NDLI: Observing secretory granules with a multiangle evanescent wave microscope.

Content Provider	PubMed Central
Author	Rohrbach, A.
Abstract	In total internal reflection fluorescence microscopy (TIRFM), fluorophores near a surface can be excited with evanescent waves, which decay exponentially with distance from the interface. Penetration depths of evanescent waves from 60 nm to 300 nm were generated by varying the angle of incidence of a laser beam. With a novel telecentric multiangle evanescent wave microscope, we monitored and investigated both single secretory granules and pools of granules in bovine chromaffin cells. By measuring the fluorescence intensity as a function of penetration depth, it is possible through a Laplace transform to obtain the fluorophore distribution as a function of axial position. We discuss the extent to which it is possible to determine distances and diameters of granules with this microscopy technique by modeling the fluorescent volumes of spheres in evanescent fields. The anisotropic near-field detection of fluorophores and the influence of the detection point-spread function are considered. The diameters of isolated granules between 70 nm and 300 nm have been reconstructed, which is clearly beyond the resolution limit of a confocal microscope. Furthermore, the paper demonstrates how evanescent waves propagate along surfaces and scatter at objects with a higher refractive index. TIRFM will have a limited applicability for quantitative measurements when the parameters used to define evanescent waves are not optimally selected.
Related Links	http://dx.doi.org/10.1016/s0006-3495(00)76808-1
Ending Page	2654
Page Count	14
Starting Page	2641
File Format	PDF
ISSN	00063495
e-ISSN	15420086
Journal	Biophysical Journal
Issue Number	5
Volume Number	78
Language	English
Publisher Date	2000-05-01
Access Restriction	Open
Subject Keyword	Biophysics Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Biophysics

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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