Investigation of single biological cells and chromosomes by confocal Raman microspectroscopy

Gerwin J. Puppels; Cees Otto; Frits F. M. de Mul; Jan Greve

doi:10.1117/12.57343

1 May 1991 Investigation of single biological cells and chromosomes by confocal Raman microspectroscopy

Gerwin J. Puppels, Cees Otto, Frits F. M. de Mul, Jan Greve

Proceedings Volume 1403, Laser Applications in Life Sciences; (1991) https://doi.org/10.1117/12.57343
Event: Laser Applications in Life Sciences, 1990, Moscow, Russian Federation

Abstract

A sensitive confocal Raman microspectrometer (CRM) has been developed in our laboratory enabling the study of single living cells and chromosomes [1,2]. Characteristics of the CR14 are; efficient signal collection (by a high power microscope objective), confocal detection achieved by positioning a small pinhole in the image plane of the microscope objective, a high signal throughput of the spectrograph (including a Chevron type dielectric band pass filter set for laser 1 ight suppression , with a 80-90% transmission of Raman signal [3] ) and virtually photon-noise limited signal detection by a liquid nitrogen cooled CCD-camera (quantum efficiency 40% at 700 nm). Laser light of 660 nm from a DCM-operated dye laser is used for excitation. It prevents degradation of the samples in the focused beam, a phenomenon observed when using the 514.5 nm line of an argon-ion laser. The degradation in that case is ascribed to as yet unidentified photo-chemical processes since the possibilities of excessive heating of the samples and multiphoton absorption could be ruled out [51 . When using 660 nm laser light Raman spectra can be obtained even of single fixed metaphase chromosomes in air without any signs of damage [6] . Radial and axial spatial resolution of the CRM in the standard configuration are 0.45 m and 1.3 un respectively [4]. This enables the recording of Raman spectra of specific cellular or chromosomal regions in situ.

Citation Download Citation

Gerwin J. Puppels, Cees Otto, Frits F. M. de Mul, and Jan Greve "Investigation of single biological cells and chromosomes by confocal Raman microspectroscopy", Proc. SPIE 1403, Laser Applications in Life Sciences, (1 May 1991); https://doi.org/10.1117/12.57343

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