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Applied Optics

Applied Optics

APPLICATIONS-CENTERED RESEARCH IN OPTICS

  • Vol. 38, Iss. 28 — Oct. 1, 1999
  • pp: 5995–6003

Imaging Properties in Two-Photon Excitation Microscopy and Effects of Refractive-Index Mismatch in Thick Specimens

Cees J. de Grauw, Jurrien M. Vroom, Hans T. M van der Voort, and Hans C. Gerritsen  »View Author Affiliations


Applied Optics, Vol. 38, Issue 28, pp. 5995-6003 (1999)
http://dx.doi.org/10.1364/AO.38.005995


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Abstract

The detrimental effects of a refractive-index mismatch on the image formation in a two-photon microscope were investigated. Point-spread functions (PSF’s) were recorded with an oil-immersion objective numerical aperture (NA) of 1.3 and a water-immersion objective NA of 1.2 in an aqueous sample at different depths. For the oil-immersion objective the enlargement of the PSF volume with increasing depth yields an axial and a lateral loss in resolution of approximately 380% and 160%, respectively, at a 90-μm depth in the sample. For the water-immersion objective no resolution decrease was found. Measurements on a thick aqueous biofilm sample shows the importance of matching the refractive index between immersion fluid and sample. With a good match, no loss in image resolution is observed.

© 1999 Optical Society of America

OCIS Codes
(110.0180) Imaging systems : Microscopy
(120.5710) Instrumentation, measurement, and metrology : Refraction
(180.2520) Microscopy : Fluorescence microscopy
(190.4180) Nonlinear optics : Multiphoton processes

Citation
Cees J. de Grauw, Jurrien M. Vroom, Hans T. M van der Voort, and Hans C. Gerritsen, "Imaging Properties in Two-Photon Excitation Microscopy and Effects of Refractive-Index Mismatch in Thick Specimens," Appl. Opt. 38, 5995-6003 (1999)
http://www.opticsinfobase.org/ao/abstract.cfm?URI=ao-38-28-5995


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