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