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Confocal Raman Microscopy of Chromosomes, Cells and Eye-Lenses

Published online by Cambridge University Press:  02 July 2020

J. Greve ,
N.M. Sijtsema ,
C.J. de Grauw ,
H. Duindam ,
S. Wouters ,
C. Otto  and
G.J. Vrensen
J. Greve
Affiliation:
Department of Applied Physics, Biomédical Technology Institute, University of Twente, P.O Box217, 7500AE Enschede, The Netherlands
N.M. Sijtsema
Affiliation:
Department of Applied Physics, Biomédical Technology Institute, University of Twente, P.O Box217, 7500AE Enschede, The Netherlands
C.J. de Grauw
Affiliation:
Department of Applied Physics, Biomédical Technology Institute, University of Twente, P.O Box217, 7500AE Enschede, The Netherlands
H. Duindam
Affiliation:
Department of Applied Physics, Biomédical Technology Institute, University of Twente, P.O Box217, 7500AE Enschede, The Netherlands
S. Wouters
Affiliation:
Department of Applied Physics, Biomédical Technology Institute, University of Twente, P.O Box217, 7500AE Enschede, The Netherlands
C. Otto
Affiliation:
Department of Applied Physics, Biomédical Technology Institute, University of Twente, P.O Box217, 7500AE Enschede, The Netherlands
G.J. Vrensen
Affiliation:
Department of Morphology, The Netherlands Ophthalmic Research Institute, P.O. Box 12141, 1100AC Amsterdam, The Netherlands
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Extract

A confocal scanning Raman microscope was constructed for spectroscopy and microscopy of biological samples (Fig. 1). The microscope contains an illumination system in which a focused krypton laser beam, of which the 647.1 nm line is used to reduce damage, is scanned over the sample using a scanning mirror which moves around two orthogonal axes. Raman scattered light is collected by a water immersion objective which directs the light on the scanning mirror. Spectral analysis takes place in a monochromator with two exit ports: one for spectroscopical purposes the other one for imaging. An image is made by scanning the light, present in the Raman band passed by the monochromator, over a CCD using a second scanning mirror which moves synchronously with the first mirror. The spatial resolution is of the order of 0.3 × 0.3 × 1.2 μm3 (x,y,z).

With this microscope we studied the following samples:

Type
Optical Microanalysis
Information
Microscopy and Microanalysis , Volume 3 , Issue S2: Proceedings: Microscopy & Microanalysis '97, Microscopy Society of America 55th Annual Meeting, Microbeam Analysis Society 31st Annual Meeting, Histochemical Society 48th Annual Meeting, Cleveland, Ohio, August 10-14, 1997 , August 1997 , pp. 821 - 822
Copyright
Copyright © Microscopy Society of America 1997

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References

1.Sijtsema, N.M.et al., “Development of a Confocal Direct Imaging Raman Microscope (CDIRM)”,to be submitted.Google Scholar
2.Duindam, J.J.et al., “Cholesterol, Phospholipid and Protein Changes in Focal Opacities in the Human Eye Lens”, Submitted to IOVS.Google Scholar
3.de Grauw, C.J.et al., “Chromatin structure in bands and interbands of polytene chromosomes imaged by atomic force microscopy”, Submitted to Journal of Structural Biology.Google Scholar