Open AccessHyperspectral two-photon excitation microscopy using visible wavelength
Author(s) -
Takanori Kubo,
Kenta Temma,
Nicholas I. Smith,
Kuo Chang Lu,
Takehisa Matsuda,
Takeharu Nagai,
Katsumasa Fujita
Publication year - 2020
Publication title -
optics letters/optics index
Language(s) - Uncategorized
Resource type - Journals
SCImago Journal Rank - 1.524
H-Index - 272
eISSN - 1071-2763
pISSN - 0146-9592
DOI - 10.1364/ol.413526
Subject(s) - hyperspectral imaging , optics , microscopy , materials science , femtosecond , two photon excitation microscopy , fluorescence , wavelength , microscope , laser , physics , remote sensing , geology
We demonstrate hyperspectral imaging by visible-wavelength two-photon excitation microscopy using line illumination and slit-confocal detection. A femtosecond pulsed laser light at 530 nm was used for the simultaneous excitation of fluorescent proteins with different emission wavelengths. The use of line illumination enabled efficient detection of hyperspectral images and achieved simultaneous detection of three fluorescence spectra in the observation of living HeLa cells with an exposure time of 1 ms per line, which is equivalent to about 2 µs per pixel in point scanning, with 160 data points per spectrum. On combining linear spectral unmixing techniques, localization of fluorescent probes in the cells was achieved. A theoretical investigation of the imaging property revealed high-depth discrimination property attained through the combination of nonlinear excitation and slit detection.