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