Related Experiment Video
Updated: Jul 6, 2026

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers
Published on: April 9, 2014
Imaging properties in two-photon excitation microscopy and effects of refractive-index mismatch in thick specimens
C J de Grauw1, J M Vroom, H T van der Voort
1Molecular Biophysics, Utrecht University, P.O. Box 80.000, 3508 TA Utrecht, The Netherlands. k.degrauw@phys.uu.nl
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.
Related Concept Videos
Imaging Biological Samples with Optical Microscopy
In optical microscopy, the specimen to be viewed is placed on a glass slide and clipped on the stage...
Phase Contrast and Differential Interference Contrast Microscopy
In-phase-contrast microscopes, interference between light directly passing through a cell and light refracted by cellular components is used to create high-contrast, high-resolution images without staining. It is the oldest and simplest type of microscope that creates an image by altering the wavelengths of light rays passing through the specimen. Altered wavelength paths are created using an annular stop in the condenser. The annular stop produces a hollow cone of...
Three-Dimensional Microscopy in Microbiology
Super-resolution Fluorescence Microscopy
Total Internal Reflection Fluorescence Microscopy
Confocal Fluorescence Microscopy

