Developments and applications of confocal theta microscopy

Frank-Martin Haar; Jim Swoger; Ernst H.K. Stelzer

doi:10.1117/12.347589

6 May 1999 Developments and applications of confocal theta microscopy

Frank-Martin Haar, Jim Swoger, Ernst H.K. Stelzer

Proceedings Volume 3605, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing VI; (1999) https://doi.org/10.1117/12.347589
Event: BiOS '99 International Biomedical Optics Symposium, 1999, San Jose, CA, United States

Abstract

Confocal theta microscopy improves the resolution of confocal laser scanning microscopes by solving the problem of the inferior axial resolution instrumentally. The specimens are observed at an angle theta relative to the illumination axis (ideally 90 degrees). The resulting observation volume, defined by the product of illumination and detection point spread functions (PSFs), is reduced by 2.5 and has an isotropic shape. Single-Lens Theta Microscopy (SLTM) is a technical variation of confocal theta microscopy. It is designed to be easily adapted to any common confocal laser scanning microscope. It is based on the use of a mirror unit between the microscope objective lens and its focal plane. This mirror unit deflects the incoming and outcoming light in such a way, that the detection axis is perpendicular to the illumination axis. With SLTM different kinds of other microscopical techniques (such as 4Pi microscopy) are possible. The quantitative evaluation of physical test systems underline the feasibility of SLTM and prove the excellent resolution. The extensions of the experimentally determined point spread functions fit well with the predicted theoretical values. The technique was applied to the investigation of GFP labelled organelles in HeLa cells as well as for the analysis of embryos.

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Frank-Martin Haar, Jim Swoger, and Ernst H.K. Stelzer "Developments and applications of confocal theta microscopy", Proc. SPIE 3605, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing VI, (6 May 1999); https://doi.org/10.1117/12.347589

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KEYWORDS

Confocal microscopy

Microscopes

Mirrors

Microscopy

Capillaries

Objectives

Point spread functions

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