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A white light confocal microscope for spectrally resolved multidimensional imaging
Author(s) -
FRANK J. H.,
ELDER A. D.,
SWARTLING J.,
VENKITARAMAN A. R.,
JEYASEKHARAN A. D.,
KAMINSKI C. F.
Publication year - 2007
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.1365-2818.2007.01803.x
Subject(s) - supercontinuum , microscope , confocal , optics , 4pi microscope , materials science , microscopy , laser , spectrometer , prism , fluorescence lifetime imaging microscopy , chromatic aberration , confocal microscopy , spectral imaging , fluorescence , wavelength , fluorescence microscope , optoelectronics , chromatic scale , physics , photonic crystal fiber , multiphoton fluorescence microscope
Summary Spectrofluorometric imaging microscopy is demonstrated in a confocal microscope using a supercontinuum laser as an excitation source and a custom‐built prism spectrometer for detection. This microscope system provides confocal imaging with spectrally resolved fluorescence excitation and detection from 450 to 700 nm. The supercontinuum laser provides a broad spectrum light source and is coupled with an acousto‐optic tunable filter to provide continuously tunable fluorescence excitation with a 1‐nm bandwidth. Eight different excitation wavelengths can be simultaneously selected. The prism spectrometer provides spectrally resolved detection with sensitivity comparable to a standard confocal system. This new microscope system enables optimal access to a multitude of fluorophores and provides fluorescence excitation and emission spectra for each location in a 3D confocal image. The speed of the spectral scans is suitable for spectrofluorometric imaging of live cells. Effects of chromatic aberration are modest and do not significantly limit the spatial resolution of the confocal measurements.