title: Imaging properties in two-photon excitation microscopy and effects of refractive-index mismatch in thick specimens.
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-microm 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.
authors.affiliation: Molecular Biophysics, Utrecht University, P.O. Box 80.000, 3508 TA Utrecht, The Netherlands. k.degrauw@phys.uu.nl
authors.forename: C J
authors.initials: CJ
authors.lastname: de Grauw
authors.forename: J M
authors.initials: JM
authors.lastname: Vroom
authors.forename: H T
authors.initials: HT
authors.lastname: van der Voort
authors.forename: H C
authors.initials: HC
authors.lastname: Gerritsen