A1 Refereed original research article in a scientific journal
Two- and multiphoton detection as an imaging mode and means of increasing the resolution in far-field light microscopy: A study based on photon-optics
Authors: Hell S., Soukka J., Hanninen P.
Publication year: 1995
Journal:: Bioimaging
Journal name in source: Bioimaging
Volume: 3
Issue: 2
First page : 64
Last page: 69
ISSN: 0966-9051
DOI: https://doi.org/10.1002/1361-6374(199506)3:2<64::AID-BIO2>3.0.CO;2-O
Web address : http://api.elsevier.com/content/abstract/scopus_id:0029152986
Abstract
A photon-optics interpretation of the image formation in a scanning single or two-photon excitation fluorescence microscope is given. This interpretation predicts the possibility of two-photon or multiphoton imaging modes based on simultaneous detection of two or more photons. We point out that by simultaneous detection of n photons stemming from the same point, the detection point-spread-function is raised to the nth power. In a two-photon detection microscope, pairs of photons rather than single photons are taken as the signal. We discuss the fundamental requirements of two-photon detection microscopy and the potential and limitations of this imaging mode. Two-photon detection leads to a reduction of the spatial extent of the detection point spread function and therefore to an increase of resolution, but also to a reduction of the total signal. The theoretically predicted narrowing of the point spread function as well as the reduction of the detected signal is confirmed in an experiment simulating the two-photon detection situation. In addition, we show a precise recording of the point-spread function of a 1.4 numerical aperture oil immersion lens at a wave length of 750 nm and the corresponding point-spread-function of a two-photon detection imaging mode.
A photon-optics interpretation of the image formation in a scanning single or two-photon excitation fluorescence microscope is given. This interpretation predicts the possibility of two-photon or multiphoton imaging modes based on simultaneous detection of two or more photons. We point out that by simultaneous detection of n photons stemming from the same point, the detection point-spread-function is raised to the nth power. In a two-photon detection microscope, pairs of photons rather than single photons are taken as the signal. We discuss the fundamental requirements of two-photon detection microscopy and the potential and limitations of this imaging mode. Two-photon detection leads to a reduction of the spatial extent of the detection point spread function and therefore to an increase of resolution, but also to a reduction of the total signal. The theoretically predicted narrowing of the point spread function as well as the reduction of the detected signal is confirmed in an experiment simulating the two-photon detection situation. In addition, we show a precise recording of the point-spread function of a 1.4 numerical aperture oil immersion lens at a wave length of 750 nm and the corresponding point-spread-function of a two-photon detection imaging mode.
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