Premium
Refractive‐index‐induced aberrations in two‐photon confocal fluorescence microscopy
Author(s) -
JACOBSEN H.,
HÄNNINEN P.,
SOINI E.,
HELL S. W.
Publication year - 1994
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1994.tb03519.x
Subject(s) - oil immersion , optics , numerical aperture , refractive index , microscopy , microscope , confocal , brightness , intensity (physics) , confocal microscopy , aperture (computer memory) , wavefront , materials science , physics , wavelength , acoustics
The effect of refractive index mismatch on the image quality in two‐photon confocal fluorescence microscopy is investigated by experiment and numerical calculations. The results show a strong decrease in the image brightness using high‐aperture objectives when the image plane is moved deeper into the sample. When exciting at 740 nm and recording the fluorescence around 460 nm in a glycerol‐mounted sample using a lens of a numerical aperture of 1·4 (oil immersion), a 25% decrease in the intensity is observed at a depth of 9 μm. In an aqueous sample, the same decrease is observed at a depth of 3 μm. By reducing the numerical aperture to 1·0, the intensity decrease can be avoided at the expense of the overall resolution and signal intensity. The experiments are compared with the predictions of a theory that takes into account the vectorial character of light and the refraction of the wavefronts according to Fermat's principle. Advice is given concerning how the effects can be taken into account in practice.