Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells
ABSTRACT 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.
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ABSTRACT: Linear Structured Illumination is a powerful technique for increasing the resolution of a fluorescence microscope by a factor of two beyond the diffraction limit. Previously this technique has only been used to image fixed samples because the implementation, using a mechanically rotated fused silica grating, was too slow. Here we describe a microscope design, using a ferroelectric spatial light modulator to structure the illumination light, capable of linear structured illumination at frame rates up to 11Hz. We show live imaging of GFP labeled Tubulin and Kinesin in Drosophila S2 cells.
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ABSTRACT: Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2).Biophysical Journal 03/2014; 106(5):1020-1032. DOI:10.1016/j.bpj.2013.12.049 · 3.83 Impact Factor
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ABSTRACT: The ability to image beyond the diffraction limit is the central tenet of the burgeoning field of superresolution fluorescence microscopy, also referred to as optical nanoscopy. The advent of superresolution has revolutionized biological fluorescence microscopy and the field at large. However, much of that excitement has been tempered by prohibitive imaging requirements. Achieving superresolution entails certain sacrifices, namely imaging speed, choice of fluorophore, ease of multicolor and three-dimensional imaging, and generally more complex instrumentation as compared to standard widefield imaging techniques. Several techniques utilizing structured illumination occupy an intriguing middle ground between the ease of use associated with traditional fluorescence microscopies and the unprecedented resolving power of modern superresolution methods, resulting in undeniably robust imaging techniques. Presented here is a review of the conceptual basis of structured illumination and its implementation, including its performance in comparison to other nanoscopies and the most recent developments in the field.ChemPhysChem 03/2014; 15(4). DOI:10.1002/cphc.201301086 · 3.36 Impact Factor