Fluorescence Microscopy

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Fluorescence Microscopy

Hans J Tanke, Leiden University Medical Center, Leiden, The Netherlands

Published online: January 2006

DOI: 10.1038/npg.els.0005780

Full Article on Wiley Online Library

Abstract
Images
References

Fluorescence microscopy is applied to visualize gene and gene transcripts as stained in situ by methods such as fluorescence in situ hybridization. Instrumentation includes the use of cameras and interferometers for recording multicolor microscopic images, and confocallaser scanning microscopes for optical sectioning and subsequent reconstruction of three-dimensional images of (living) cells.

Keywords: fluorescence microscopy; CCD camera; confocal laser scanning; spectral analysis; optical filters; living cells

	Figure 1. Principle and correction of spherical and chromatic aberration.
	Figure 2. Transmission characteristic of a triple bandpass filter for simultaneous visualization of blue, green and red lines (see also Reichman, 2000).
	Figure 3. Schematic representation of an epi-fluorescence microscope equipped with a CCD camera for digital recording of images.
	Figure 4. Principle of spectral analysis based on interferometry as used in the SKY system for spectral karyotyping (see also Garini et al., (1996); Schröck et al., (1996) ). Interferograms are generated using a Sagnac interferometer and converted by Fourier analysis into spectral information, which can be used for color classification of, for instance, FISH-stained chromosomes.
	Figure 5. Principle of optical sectioning by confocal illumination. Only emission rays from the focal plane pass the pinhole, whereas out-of-focus emission is blocked.
	Figure 6. Optical ray path of a confocal laser scanning microscope (Leica TCS SP2). Note the application of two alternatives for optical filtering: (1) use of a prism and variable pinhole (left inset) and (2) an acousto-optical beam splitter (right inset).

References
	Dyba M and Hell SW (2002) Focal spots of size lambdas/23 open up far-field fluorescence microscopy of 33 nm axial resolution. Physical Review Letters 88: 163–901.
	Garini Y, Macville M, du Manoir S, et al. (1996) Spectral karyotyping. Bioimaging 4: 65–72.
	book Haugland R (ed.) (2002) Handbook of Fluororescent Probes and Research Products. Eugene: Molecular Probes Inc.
	Hiraoko Y, Sedat JW and Agard DA (1987) The use of a charge-coupled device for quantitative optical microscopy of biological structures. Science 238: 36–41.
	Lewis A, Isaacson M, Harootunian A and Murray A (1984) Development of a 500 Å spatial resolution light microscope. Ultramicroscopy 13: 227.
	other Reichman J (2000) Handbook of Optical Filters for Fluorescence Microscopy. Chroma Technologies.
	Schröck E, du Manoir S, Veldman T, et al. (1996) Multicolor spectral karyotyping of human chromosomes. Science 273: 494–497.
	Speicher MR, Ballard SG and Ward DC (1996) Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nature Genetics 12: 368–375.
	Tsien RY (1998) The green fluorescent protein. Annual Reviews of Biochemistry 67: 509–544.
Further Reading
	book Bradbury S, Evenett P, Haselmann H and Piller H (1989) Microscopy Handbooks 15: RMS Dictionary of Light Microscopy. New York, NY: Oxford University Press.
	book Diaspro A (ed.) (2002) Confocal and Two-photon Microscopy: Foundations, Applications and Advances. New York, NY: Wiley Liss Inc.
	book Herman H (1998) "Fluorescence microscopy". Microscopy Handbooks 40, 2nd edn. New York, NY: Springer-Verlag.
	book Robinson JP, Darzynkiewicz Z, Dean PN, et al.(eds.) (2000) Current Protocols in Cytometry, chap. 2. New York, NY: John Wiley.
	book Tanke HJ (1999) "Fluorescence microscopy for quantitative fluorescence in situ hybridisation analysis". In: Andreeff M and Pinkel D (eds.) Introduction to Fluorescence in situ Hybridisation, pp. 33–52. New York, NY: Wiley Liss Inc.

Article Title:	Fluorescence Microscopy
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