Journal ArticleDOI
Super-resolution measurements with evanescent-wave fluorescence-excitation using variable beam incidence
TLDR
Three-dimensional reconstruction of the fluorophore distribution from angle-resolved image stacks results in topographical information with an axial resolution of tens of nanometers to study the axial position of dye-labeled subcellular storage organelles ('vesicles') in the "footprint" region of living neuroendocrine cells grown on the interface.Abstract:
The evanescent wave (EW) elicited by total internal reflection of light selectively excites fluorophores in an optical slice above a reflecting dielectric interface. EW excitation eliminates out-of-focus fluorescence present in epiillumination microscopy, and--close to the coverslip--can offer a fivefold enhancement of axial optical sectioning compared to confocal and two-photon microscopy. The decay length of the evanescent field is a function of the refractive indices and light wavelength involved, and is modulated by the beam angle. EW microscopy was used to study the distribution and concentration of fluorophores at or near the interface in the presence of high concentrations in bulk solution. We modified an upright microscope to accommodate the condenser optics needed for EW excitation. Systematic variations of the angle of incidence were attained using an acousto-optical deflector, telecentric optics, and a hemicylindrical prism. The three-dimensional reconstruction of the fluorophore distribution from angle-resolved image stacks results in topographical information with an axial resolution of tens of nanometers. We applied this technique to study the axial position of dye-labeled subcellular storage organelles ('vesicles') of approximately 300 nm diameter in the "footprint" region of living neuroendocrine cells grown on the interface.read more
Citations
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Journal ArticleDOI
Total internal reflection fluorescence microscopy in cell biology.
TL;DR: This review describes a microscopy technique based on total internal reflection fluorescence which is well suited for optical sectioning at cell‐substrate regions with an unusually thin region of fluorescence excitation.
Book ChapterDOI
Total internal reflection fluorescence microscopy in cell biology.
TL;DR: This review describes a microscopy technique based on total internal reflection fluorescence which is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation.
Journal ArticleDOI
A new wave of cellular imaging.
Derek Toomre,Joerg Bewersdorf +1 more
TL;DR: This work examines the optical principles and design of these super-resolution nanoscopy techniques and their ability to see more detail with greater sensitivity--down to single molecules with tens of nanometers resolution.
Journal ArticleDOI
Selective imaging of surface fluorescence with very high aperture microscope objectives
TL;DR: Three approaches to selective surface fluorescence detection are described and the first two are elaborations of "prismless" total internal reflection fluorescence (TIRF), one approach with a laser illumination and the second with arc lamp illumination.
Book ChapterDOI
Chapter 7: Total internal reflection fluorescence microscopy.
TL;DR: This chapter reviews the history, current applications in cell biology and biochemistry, basic optical theory, combinations with numerous other optical and spectroscopic approaches, and a range of setup methods, both commercial and custom.
References
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Journal ArticleDOI
Internal Reflection Spectroscopy
N. J. Harrick,Joseph G. Hoffman +1 more
TL;DR: In this paper, it is shown that if the surface is flat and smooth, the nature of the reflection is called specular, i.e., mirror-like, and obeys the simple law that the angle of incidence equals the angles of reflection.
Journal ArticleDOI
Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution
TL;DR: This approach can be used directly to image single fluorescently labelled myosin molecules and detect individual ATP turnover reactions and can be applied to the study of many types of enzymes and biomolecules.
Journal ArticleDOI
Direct observation of single kinesin molecules moving along microtubules
TL;DR: A new assay is reported in which the processive movement of individual fluorescently labelled kinesin molecules along a microtubule can be visualized directly; this observation is achieved by low-background total internal reflection fluorescence microscopy in the absence of attachment of the motor to a cargo (for example, an organelle or bead).
Journal ArticleDOI
Cell-to-substrate contacts in living fibroblasts: an interference reflexion study with an evaluation of the technique
C. S. Izzard,Linda R. Lochner +1 more
TL;DR: The closeness of contact between cultured chick heart fibroblasts and glass substrates has been examined by interference reflexion microscopy and the common identity is discussed of the focal contacts and associated cytoplasmic fibres described here in living cells with the regions of closest apposition to the substrate.
Journal ArticleDOI
Transport, docking and exocytosis of single secretory granules in live chromaffin cells
TL;DR: It is concluded that a large pool of docked granules turns over slowly, that granules move actively to their docking sites, that docking is reversible, and that the ‘rapidly releasable pool’ measured electrophysiologically represents a small subset of docking granules.
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