Hostname: page-component-586b7cd67f-dsjbd Total loading time: 0 Render date: 2024-11-27T01:46:33.691Z Has data issue: false hasContentIssue false

Imaging Dynamic Events in Living Tissue using Water Immersion Objectives

Published online by Cambridge University Press:  02 July 2020

M. Brenner*
Affiliation:
Nikon Inc., BioScience Department, 1300 Walt Whitman Road, Melville, NY11747
Get access

Extract

The use of water immersion objectives can provide a significant benefit to research involving the study of dynamic events in living tissue. Optical and quantitative imaging techniques like Confocal, Differential Interference Contrast (DIC), and Epi-fluorescence microscopy are dependent on the ability of high magnification, high numerical aperture immersion objectives to provide a high resolution image. The need to image the specimen not only at the surface next to the cover glass, but deeper down to 200 microns or more, can be greatly assisted by the use of highly corrected water immersion objectives. With oil immersion objectives, the deeper into the specimen and aqueous media one images, the more spherical aberration occurs.

Spherical aberration is the main optical problem in imaging living cells with oil immersion, and spherical aberration increases proportionately with depth into the aqueous media and cellular material. The destructive effect of spherical aberration is seen as a loss of intensity and contrast, inability to collect and resolve small spatial frequencies, and reduced accuracy of reproduction in an optical system, all in direct proportion to the distance from the cover glass. It is possible to compensate mathematically for these distortions and extend resolution with deconvolution or image restoration software. These methods require accurate measurement of the point-spread function; if resolution varies with depth, then measurements must be taken at different depths, which complicates the process.

Type
Recent Advances in Confocal Microscopy
Copyright
Copyright © Microscopy Society of America

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

1.Inoue, S.Foundations of confocal scanned imaging in light microscopy. In: Pawley, J, ed. Handbook of biological confocal microscopy. Revised edition. New York and London: Plenum Press, 1990:12.Google Scholar
2.Hell, S, Reiner, G, Cremer, C, Stelzer, EHK. Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index. J Microsc 1991; 169:391405.CrossRefGoogle Scholar
3.Oldenbourg, R, Terada, H, Tiberio, R, Inoue, S.Image sharpness and contrast transfer in coherent confocal microscopy. J Microsc 1993; 172:31-9.CrossRefGoogle ScholarPubMed
4.Carrington, W, Fogarty, K, Lifschitz, L, Fay, F.3D imaging on confocal and wide-field microscopes. In: Pawley, J, ed. Handbook of biological confocal microscopy. Revised edition. New York and London: Plenum Press, 1990:156.Google Scholar