Confocal Laser Scanning Light Microscopy

Abstract

The confocal laser scanning microscope is an improved design of light microscope used for imaging fixed or living tissues that are usually labelled with one or more fluorescent probes.

Keywords: microscopy; confocal; fluorescence; 3D reconstruction; laser

Figure 1.

Schematic diagram of a CLSM system. Light from a laser source (1) is attenuated by neutral density filters usually mounted in a filter wheel (2) and passed through a fibre‐optic cable to an exciter filter (3a), which allows only the wavelength of interest to pass through it to a dichromatic mirror (3b), which reflects the light to the scanning unit (4). The scanning unit produces a scanned beam into the back focal plane of an objective lens (5), which focuses the light into the specimen (6). The specimen, if fixed, is mounted on a glass slide in a glycerol medium containing some form of antifade reagent, or if living, in a cell chamber, and in both cases covered with a coverslip of correct thickness for the objective lens. The laser light excites the fluorochrome staining the specimen, and the emitted light passes back through the objective lens and the scanner; since it is of longer wavelength than the excitation light, it passes through the dichromatic mirror (3b). A barrier filter (3c) allows only the wavelength emitted from the fluorophore to pass through. This light is eventually focused at the pinhole (7), placed in front of the PMT (8). All of the optical elements are contained in the scan head (9), which is mounted on the microscope stand. The output of the PMT is built up into an image in the digital imaging system (10) and displayed as an image on a high‐resolution video monitor (11, 11a) or exported to a digital reproduction or storage device (12). The two images on the video monitors (11, 11a) are single optical sections of immunofluorescently labelled microtubules in a two‐cell Caenorhabditis elegans embryo collected with the pinhole at an optimal setting (11), and with the pinhole opened wide (11a). Image 11a simulates the image that would be produced by a conventional epifluorescence microscope. The stage of the microscope can be moved using a stepper motor to produce Z‐series (13).

Figure 2.

Multiple‐labelling applications. A Drosophila embryo at the cellular blastoderm stage has been permeabilized, fixed and labelled with three antibody probes to the transcription factors – (a) hairy, (b) Kruppel and (c) giant – and subsequently with three secondary antibodies labelled with fluorescein, rhodamine and cyanine 5, respectively. The three greyscale images were collected as single optical sections using the CLSM, and were colourized in green, red and blue, respectively, and merged to give a three‐colour image (d). Any colocalization of the gene products in the nuclei is immediately seen as an additive colour change; for example, the two stripes of hairy expression in the red Kruppel expression domain are yellow rather than green. More details of the nuclei are observed when an objective lens of higher NA is used: compare image (d) using a ×16 lens NA 0.5 with image (e) collected using a ×60 NA 1.4 objective lens.

Figure 3.

3D reconstruction. Five sample images from a serial optical series (Z‐series) of 30 images collected through a pollen grain using autofluorescence of the cell wall for imaging (a–e). An optical projection of the entire dataset is shown in (f).

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References

Brelje TC, Wessendorf MW and Sorenson RL (1993) Multicolour laser scanning confocal immunofluorescence microscopy: practical applications and limitations. Methods in Cell Biology 38: 98–177.

Denk W, Strickler JH and Webb WW (1990) Two‐photon laser scanning fluorescence microscopy. Science 248: 73–76.

Errington RJ, Fricker MD, Wood JL, Hall AC and White NS (1997) Four‐dimensional imaging of living chondrocytes in cartilage using confocal microscopy: a pragmatic approach. American Journal of Physiology 272: C1040–C1051.

Haugland RP (1999) Handbook of Fluorescent Probes and Research Chemicals, 7th edn. Eugene, OR: Molecular Probes Inc.

Mohler WA and White JG (1998) Stereo‐4‐D reconstruction and animation from living fluorescent specimens. BioTechniques 24: 1006–1012.

Paddock SW (ed.) (1998) Confocal microscopy: methods and protocols. Methods in Molecular Biology, vol. 122. Totowa, NJ: Humana Press.

Paddock SW, Hazen EJ and DeVries PJ (1997) Methods and applications of three colour confocal imaging. BioTechniques 22: 120–126.

Terasaki M and Dailey ME (1995) Confocal microscopy of living cells. In: Pawley JB (ed.) Handbook of Biological Confocal Microscopy, 2nd edn, pp. 327–346. New York: Plenum Press.

White JG, Amos WB and Fordham M (1987) An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. Journal of Cell Biology 105: 41–48.

White JG, Amos WB, Durbin R and Fordham M (1990) Development of a confocal imaging system for biological epifluorescence applications. In: Optical Microscopy for Biology, pp. 1–18. New York: Wiley.

White NS (1995) Visualisation systems for multidimensional CLSM. In: Pawley JB (ed.) Handbook of Biological Confocal Microscopy, 2nd edn, pp. 211–254. New York: Plenum Press.

Further Reading

Inoue S and Spring KS (1997) Video Microscopy: The Fundamentals, 2nd edn. New York: Plenum Press.

Matsumoto B (1993) Cell biological applications of confocal microscopy. Methods in Cell Biology, vol. 38. San Diego: Academic Press.

Minsky M (1988) Memoir on inventing the confocal scanning microscope. Scanning 10: 128–138.

Pawley JB (1995) Handbook of Biological Confocal Microscopy, 2nd edn. New York: Plenum Press.

Piston DW (1999) Imaging living cells and tissues by two‐photon excitation microscopy. Trends in Cell Biology 9: 66–69.

Potter SM (1996) Vital imaging: two photons are better than one. Current Biology 6: 1595–1598.

Sheppard CJR and Shotten DM (1997) Confocal Laser Scanning Microscopy, Royal Microscopical Society Handbook Series no. 38 Oxford: Bios.

Stevens JK, Mills LR and Trogadis JE (1994) Three‐dimensional confocal microscopy: volume investigation of biological systems. Cell Biology: A Series of Monographs. San Diego: Academic Press.

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Paddock, Stephen(Apr 2001) Confocal Laser Scanning Light Microscopy. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0002993]