Phase-contrast microscopy

Phase-contrast microscope

A phase-contrast microscope
Uses	Microscopic observation of unstained biological material
Inventor	Frits Zernike
Manufacturer	Zeiss, Nikon, Olympus and others
Related items	Differential interference contrast microscopy, Hoffman modulation-contrast microscopy, Quantitative phase-contrast microscopy

Phase-contrast microscopy is an optical-microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations.

When light waves travel through a medium other than vacuum, interaction with the medium causes the wave amplitude and phase to change in a manner dependent on properties of the medium. Changes in amplitude (brightness) arise from the scattering and absorption of light, which is often wavelength-dependent and may give rise to colors. Photographic equipment and the human eye are only sensitive to amplitude variations. Without special arrangements, phase changes are therefore invisible. Yet, phase changes often carry important information.

The same cells imaged with traditional bright-field microscopy (left) and with phase-contrast microscopy (right)

Phase-contrast microscopy is particularly important in biology. It reveals many cellular structures that are not visible with a simpler bright-field microscope, as exemplified in the figure. These structures were made visible to earlier microscopists by staining, but this required additional preparation and killed the cells. The phase-contrast microscope made it possible for biologists to study living cells and how they proliferate through cell division.^[1] After its invention in the early 1930s,^[2] phase-contrast microscopy proved to be such an advancement in microscopy, that its inventor Frits Zernike was awarded the Nobel prize (physics) in 1953.^[3]

Working principle

The basic principle to make phase changes visible in phase-contrast microscopy is to separate the illuminating background light from the specimen scattered light, which make up the foreground details, and to manipulate these differently.

The ring-shaped illuminating light (green) that passes the condenser annulus is focused on the specimen by the condenser. Some of the illuminating light is scattered by the specimen (yellow). The remaining light is unaffected by the specimen and forms the background light (red). When observing an unstained biological specimen, the scattered light is weak and typically phase-shifted by −90° relative to the background light. This leads to the foreground (blue vector) and background (red vector) having nearly the same intensity, resulting in a low image contrast (a).

In a phase-contrast microscope, the image contrast is improved in two steps. The background light is phase-shifted by −90° by passing it through a phase-shift ring. This eliminates the phase difference between the background and the scattered light, leading to an increased intensity between foreground and background (b). To further increase contrast, the background is dimmed by a gray filter ring (c). Some of the scattered light will be phase-shifted and dimmed by the rings. However, the background light is affected to a much greater extent, which creates the phase-contrast effect.

The above describes negative phase contrast. In its positive form, the background light is instead phase-shifted by +90°. The background light will thus be 180° out of phase relative to the scattered light. This results in that the scattered light will be subtracted from the background light in (b) to form an image where the foreground is darker than the background, as shown in the first figure.^{[4][5][6][7][8][9]}

Related methods

Cultured cells imaged by DIC microscopy

A quantitative phase-contrast microscopy image of cells in culture. The height and color of an image point correspond to the optical thickness, which only depends on the object's thickness and the relative refractive index. The volume of an object can thus be determined when the difference in refractive index between the object and the surrounding media is known.

The success of the phase-contrast microscope has led to a number of subsequent phase-imaging methods. In 1952 Georges Nomarski patented what is today known as differential interference contrast (DIC) microscopy.^[10] It enhances contrast by creating artificial shadows, as if the object is illuminated from the side. But, to achieve this, DIC microscopy uses polarized light, making it unsuitable when the object or its container alter polarization. With the growing use of polarizing plastic containers in cell biology, DIC microscopy is increasingly replaced by Hoffman modulation contrast microscopy, invented by Robert Hoffman in 1975.^[11]

Traditional phase-contrast methods enhance contrast optically, blending brightness and phase information in single image. Since the introduction of the digital camera in the mid-1990s, several new digital phase-imaging methods have been developed, collectively known as quantitative phase-contrast microscopy. These methods digitally create two separate images, an ordinary bright-field image and a so-called phase-shift image. In each image point, the phase-shift image displays the quantified phase shift induced by the object, which is proportional to the optical thickness of the object.^[12][13]

See also

• Live cell imaging
• Phase-contrast imaging
• Phase-contrast X-ray imaging

References

1. "The phase contrast microscope". Nobel Media AB.
2. Zernike, F. (1955). "How I Discovered Phase Contrast". Science 121 (3141): 345–349. doi:10.1126/science.121.3141.345. PMID 13237991.
3. "The Nobel Prize in Physics 1953". Nobel Media AB.
4. "Phase contrast microscopy". Phase Holographic Imaging AB.
5. "Introduction to Phase Contrast Microscopy". Nikon MicroscopyU.
6. "Phase Contrast". Leica Science Lab.
7. Frits Zernike (1942). "Phase contrast, a new method for the microscopic observation of transparent objects part I". Physica 9 (7): 686–698. Bibcode:1942Phy.....9..686Z. doi:10.1016/S0031-8914(42)80035-X.
8. Frits Zernike (1942). "Phase contrast, a new method for the microscopic observation of transparent objects part II". Physica 9 (10): 974–980. Bibcode:1942Phy.....9..974Z. doi:10.1016/S0031-8914(42)80079-8.
9. Oscar Richards (1956). "Phase Microscopy 1954-56". Science 124 (3226): 810–814. Bibcode:1956Sci...124..810R. doi:10.1126/science.124.3226.810.
10. US2924142, Georges Nomarski, "INTERFERENTIAL POLARIZING DEVICE FOR STUDY OF PHASE OBJECTS" 
11. US4200354, Robert Hoffman, "Microscopy systems with rectangular illumination particularly adapted for viewing transparent object" 
12. Kemmler, M.; Fratz, M.; Giel, D.; Saum, N.; Brandenburg, A.; Hoffmann, C. (2007). "Noninvasive time-dependent cytometry monitoring by digital holography". Journal of Biomedical Optics 12 (6): 064002. doi:10.1117/1.2804926. PMID 18163818.
13. "Quantitative phase contrast microscopy". Phase Holographic Imaging AB.

External links

• Optical Microscopy Primer — Phase Contrast Microscopy by Florida State University