This is the concept behind phase-contrast. Phase-contrast microscopy was first described by Dutch physicist Frits Zernike in the s, for which he later was awarded the Nobel Prize in Physics Phase contrast is a technique that exploits the ability of some microscope samples to alter the OPL of light passing through it, adding contrast through the interference of light of different phases. Transparent unstained samples such as cells do not absorb light and are called phase objects.
When light passes through a sample area with no phase object , there is no significant change in the RI or OPL, so no significant diffraction occurs Fig. This light that is not diffracted is often referred to as direct or zero-order light as it continues unmodified through the sample.
On the other hand, when light passes through an area of the sample with a phase object such as cellular structures , small changes in the RI will diffract and scatter some light and cause changes to the OPL, depending on the thickness and RI of each structure. The thicker the structure, the greater the diffraction of the light.
The diffracted light is a small proportion of the total light that has passed through the sample. This diffracted light that passed through a phase object arrives at the detector out of phase with the direct light that did not pass through a phase object. This small phase shift is not enough to cause significant interference between direct and diffracted light, which along with the poor absorption of transparent structures means there is little amplitude difference between areas where such structures are present and where they are not.
Phase-contrast microscopy is a method that manipulates this property of phase objects to introduce additional interference between the direct and diffracted. This technique transforms differences in phase into differences in brightness , increasing contrast in images of non-absorbing samples. There are two main issues when implementing phase-contrast microscopy: How to phase shift the scattered light or the direct light but not both , and how to get light with well-ordered phase to illuminate the sample.
It was known that constraining light through a small pinhole generated an expanding light wave with well-organized phase but at the expense of a great loss of intensity. This circular wave was easily converted to a flat wave with a lens. Phase-contrast compromises between light intensity and uniform phase by using a circular ring annulus of illumination. This annulus acted similarly to a ring of pinholes , with any particular direction around the ring having the same phase, even though the phase would vary irregularly around the ring.
To phase shift either the scattered or direct light, a phase-shifting optic like a glass disk is placed in the light path where it would predominantly affect the direct light. As the light hits the sample the phase and direction of the diffracted solid lines to the right of the sample and direct dashed lines light changes.
The objective lens takes the scattered light and focuses it to ordered waves, while the direct light is focused to the optical center, where the phase-shifting material is placed. This brings the scattered light and direct light back into phase , allowing for the generation of contrast through interference upon arrival at the detector. A phase-contrast microscope with annular illumination is depicted in Fig.
Implemented in a modern infinity-corrected microscope, the phase-shifting ring is located at the objective rear focal plane. The two components required to convert a traditional bright field microscope into a phase-contrast microscope are the annular diaphragm placed in the condenser back aperture, and the optically matched internal phase plate. The phase plate is introduced into the light path at the rear focal plane, often permanently etched to one of the internal lens elements of the objective such as the tube lens from Fig.
This phase plate decreases the direct light to more closely match the intensity of the diffracted light, and thus further reduce the contribution of background light to the image. Light passing through the sample is refracted or diffracted due to features with different refractive indices, creating new optical paths. Almost all of these new optical paths will not pass through the attenuating areas of the phase plate but instead will pass through the non-attenuating center of the phase ring.
On the other hand, halo effects can often emphasize contrast differences between the specimen and its surrounding background and can increase the visibility of thin edges and border details in many specimens.
This effect is particularly helpful in negative phase contrast, which produces a dark halo surrounding low frequency image detail. In many cases it is possible to reduce the degree of phase shift and diffraction, resulting in reduced halo size around the specimen.
The easiest remedy for removing or attenuating the intensity of halos is to modify the refractive index of the observation medium with higher refractive index components, such as glycerol, mannitol, dextran, or serum albumin. In some cases, changing the refractive index of the medium can even produce a reversal in image contrast, turning dark specimen features bright without significantly disturbing the background intensity.
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. These objectives are termed apodized phase contrast objectives, and enable structures of phase objects having large phase differences to be viewed and photographed with outstanding clarity and definition of detail.
In most cases, subcellular features such as nucleoli can be clearly distinguished as having dark contrast with apodized objectives, but these same features have bright halos or are imaged as bright spots using conventional phase contrast optics. With the apodized optics, contrast is reversed due to the large amplitude of diffracted light relative to that of the direct light passing through the specimen.
In practice, halo reduction and an increase in specimen contrast with apodized optical systems can be achieved by the utilization of selective amplitude filters located adjacent to the phase film in the phase plates built into the objective at the rear focal plane. These amplitude filters consist of neutral density thin films applied to the phase plate surrounding the phase film.
The transmittance of the phase shift ring in the classical phase plate is approximately 25 percent, while the pair of adjacent rings surrounding the phase shift ring in the apodized plate have a neutral density with 50 percent transmittance.
The width of the phase film in both plates is the same. These values are consistent with the transmittance values of phase shifting thin films applied to standard plates in phase contrast microscopes. Shade-off is another very common optical artifact in phase contrast microscopy, and is often most easily observed in large, extended phase specimens. It would normally be expected that the image of a large phase specimen having a constant optical path length across the diameter would appear uniformly dark or light in the microscope.
Unfortunately, the intensity of images produced by a phase contrast microscope does not always bear a simple linear relationship to the optical path difference produced by the specimen. Other factors, such as absorption at the phase plate and the amount of phase retardation or advancement, as well as the relative overlap size of the phase plate and condenser annulus also play a critical role.
The intensity profile of a large, uniformly thick positive phase contrast specimen often gradually increases from the edges to the center, where the light intensity in the central region can approach that of the surrounding medium the reverse is true for negative phase specimens.
This effect is termed shade-off, and is frequently observed when examining extended planar specimens, such as material slabs glass or mica , replicas, flattened tissue culture cells, and large organelles. The effects of halo and shade-off artifacts in both positive and negative phase contrast are presented in Figure 8 for a hypothetical extended phase specimen having rectangular geometry and a higher refractive index than the surrounding medium Figure 8 a.
The intensity profile recorded across a central region of the specimen is illustrated in Figure 8 b. In positive phase contrast Figure 8 c , the specimen image exhibits a distinctively bright halo and demonstrates a dramatic shade-off effect, which is manifested by progressively increasing intensity when traversing from the edges to the central region of the specimen see the intensity profile in Figure 8 d.
The halo and shade-off effects have reversed intensities in negative phase contrast Figure 8 e and 8 f. 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. In addition, the intensity profile Figure 8 f is reversed from that observed with positive phase contrast.
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. In the central regions of a specimen, both the relative angles and the amount of diffracted light are dramatically reduced when compared to the edges. 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.
As a result, the intensity of the central specimen region remains essentially identical to that of the background. The appearance of shade-off effects in relatively flat planar specimen areas, along with the excessively high contrast produced by edges and boundaries, provides strong evidence that the phase contrast mechanism is primarily controlled by the combined phenomena of diffraction and scattering.
Halo and shade-off artifacts depend on both the geometrical and optical properties of the phase plate and the specimen being examined. In particular, the width and transmittance of the phase plate material play a critical role in controlling these effects the phase plate width is typically about one-tenth the total aperture area of the objective.
Wider phase plates having reduced transmittance tend to produce higher intensity halos and shade-off, whereas the ring diameter has a smaller influence on these effects. For a particular phase objective either positive or negative , the optical path difference and specimen size, shape, and structure have significant influence on the severity of halo and shade-off effects.
In addition, these effects are heavily influenced by the objective magnification, with lower magnifications producing better images. Phase contrast is an excellent method for enhancing the contrast of thin, transparent specimens without loss of resolution, and has proven to be a valuable tool in the study of dynamic events in living cells. 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.
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. The technique of phase contrast is widely applied in biological and medical research, especially throughout the fields of cytology and histology.
As such, the methodology is utilized to examine living cells, tissues, and microorganisms that are transparent under brightfield illumination. Phase contrast enables internal cellular components, such as the membrane, nuclei, mitochondria, spindles, mitotic apparatus, chromosomes, Golgi apparatus, and cytoplasmic granules from both plant and animal cells and tissues to be readily visualized. In addition, phase contrast microscopy is widely employed in diagnosis of tumor cells and the growth, dynamics, and behavior of a wide variety of living cells in culture.
Specialized long-working distance phase contrast optical systems have been developed for inverted microscopes employed for tissue culture investigations.
Other areas in the biological arena that benefit from phase contrast observation are hematology, virology, bacteriology, parasitology, paleontology, and marine biology. Industrial and chemical applications for phase contrast include mineralogy, crystallography, and polymer morphology investigations. 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.
In fact, quantitative refractometry is often utilized to obtain refractive index values and for identification purposes. Other commercial products scrutinized by phase contrast optical techniques include clays, fats, oils, soaps, paints, pigments, foods, drugs, textiles, and other fibers. Incident light phase contrast microscopy, although largely supplanted by differential interference contrast techniques, is useful for examination of surfaces, including integrated circuits, crystal dislocations, defects, and lithography.
A good example is the stacking faults in silicon epitaxial wafers, which are of tremendous significance to the semiconductor industry. In reflected light phase contrast systems, an image of the illuminating annulus is projected into the rear focal plane of the objective, where the phase plate is normally located.
In addition, the phase plate is not positioned within the objective, but an image of the rear focal plane is formed by an auxiliary lens system that avoids reflections and scattering generated by the phase plate. It should be noted that in reflected light phase contrast microscopy, phase differences arise from relief on the specimen surfaces, rather than phase gradients within the specimen.
Reduction in halo and shading-off artifacts remains a primary concern in phase contrast microscopy. Apodized phase plates are useful for reducing the severity of halo, and specialized variable phase contrast systems can be fine-tuned to control these effects in order to optimize image quality and the fidelity of information obtained by the technique.
There is also considerable interest in development of advanced phase contrast systems that provide accurate measurements of phase specimens having large optical path differences, as well as combined observations with other contrast-enhancing techniques.
In particular, phase contrast is often utilized with fluorescence imaging to determine the locations of fluorophores, and shows promise for enhancing contrast in scanning optical microscopy. Douglas B. Michael W. 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.
Interactive Tutorial - Specimen Optical Path Length Variations Explore the effects of changes to refractive index and thickness on optical path length, and discover how two specimens can have different combinations of these variables but still display the same path length. Wave Interactions in Phase Contrast Microscopy Phase relationships between the surround, diffracted, and particle S , D , and P waves in the region of the specimen at the image plane for brightfield microscopy in the absence of phase contrast optical accessories are presented in Figure 3.
The Phase Contrast Microscope The most important concept underlying the design of a phase contrast microscope is the segregation of surround and diffracted wavefronts emerging from the specimen, which are projected onto different locations in the objective rear focal plane the diffraction plane at the objective rear aperture.
Interactive Tutorial - Optical Pathways in the Phase Contrast Microscope Examine the light pathways through a phase contrast microscope and learn how these systems dissect the incident electromagnetic wave into a surround S , diffracted D , and resultant particle P component. Interactive Tutorial - Positive and Negative Phase Contrast This interactive tutorial explores relationships between the surround S , diffracted D , and resulting particle P waves in brightfield as well as positive and negative phase contrast microscopy.
Interpretation of Phase Contrast Images Images produced by phase contrast microscopy are relatively simple to interpret when the specimen is thin and distributed evenly on the substrate as is the case with living cells grown in monolayer tissue culture.
Interactive Tutorial - Shade-Off and Halo Phase Contrast Artifacts Explore shade-off and halo artifacts, where the observed intensity does not directly correspond to the optical path difference refractive index and thickness values between the specimen and the surrounding medium.
Conclusions Phase contrast is an excellent method for enhancing the contrast of thin, transparent specimens without loss of resolution, and has proven to be a valuable tool in the study of dynamic events in living cells. Contributing Authors Douglas B. It is this characteristic that enables background light to be separated from specimen diffracted light.
When the light is focused on the image plane, the diffracted and background light cause destructive or constructive interference which decreases or increases the brightness of the areas that contain the sample, in comparison to the background light.
Light from a tungsten-halogen lamp goes through the condenser annulus in the substage condenser before it reaches the specimen. This allows the specimen to be illuminated by parallel light that has been defocused. Some of the light that passes through the specimen will not be diffracted bright yellow in the picture. These light waves form a bright image on the rear aperture of the objective. The light waves that are diffracted by the specimen pass the diffracted plane and focus on the image plane only.
This allows the background light and the diffracted light to be separated. When the light is focused on the image plane, the diffracted and background light will cause destructive or constructive interference, which changes the brightness of the areas that contain the sample in comparison to the background light. All of the components required for phase contrast need to be aligned and centred. Some phase sliders are pre-centred, we therefore recommend that you check beforehand.
Details of how to centre and align phase components are below:. If possible, set up Koehler illumination on your microscope. Put the phase contrast telescope into focus, so that the phase plate and phase ring are in focus. Put the lowest magnification phase objective and corresponding phase annulus in place. For example, a 10x Ph1 objective with a Ph1 phase annulus. Using the adjustment screws on the condenser, centre the phase plate and phase ring so the segmented circle of light sits on the black ring.
Once all objectives have been aligned and centred, remove the phase contrast centring telescope and replace this with the eyepieces. It is, however, recommended to regularly check that the set-up is centred using the phase contrast telescope. For more information about phase contrast, please contact our team of experts at Scientifica, at [email protected]. Banner image: Confluente Rhabdomyosarcoma RD cell line under an inverted phase contrast microscope.
The image appears bright on a darker background. This type of phase contrast is described as negative or bright contrast. Because the undeviated light of the zeroth order is much brighter than the faint diffracted light, a thin absorptive transparent metallic layer is deposited on the ring to bring the direct and diffracted light into better balance of intensity in order to increase contrast.
Such a green filter also helps achromatic objectives produce their best images, since achromats are spherically corrected for green light. The accessories needed for phase contrast work are a substage phase contrast condenser equipped with annuli and a set of phase contrast objectives, each of which has a phase plate installed.
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. Each phase objective has a darkened ring on its back lens. Such objectives can also be used for ordinary brightfield transmitted light work with only a slight reduction in image quality.
Practice aligning a phase contrast microscope and discover how improper alignment affects specimen appearance. The phase outfit, as supplied by the manufacturer, usually includes a green filter and a phase telescope.
The latter is used to enable the microscopist to alight the condenser annulus to superimpose it onto the ring of the phase plate. A set of centering screws in the substage condenser allows manipulation of the annulus to align it while observing the back focal plane of the objective with the phase telescope or through a Bertrand lens. To set up a phase microscope cheek lining cells are a readily available test material , focus the specimen with the 10X phase objective.
This critical step is to assure the proper alignment of the microscope's objective, condenser, and field diaphragm. After the microscope is properly aligned, open up the condenser aperture diaphragm and swing the turret of the condenser into the 10 position this usually automatically opens the aperture diaphragm.
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