Confocal Microscopy

Confocal microscopy is now a well-established tool for the examination of subcellular structure and function and complements light and electron microscopy. Because of its high temporal resolution, confocal microscopy allows for the visualization of living as well as fixed tissues and cells, and therefore dynamic processes can be examined and analyzed quantitatively as they actually occur. Confocal microscopy offers several advantages over conventional light microscopy, among which are an increase in contrast, resolution, and clarity (1). Conventional light microscopy provides a two-dimensional (2-D) image of the specimen in the focal plane of the objective lens, but this image is contaminated by out-of-focus images of the specimen above and below the focal plane. Confocal microscopy provides a 2-D image in the focal plane without the out-of-focus information. Furthermore, the resolution of images from a confocal microscope is improved by a factor of 1.4–1.75  (2) . Through computer control of the focus and acquisition of images, modern confocal light microscopes can collect a series of 2-D images (or “optical sections”) through the specimen producing a three-dimensional (3-D) image. Four-dimensional imaging (4-D), defined as 3-D imaging over time, is a recently developed extension in which 3-D images are recorded at periodic time intervals (3).

The basic principle of the confocal microscope is to eliminate the scattered, reflected, or fluorescent light from out-of-focus planes by making the illumination, specimen, and detector all have the same focus, ie, they are confocal. In effect, this microscope will image only the very thin optical section on which the beam is focused. Matched pinholes are used, one at the light source which is imaged onto the specimen to function as a probe that is scanned over the specimen, and one at the detector to capture only a narrow plane of focus. Thus, the out-of-focus blur from areas above and below the focal plane are eliminated. The matched pinhole apertures improve the lateral resolution over conventional light microscopes by a factor of 1.4 with the use of circular apertures and 1.75 with annular apertures (2). Confocal microscopes are often designed to scan in a raster pattern over the sample in which the microscope illuminates one spot at a time, scanning the spot along parallel lines in the focal plane. Lasers are an ideal illumination source for raster scanning because they provide an intense beam of monochromatic radiation that can be condensed onto a small spot. Hence many confocal microscopes are laser scanning (LSCM).

Applications of LSCM include (i) determining the location of proteins, lipids and nucleic acids, cytoskeletal structures and organelles within cells (4, 5), using fluorescent dyes, antibodies, phalloidin, and lectins, (ii) observing ionic fluctuations, such as calcium, magnesium, and pH, in cells and organelles (6), and, (iii) measuring membrane potential using fluorescent dyes (7). The power of 4-D imaging in molecular biology was illustrated by the noninvasive monitoring by LSCM of mitotic events, and cleavage and migration patterns of fertilized sea urchin eggs labeled with DiOC⁶ (8). Macromolecules and subunits can be characterized by immunocytochemical fluorescence probes, which involves the use of antibodies labeled with fluorophores (9). When more than one fluorophore is used, 3-D multilabel (multicolor) imaging can be used to map the 3-D relationship of the labeled structures. For example, z-series collected from two different channels, fluorescein and rhodamine, can be merged into a single reconstruction. By rotating the rendered volume, particular nuances of the structural relationships highlighted by the bound fluorophores may be revealed (10, 11). It is not only important to know that a particular compound is present in a specific cell type or subcellular component, but it is also important to detect and quantify changes in local concentrations of such compounds. Quantitative immunocytochemistry utilizes the principles of stereology and statistical analyses (12) and can be coupled to LSCM to detect differences in immunoreactivity. This method has been used to measure the distribution of integral membrane proteins in the vertebrate retina and to determine the distribution of transport vesicles (13).

Many of the advantages of a confocal light microscope can be achieved with a standard light microscope equipped with a digital camera interfaced to a computer, which controls the microscope stage and focus control as well as run rapid deconvolution algorithms (14). With knowledge of the 3-D point spread function of the microscope, the deconvolution algorithms remove out-of-focus signal to produce images comparable in quality to LSCM images. The advantages of the deconvolution confocal microscope are that (i) the system expense is a fraction of the cost for a LSCM system, and ) ii) lower illumination levels can be used, since all of the light emitted from the specimen is used in forming the 3-D image. Lower illumination minimizes the problem of photobleaching of fluorophores used to label the specimen. A disadvantage of this approach is that the full 3-D deconvolution process can take significantly more time to produce a 3-D image than does a confocal microscope.

References

1. A. Boyde (1990) "Confocal optical microscopy",In Modern Microscopies (P. J. Duke and A. G. Michette, eds.), Plenum Press, New York, pp. 185–204. 

2. E. M. Slayter and H. S. Slayter (1992) Light and Electron Microscopy, Cambridge University Press, Cambridge, UK. 

3. S. A. Stricker, S. Paddock, and G. Schatten (1990) J. Cell Biol. 111, 113a. 

4. A. H. Cornell-Bell et al. (1993) "Membrane glycolipid trafficking in living polarized pancreatic acinar cells: Assessment by confocal microscopy", In Methods in Cell Biology, Vol. 38: Cell Biological Applications of Confocal Microscopy (B. Matsumoto, ed.), Academic Press, San Diego, pp. 222–241. 

5. I. L. Hale and B. Matsumoto (1993) "Resolution of subcellular detail in thick tissue sections: Immunohistochemical preparation and fluorescence confocal microscopy", In Methods in Cell Biology, Vol. 38: Cell Biological Applications of Confocal Microscopy (B. Matsumoto, ed.), Academic Press, San Diego, pp. 290–325. 

6. A. Boyde (1995) "Confocal optical microscopy", In Image Analysis in Histology: Conventional and Confocal Microscopy (R. Wootton et al., eds.), Cambridge University Press, Cambridge, UK, pp. 151–196. 

7. L. M. Loew (1993) "Confocal microscopy of potentiometric fluorescent dyes", In Methods in Cell Biology, Vol. 38: Cell Biological Applications of Confocal Microscopy (B. Matsumoto, ed.), Academic Press, San Diego, pp. 195–210. 

8. S. A. Stricker et al. (1992) Dev. Biol. 149, 370–380. 

9. T. C. Brelje, M. W. Wessendorf, and R. L. Sorenson (1993) "multicolor laser scanning confocal immunofluorescence microscopy: Practical application and limitations", In Methods in Cell Biology, Vol. 38: Cell Biological Applications of Confocal Microscopy (B. Matsumoto, ed.), Academic Press, San Diego, pp. 98–182. 

10. W. Galbraith et al. (1989) Soc. Photo-Opt. Instrum. Eng. 1063, 19–20. 

11. M. W. Wessendorf (1990) In Handbook of Chemical Neuroanatomy, Vol. 8 (A. Bjorklund et al., eds.), Elsevier, Amsterdam, pp. 1–45. 

12. J. T. McBride (1995) "Quantitative immunocytochemistry", In Image Analysis in Histology: Conventional and Confocal Miscroscopy (R. Wootton et al., eds.), Cambridge University Press, Cambridge, UK, pp. 339–354. 

13. B. Matsumoto and I. L. Hale (1993) In Methods in Neuroscience, Vol. 15 (P. C. Hargrave, ed.), Academic Press, San Diego, pp. 54–71. 

14. D. Agard (1984) Ann. Rev. Biophys. Bioeng. 13, 191–219.