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Confocal Microscopy: Principles and Practices
Publication Name:
Current Protocols in Cytometry
Unit Number:
Unit 2.8
DOI:
10.1002/0471142956.cy0208s05
Online Posting Date:
May, 2001
Abstract
The author presents a well rounded and complete overview of the topic, covering optics, detectors, image collection, and analysis, and designed to take the uninitiated person through an excellent understanding of this very actively growing technology.
Figures
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Figure 2.8.1 Confocal microscope design of Minsky. The condenser forms an image of the illumination pinhole onto a spot in the object. The objective lens forms an image of the spot in the object onto the detector pinhole. The two pinholes and the spot in the object are confocal. Any other spot in the object would be poorly illuminated and its emission would not pass efficiently through the pinhole. -
Figure 2.8.2 An epi-illuminated laser scanning confocal microscope. Rotating mirrors are inserted between the laser and the object to permit scanning of the object in three dimensions at high speed. Since the illuminating and fluorescent light both pass through the same lens and are reflected from the same scanner mirrors, only one pinhole is required. -
Figure 2.8.3 Lily pollen labeled with acridine orange. The left image was taken with an aperture (pinhole) of 1600 µm diameter to simulate nonconfocal operation and the right image was taken with a 40-µm-diameter aperture. The improvement with confocal operation is dramatic. These images were provided through the courtesy of Edwin de Feijter, Insight Biomedical Imaging (Lansing, Mich.). -
Figure 2.8.4 A live human teratocarcinoma cell labeled with the membrane probe NBD-phosphatidylcholine. Images were collected as in Figure -
Figure 2.8.5 Rod photoreceptors from the frog Xenopus laevis labeled by the lectin wheat germ agglutinin conjugated to the fluorochrome Cy3. Images were collected as in Figure -
Figure 2.8.6 Schematic of operation of the xy scanner. Two galvanometer mirrors are used to scan the object. The mirror drivers are under computer control and scan a single line or a rectangular pattern. They can also be directed to a single spot for repeated measurements over time. -
Figure 2.8.7 A confocal microscope that includes a CCD camera. The front side of the mirror scans a slit shaped beam over the object, while the reverse side directs the reflected (or fluorescent) light onto a CCD camera, which collects the image. -
Figure 2.8.8 A tandem scanning confocal microscope. In this device the illuminating light is focused onto a small hole (pinhole) in a disk. The emitted light (scatter or fluorescence) is focused onto a complementary hole on the opposite side of the disk and into a camera, producing a confocal arrangement. The disk contains thousands of these hole pairs, of which only a few are shown. The disk is rotated at high speed such that the holes are scanned across the object, forming a complete confocal image of the scanned area at video rates. -
Figure 2.8.9 Reconstruction of a human-hamster hybrid cell nucleus containing three human chromosomes, showing views at (A) 45°, (B) 90°, and (C) 135°.
Videos
Literature Cited
| Literature Cited | |
| Amos, W.B., White, J.G., and Fordham, M. 1987. Use of confocal imaging in the study of biological structures. Appl. Opt. 26:3239-3243. | |
| Boyde, A. 1985. Stereoscopic images in confocal (tandem scanning) microscopy. Science 230:1270-1272. | |
| Brakenhoff, G.J. and Visscher, K. 1990. Novel confocal imaging and visualization techniques. In Transactions of the Royal Microscopical Society, Vol. 1. (H.Y. Elder, ed.) pp. 247-250. Adam Hilger, Bristol. | |
| Cavanagh, H.D., Petroll, W.M., and Jester, J.V. 1993. The application of confocal microscopy to the study of living systems. Neurosci.Biobehav. Rev. 17.4:483-498. | |
| D Cogswell, C.J. and Sheppard, C.J.R. 1990. Confocal brightfield imaging techniques using an on-axis scanning optical microscope. In Confocal Microscopy (T. Wilson, ed.) pp. 213-243 Academic Press, London. | |
| Cogswell, C.J. and Sheppard, C.J.R. 1992. Confocal differential interference contrast (DIC) microscopy: Including a theoretical analysis of conventional and confocal DIC imaging. J. Microscopy 165:81-101. | |
| Denk, W., Strickler, J.H., and Webb, W.W. 1990. Two-photon laser scanning fluorescence microscopy. Science 248:73-76. | |
| Draaijer, A. and Houpt, P.M. 1987. A real-time confocal laser scanning microscope (CLSM). Proc. SPIE 809:85-88. | |
| Florijn, R.J., Slats, J., Tanke, H.J., and Rapp, A.K. 1995. Analysis of antifading reagents for fluorescence microscopy. Cytometry 19:177-182. | |
| Kino, G.S. 1989. Efficiency in Nipkow disk microscopes. In The Handbook of Biological Confocal Microscopy (J. Pawley, ed.) pp.93-97. IMR Press, Madison, Wis. | |
| Matsumoto, B. 1993. Cell Biological Applications of Confocal Microscopy. Methods in Cell Biology,Volume 38. Academic Press, New York. | |
| Minsky, M. 1957. Microscopy Apparatus. U.S. Patent no. 3013467 (awarded 1961). | |
| Minsky, M. 1988. Memoir on inventing the confocal scanning microscope. Scanning 10:128-138. | |
| Nipkow, P. 1984. German Patent no. 30105. | |
| Petran, M., Hadravsky, M., Egger, D., and Galambos, R. 1968. Tandem-scanning reflected light microscope. J. Opt. Soc. Amer. 58:661-664. | |
| Wilson, T. (ed.) 1990. Confocal Microscopy. Academic Press, London. | |
| Key References | |
| Elder, H.Y. (ed.) 1990. Transactions of the Royal Microscopical Society, Vol. 1. Adam Hilger, Bristol. | |
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Contains papers presented at Micro 90, a conference of the Society held in 1990 at Shortlands, Hammersmith, London. The 21 chapters cover virtually all aspects of microscopy. In particular, it contains excellent papers on the physics and configuration of confocal microscopes, as well as on methods of staining and preparation of samples and on three-dimensional image processing and applications. | |
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Matsumoto, B.
1993. See | |
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Contains 13 chapters with extensive discussions of a variety of applications that utilize confocal microscopy. | |
| Pawley, J. (ed.) 1989. The Handbook of Biological Confocal Microscopy. IMR Press, Madison, Wis. | |
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Consists of 19 papers covering the practical aspects of confocal microscopy; an excellent general reference to the topic. | |
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Wilson, T.
1990. See | |
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Contains 15 chapters covering all aspects of confocal microscopy, primarily from the theoretical point of view. | |
