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Sub-100nm Axial Resolution in 3D Widefield Optical Microscopy Using Two Opposing Objective Lenses

Published online by Cambridge University Press:  02 July 2020

Mats G.L. Gustafsson ,
David A. Agard  and
John W. Sedat
Mats G.L. Gustafsson
Affiliation:
Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA94143
David A. Agard
Affiliation:
Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA94143
John W. Sedat
Affiliation:
Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA94143
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Extract

It has been understood since the days of Ernst Abbe that an optical microscope can reach higher resolution the wider the set of angles from which it can observe the specimen — this angular range, of course, is what is indicated by the Numerical Aperture (NA) of the objective lens. Over more than a century of objective lens development, this light collecting angle has been increased closer and closer to the full 180° angle available above a microscope slide. There is, however, a second, equally large and easily accessible set of observation angles available on the other side of the specimen slide, and a standard microscope makes no use whatsoever of light emitted in these directions. One might then ask whether there is not a substantial resolution advantage to be had by devising a microscope that does make use of this "back side" light. The answer, in fact, is yes.

Type
Light Microscopy: Recent Advances
Information
Microscopy and Microanalysis , Volume 3 , Issue S2: Proceedings: Microscopy & Microanalysis '97, Microscopy Society of America 55th Annual Meeting, Microbeam Analysis Society 31st Annual Meeting, Histochemical Society 48th Annual Meeting, Cleveland, Ohio, August 10-14, 1997 , August 1997 , pp. 801 - 802
Copyright
Copyright © Microscopy Society of America 1997

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References

1Gustafsson, M.G.L.et al., Proc. SPIE 2412(1995)147.10.1117/12.205334CrossRefGoogle Scholar
2Agard, et al., Meth. Cell Biol. 30(1989)353.10.1016/S0091-679X(08)60986-3CrossRefGoogle Scholar