I want to end by noting that in actuality, if you just want to see phase effects due to light passing through a phase object, you don't need any special instruments. In order to reduce the amplitude of the surround wavefront to a value closer to that of the diffracted wave and enforce interference at the image plane , the phase plate in the objective is increased in opacity by application of a semi-transparent metallic neutral density coating. To create the colour intensities, the specimen is first stained with suitable dyes which will impart specific colour. Zernike succeeded in devising a method--now known as Phase Contrast microscopy--for making unstained, phase objects yield contrast images as if they were amplitude objects. In some phase contrast objectives, the thin phase plate contains a ring etched into the glass that has reduced thickness in order to differentially advance the phase of the surround S wave by a quarter-wavelength. In this tutorial the principle of phase contrast imaging is described taking the example of an inverted research microscope. However, when the lights pass through an unstained cell, it encounters regions in the cells with different refractive indexes and thickness.
The new system took advantage of both direct and diffracted light to increase the quality and definition of transparent samples. Zernike manipulated the rings and light source, ultimately reducing the light wavelength by a ½ phase. Zernike experimented with the speed of the light path directed onto a specimen; he discovered interference patterns resulting in the image appearing darker or lighter. The amplitude profiles of the destructively interfering waves for positive phase contrast are depicted in the upper graph of Figure 6. The phase plate configurations, wave relationships, and vector diagrams associated with the generation of positive and negative phase contrast images are presented in Figure 6. Image adapted from: Willey, J. Although these specimens can be observed and recorded with darkfield and oblique illumination, or by defocusing a brightfield microscope, this methodology has proven unreliable in providing critical information about cellular structure and function.
These structures were made visible to earlier microscopists by , but this required additional preparation and thus killing the cells. If the refractive index of the specimen is greater than that of the surrounding medium, the wave is reduced in velocity while passing through the specimen and is subsequently retarded in relative phase when it emerges from the specimen. The phase plate is a transparent glass disc with one or few channels. The problem is compounded by the fact that the width of the zeroth-order surround wavefront projected onto the phase plate by the condenser annulus is smaller than the actual width of the phase plate ring. The result is that the image at the eyepiece level is so lacking in contrast as to make the details almost invisible.
The shade-off phenomenon is also commonly termed the zone-of-action effect, because central zones having uniform thickness in the specimen diffract light differently than the highly refractive zones at edges and boundaries. The interference of the two parts at recombination is sensitive to their optical path difference i. An annular aperture in the diaphragm placed in the focal plane of the sub-stage condenser controls the illumination of the object. Ø Provide high contrast images highlighting the fine details of the cells. Image quality, when used under suitable conditions, is outstanding in resolution and almost entirely free of artifacts unlike phase contrast. Today, many color films reproduce black, white, and grayscales very effectively, especially the tungsten-balanced transparency films from Fuji, Kodak, and Agfa. Each phase objective has a darkened ring on its back lens.
Optical Components of Phase Contrast Microscope The phase contrast microscope is similar to an ordinary compound microscope in its optical composition. To create the colour intensities, the specimen is first stained with suitable dyes which will impart specific colour. Ø Enable the study of cytoskeleton dynamics during cell division, phagocytosis etc. If the phase retardation introduced by the specimen is large enough a phase shift of the diffracted wave by approximately a half-wavelength , interference between the diffracted waves and the surround waves becomes constructive, rendering these specimens brighter than the surrounding background. As a result, undeviated light passing through the phase ring travels a shorter distance in traversing the glass of the objective than does the diffracted light. For example, some internal cellular organelles and components often have a lower refractive index than that of the surrounding cytoplasm, while others have a higher refractive index. A phase-contrast microscope Uses Microscopic observation of unstained biological material Inventor Manufacturer , , , and others Related items , , Phase-contrast microscopy is an technique that converts in light passing through a transparent specimen to brightness changes in the image.
It is so because the living cells are not usually coloured i. Because diffracted wavefronts originating from the central specimen areas have only a marginal spatial deviation from the zeroth-order non-deviated surround wavefronts but are still retarded in phase by a quarter-wavelength , they are captured by the phase plate in the objective rear focal plane, along with the surround light. In addition, the intensity profile Figure 8 f is reversed from that observed with positive phase contrast. Applications of phase contrast microscopy in biological research are numerous. Prior to the introduction of phase contrast optical systems, cells and other semi-transparent specimens were rendered visible in brightfield microscopy by artificial staining techniques. The MicroscopeMaster website is for educational purposes only. The sub-stage annular diaphragm helps to create a narrow, hollow cone or ring of light to illuminate the object.
The advanced surround wavefront is now able to participate in destructive interference with the diffracted D waves at the intermediate image plane. In addition, examples of specimens imaged by these techniques are also illustrated. A dark halo surrounds the specimen image when viewed with negative phase contrast optics Figure 8 e , and the shade-off transition ranges from bright at the edges to darker gray levels in the center. History of Phase Contrast Microscope The Phase Contrast Microscope was developed by Zernike in early 1930s. The condenser usually has a brightfield position with an aperture diaphragm and a rotating turret of annuli each phase objective of different magnification requires an annulus of increasing diameter as the magnification of the objective increases. History and Background Information Frits Zernike, a Dutch physicist and mathematician, built the first phase contrast microscope in 1938.
Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr. The halo effect can also be significantly reduced by utilizing specially designed phase objectives that contain a small ring of neutral density material surrounding the central phase ring material near the objective rear aperture. Ø Live cell imaging and live process monitoring are possible. However, in this case, both materials are sandwiched within the phase plate so that the undiffracted surround wavefront is the only species affected attenuated and retarded in phase by 90 degrees. The illuminating light also is attenuated to make it closer in intensity to the weaker diffracted light from the specimen and thus generate better contrast.
It is impossible to distinguish between high and low refractive index components in a phase contrast image without information pertaining to the relative thickness of the components. Colorless microcrystals, powders, particulate solids, and crystalline polymers, having a refractive index that differs only slightly from that of the surround immersion liquid, are often easily observed using phase contrast microscopy. The phase contrast microscope has special devices annular diaphragm and phase plate , which convert these minute phase changes into amplitude changes or brightness changes so that a contrast difference can be created in the final image. How Contrast is Created in Phase Contrast Microscopy? Some rays direct rays pass through the thinner region of the specimen and do not undergo any retardation and they directly enter into the objective lens. Two very common effects in phase contrast images are the characteristic halo and shade-off contrast patterns in which the observed intensity does not directly correspond to the optical path difference refractive index and thickness values between the specimen and the surrounding medium.
If illuminating light is phase advanced by ¼ l, then the specimen and illuminating light will be ½ l out-of-phase, resulting in destructive interference and a darker image than the background. Thus, the diffracted specimen waves passing through the phase plate remain 90-degrees a quarter-wavelength out of phase relative to the zeroth-order undeviated or surround light. In addition, the contrast range is dramatically improved. Cellular attachments become discernable, as does much of the internal structure. Interaction of Light Waves with Phase Specimens An incident wavefront present in an illuminating beam of light becomes divided into two components upon passing through a phase specimen. These scales are commonly found in the majority of bony fishes referred to as the Teleostei.