence microscopy, and its F-actin density (1580 ± 613 μm of F-actin/ μm 3), via image-based photometry. In combination with data from previous studies, we have computed the density of growing actin filament ends at the lamellipodium margin (241 ± 100/ μm) and the maximum force (1.86 ± 0.83 nN/ μm) and pressure (10.5 ± 4.8 kPa) obtainable via actin assembly. We have used cell deformability measurements (Erickson, 1980. J. Cell Sci. 44:187–200; Petersen et al., 1982. Proc. Natl. Acad. Sci. USA. 79:5327–5331) and an estimate of the force required to stall the polymerization of a single filament (Hill, 1981. Proc. Natl. Acad. Sci. USA. 78:5613–5617; Peskin et al., 1993. Biophys. J. 65:316–324) to argue that actin assembly alone could drive lamellipodial extension directly.

Development of a standing-wave fluorescence microscope with high nodal plane flatness.

This article reports about the development and application of a standing-wave fluorescence microscope (SWFM) with high nodal plane flatness. As opposed to the uniform excitation field in conventional fluorescence microscopes as SWFM uses a standing-wave pattern of laser light. This pattern consists of alternating planar nodes and antinodes. By shifting it along the axis of the microscope a set of different fluorescent structures can be distinguished. Their axial separation may just be a fraction of a wavelength so that an SWFM allows distinction of structures which would appear axially unresolved in a conventional or confocal fluorescence microscope. An SWFM is most powerful when the axial extension of the specimen is comparable to the wavelength of light. Otherwise several planes are illuminated simultaneously and their separation is hardly feasible. The objective of this work was to develop a new SWFM instrument which allows standing-wave fluorescence microscopy with controlled high nodal plane flatness. Earlier SWFMs did not allow such a controlled flatness, which impeded image interpretation and processing. Another design goal was to build a compact, easy-to-use instrument to foster a more widespread use of this new technique. The instrument developed uses a green-emitting helium-neon laser as the light source, a piezoelectric movable beamsplitter to generate two mutually coherent laser beams of variable relative phase and two single-mode fibres to transmit these beams to the microscope. Each beam is passed on to the specimen by a planoconvex lens and an objective lens. The only reflective surface whose residual curvature could cause wavefront deformations is a dichroic beamsplitter. Nodal plane flatness is controlled via interference fringes by a procedure which is similar to the interferometric test of optical surfaces. The performance of the instrument was tested using dried and fluorescently labelled cardiac muscle cells of rats. The SWFM enabled the distinction of layers of stress fibres whose axial separation was just a fraction of a wavelength. Layers at such a small distance would lie completely within the depth-of-field of a conventional or confocal fluorescence microscope and could therefore not be distinguished by these two methods. To obtain further information from the SWFM images it would be advantageous to use the images as input-data to image processing algorithms such as conceived by Krishnamurthi et al. (Proc. SPIE, 2655, 1996, 18-25). To minimize specimen-caused nodal plane distortion, the specimen should be embedded in a medium of closely matched refractive index. The proper match of the refractive indices could be checked via the method presented here for the measurement of nodal plane flatness. For this purpose the fluorescent layer of latex beads would simply be replaced by the specimen. A combination of the developed SWFM with a specimen embedded in a medium of matched refractive index and further image processing would exploit the full potential of standing-wave fluorescence microscopy.

Optical subsectioning and multiple focal-plane imaging in the standing-wave fluorescence microscope

Standing-wave fluorescence microscopy (SWFM) is a direct-imaging method through which very high axial resolution can be obtained in certain types of biological specimens. In the microscope, interference of light is used to produce a standing-wave field through the specimen, so that it is illuminated in a pattern of planar zones, rather than uniformly. Fluorescence is excited with spatial selectivity in proportion to the intensity pattern. In the instruments in use at present, two nearlycollimated s-polarized beams from a laser are directed into the specimen from opposite sides at mirrorimage angles with respect to the microscope axis (Figure). The alternating nodal and antinodal planes of the field are in this case parallel to the object focal plane, with a node spacing equal to λo/(2n cos θ), where θ is the beam angle, and n is the refractive index of the specimen. The minimum node spacing, λo/2n, is obtained when the beams are counterpropagating on-axis, and is typically 0.18 μm. In the simplest SWFM system, a mirror is placed behind the specimen such that an on-axis beam interferes with its reflection. In our dual-beam instrument, the laser output is split, and the beams directed into the specimen through a pair of opposed high-NA objectives. In both cases, the standing-wave field planes can be shifted axially through the specimen, by control of the phase of one of the beams. This can be done with high precision by use of a piezoelectric mirror drive. For SWFM to work well, it is necessary that the specimen be a weak phase object, i.e., that the refractive index heterogeneity be small.

Excitation field synthesis as a means for obtaining enhanced axial resolution in fluorescence microscopes

For fundamental reasons, fluorescence microscopes are more limited in axial, as opposed to transverse, resolution. By giving the excitation field a particular axial structure, this limitation can be partially alleviated, as in confocal scanning or two-photon scanning, or even in optical sectioning microscopy in cases where the object occupies only a small part of the field of view. Standing-wave fluorescence microscopy (SWFM) is a direct imaging method in which the specimen is excited by a 3-D field of planar interference fringes oriented parallel to the object focal plane of the microscope. By shifting the position of the nodal planes of this field relative to the specimen, structures that are normally obscured under uniform excitation become resolved. We demonstrate that, in very thin biological specimens, this optical subsectioning increases axial resolution by an order of magnitude, to 0.04 μm. In comparison to confocal scanning, SWFM resolves fine axial structure with more than 10-fold greater speed, and with similarly-reduced photobleaching. We also discuss the more general case of excitation field synthesis (EFS), in which standing wave fields differing in node spacing can be overlapped or time-multiplexed in the specimen so that the average field is non-periodic and peaked at the object focal plane. A transfer function model is given to show that for weakly-refractive specimens of arbitrary thickness, such as single cells or cell monolayers, EFS should lead to a fivefold improvement in axial resolution.