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Applied Optics

Applied 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)

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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

Original Manuscript: March 22, 1999
Revised Manuscript: June 3, 1999
Published: October 1, 1999

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)

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