A white light confocal microscope for spectrally resolved multidimensional imaging
A white light confocal microscope for spectrally resolved multidimensional imaging
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DOI：
10.1111/j.1365-2818.2007.01803.x
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发表时间：
2007-09-01
影响因子：
2
通讯作者：
Kaminski, C. F.
中科院分区：
文献类型：
--
作者：
Frank, J. H.;Elder, A. D.;Kaminski, C. F.
荧光光谱成像显微镜是在共焦显微镜中使用超连续激光作为激发源和定制的棱镜光谱仪进行检测。该显微镜系统提供共聚焦成像，具有光谱分辨的荧光激发和450至700 nm的检测。超连续谱激光器提供了一个宽光谱的光源，并与声光可调谐滤波器耦合，以提供连续可调谐的荧光激发与1 nm的带宽。可同时选择8种不同的激发波长。棱镜光谱仪提供光谱分辨检测，其灵敏度可与标准共焦系统相媲美。这种新的显微镜系统能够最佳地访问大量的荧光团，并为3D共焦图像中的每个位置提供荧光激发和发射光谱。光谱扫描的速度适用于活细胞的荧光光谱成像。色差的影响是适度的，并没有显着限制共焦测量的空间分辨率。
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.