Phase Contrast Microscopy

Abstract

Phase contrast microscopy is a method that enables us to see very transparent objects, which are otherwise almost invisible by ordinary light microscopy, in clear detail and in good contrast to their surroundings. This is achieved optically, without altering the specimen by staining or other processing.

Keywords: phase; contrast; refractive index; amplitude difference; phase difference; interference; positive phase contrast; negative phase contrast; halo artefact; ‘phase‐telescope’

Figure 1.

All parts of this figure show the same field of view of living HeLa cells (a–e) and fixed, embedded HeLa cells in thin section (f–h).

(a) Living HeLa cells in culture by phase contrast. (b) The same cells by transmitted‐light bright‐field microscopy. (c) In bright‐field mode, without phase contrast, closing the condenser diaphragm will enhance contrast to some degree, but at the expense of resolution in the image. This method is to be avoided. (d) Same image as (a), but the image has been taken with the annulus and phase plate out of alignment (see also Figures 3e,f). (e) The use of a green filter improves the quality of the phase contrast image. (f) Stained HeLa cells, together with the bright‐field image (g) for comparison with the phase contrast image (h).

Parts (h) and (i) are included for comparison of phase contrast images of living cells with those that have been fixed, embedded and sectioned thinly. The manner in which cells and tissues are fixed (if at all) and prepared will influence the resulting phase contrast image. The living cells (h) exhibit high contrast, where there is a relatively high difference of refractive index between the cells and the watery medium they are contained in. The sections of cells embedded in resin in (i) exhibit lower contrast. This is because there is a smaller difference of refractive index between the cell constituents and the background resin. Likewise, cells fixed in methanol, an extracting fixative, exhibit a higher contrast image than those fixed in paraformaldehyde, a crosslinking fixative that retains more of the cytoplasm.

Figures (a)–(e) were taken using a Zeiss Axiovert 25, inverted microscope for tissue culture using a 32 × NA 0.5 long working distance objective. Figures (f)–(h) were taken using a Zeiss Axiophot microscope equipped with a Plan Neofluar 40 × NA 1.30 oil immersion phase contrast objective.

Figure 2.

Ray diagram of the phase contrast method. The heavy lines represent the undiffracted beams, while the diffracted beams are shown by dashed lines. Adapted with permission from Plášek and Reischig .

Figure 3.

(a) and (b) show the top view of different types of phase contrast condenser, in which the various annuli are contained within a housing. This permits them to be changed quickly and efficiently as required. (c) The commonly encountered green inscription engraved on the barrel of a phase contrast objective. The correct annulus to use is denoted, shown here by the designation Ph3. (d) The underside of the condenser in (b), revealing the separate controls for centring the condenser onto the optical axis during alignment of the microscope, and those for independently aligning the annulus with the phase ring. The different sizes of annuli can also be seen. (e) and (f) show the effects on the phase contrast image of not having the annulus and phase ring in absolute alignment.

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References

Bradbury S and Bracegirdle B (1998) Introduction to Light Microscopy. Oxford: Bios Scientific Publishers.

Bradbury S and Evennett PJ (1996) Contrast Techniques in Light Microscopy. Oxford: Bios Scientific Publishers.

Oldfield R (1994) Light Microscopy: An Illustrated Guide. London: Wolfe.

Plášek J and Reischig J (1998) Transmitted‐light microscopy for biology: a physicist's point of view, part 2. Proceedings of the Royal Microscopical Society 33: 196–205.

Pluta M (1989) Phase contrast microscopy. In: Advanced Light Microscopy, vol. 2: Specialized Methods, chap. 5, pp. 1–90. Oxford: Elsevier.

Further Reading

Beck R (1989) The Development of the Phasecontrast Technique for Microscopy. In memoriam Fritz Zernike 1888–1966. Scientific and Technical Information. Vol. IX, No. 5, June 1989. Wild Leitz.

Ross KFA (1988) Phase contrast and interference microscopy. Microscopy 36: 97–123.

Zernike F (1955) How I discovered phase contrast. Science 121: 345–349.

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How to Cite close
Sanderson, Jeremy B(Aug 2001) Phase Contrast Microscopy. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0002635]