Favard C, Valisa P, Egret-Charlier M, Sharonov S, Herben C, Manfait M, Da Silva E, Vigny P
Centre de Biophysique Moléculaire, CNRS, UPR4301 conventionnée avec l'Université d'Orléans, France.
Biospectroscopy. 1999;5(2):101-15. doi: 10.1002/(SICI)1520-6343(1999)5:2<101::AID-BSPY4>3.0.CO;2-W.
With the aim to perform spectroscopic studies and spectral images inside living cells, a microspectrofluorometer has been designed for two-dimensional spectral imaging in the visible and in the near-UV region. The main advantage of the device relies on its ability to scan the laser beam along one direction of the sample. This scanning is optically coupled with one direction of a bidimensional detector, allowing an instantaneous recording of a one-dimensional spectral image. The overall scanning of the sample is achieved by means of submicrometric displacements of the stage in the perpendicular direction. The main characteristics and performances of the microspectrofluorometer in terms of sensitivity (detection of a few molecules), spatial resolution (0.5 x 0.5 x 1 microm), and spectral resolution (1 nm) are presented. Finally, applications of this new apparatus concerning in situ localization and spectral characterization of two dyes are shown with Drosophila salivary glands (ethidium bromide) and T47D tumor cells (Hoechst 33342).
为了在活细胞内进行光谱研究和光谱成像，设计了一种显微光谱荧光计，用于在可见光和近紫外区域进行二维光谱成像。该设备的主要优点在于其能够沿样品的一个方向扫描激光束。这种扫描与二维探测器的一个方向进行光学耦合，从而能够即时记录一维光谱图像。通过样品台在垂直方向上的亚微米级位移实现对样品的整体扫描。介绍了显微光谱荧光计在灵敏度（检测少数分子）、空间分辨率（0.5×0.5×1微米）和光谱分辨率（1纳米）方面的主要特性和性能。最后，展示了该新仪器在果蝇唾液腺（溴化乙锭）和T47D肿瘤细胞（Hoechst 33342）中两种染料的原位定位和光谱表征方面的应用。
