Frits Zernike Related Posts. James Gregory and the Gregorian Telescope. The Northern Expeditions of Fridtjof Nansen. Leave a Reply Cancel reply Your email address will not be published. Tweets by SciHiBlog. November December December 3. November 2. Imprint Privacy Statement. Ironically, it was the German war machine that confiscated his invention and made a series of microscopes, which ultimately demonstrated the true utility of Zernike's technique.
After the war, most microscope manufacturers rushed to produce microscopes with this enhanced mode of specimen illumination. His efforts were further rewarded by the Nobel committee with the prize in physics in Rather, it was my involvement with diffraction gratings that led to the breakthrough.
Around , our laboratory acquired a sizeable concave grating and the object's surface appeared striped due to periodic imperfections in the grating lines caused by the ruling machines of the time. Yet, when I focused a telescope on the surface from about six meters away, the stripes disappeared! A succession of experiments and calculations enabled me to explain the phenomenon.
In a simpler instance, I found that a telescope with a vertical 2-millimeter slit placed close behind its objective could observe the diffraction pattern of a vertical line-source of light.
The diffraction maxima could be observed, but their phases could not be discerned. The phases could be distinguished, however, if the diffraction image was projected onto a coherent background that could serve as a reference. I was familiar with Lord Rayleigh's simple process of making optically sound glass plate etchings using acid and utilized the technique to make what I call phase strips, glass plates with a single groove, one millimeter wide and etched to a depth of half a wavelength.
When I placed a phase strip in the spectrum of the faulty grating and inspected it with a telescope, the stripes on the grating surface were clearly visible. Recall that Ernst Abbe's theoretical description of the microscope image associates the transparent object under a microscope with a grating. Abbe studied gratings consisting of alternating opaque and transparent strips amplitude gratings , but I was more concerned with alternating thick and thin strips phase gratings.
Phase gratings generate spots of diffraction that demonstrate a phase difference of 90 degrees. For a phase object, when my phase strip is placed in the focal plane of the microscope objective, the direct image of the light source is brought into phase with the diffracted images of a phase object. The result is that the image viewed appears similar to that produced by an amplitude object. The image in the eyepiece of the microscope appears in black and white contrast, as if it were an absorbing object.
We are quick to learn-that is, to emulate what others have already done or thought-but slow to understand-that is, to realize the deeper connections. We are slowest of all, however, in conceiving new connections and in applying old ideas in a new area. In my situation, the truly new point was the fact that the diffraction pattern of the lines of gratings differ in phase from the principal line, and that the phases require projection of the diffraction image on a coherent background to be visualized.
Phase contrast microscopy takes advantage of these optical conjugate properties to enhance image contrast by modifying the microscope aperture function to introduce spatial filtration of specific image information.
Introduction of a phase plate filter in the objective rear diffraction plane enables transformation of specimen phase variations into intensity variations that can be observed in the final image. 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. When thin specimens are examined using positive phase contrast optics, which is the traditional form produced by most manufacturers, they appear darker than the surrounding medium when the refractive index of the specimen exceeds that of the medium.
Phase contrast optics differentially enhance the contrast near the edges surrounding extended specimens, such as the boundary between a cellular membrane and the bathing nutrient medium, and produce overall high-contrast images that can be roughly interpreted as density maps.
Because the amplitude and intensity of a specimen image in phase contrast is related to refractive index and optical path length, image density can be utilized as a gauge for approximating relationships between various structures. In effect, a series of internal cellular organelles having increasing density, such as vacuoles, cytoplasm, the interphase nucleus, and the nucleolus or mitotic chromosomes , are typically visualized as progressively darker objects relative to a fixed reference, such as the background.
It should also be noted that numerous optical artifacts are present in all phase contrast images, and large extended specimens often present significant fluctuations in contrast and image intensity. Symmetry can also be an important factor in determining how both large and small specimens appear in the phase contrast microscope.
Sensible interpretation of phase contrast images requires careful scrutiny and examination to ensure that artifacts are not incorrectly assigned to important structural features. 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.
Because of the varying refractive indices exhibited by these numerous intracellular structures, the interior of living cells, when viewed in a positive phase contrast microscope, can reveal an array of intensities ranging from very bright to extremely dark.
For example, pinocytotic vesicles, lipid droplets, and air vacuoles present in plants and single cell protozoans have a lower refractive index than the cytoplasm, and thus appear brighter than other components. In contrast, as discussed above, organelles that have high refractive indices nuclei, ribosomes, mitochondria, and the nucleolus appear dark in the microscope. 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.
In order to avoid confusion regarding bright and dark contrast in phase contrast images, the optical path differences occurring within the specimen preparation should be carefully considered. As discussed above, the optical path difference is derived from the product of the refractive index and the specimen object thickness, and is related to the relative phase shift between the specimen and background diffracted and surround waves.
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. For example, a small specimen having a high refractive index can display an identical optical path difference to a larger specimen having a lower refractive index.
The two specimens will have approximately the same intensity when viewed through a phase contrast optical system. In many biological experiments, conditions that produce a shrinking or swelling of cells or organelles can result in significant contrast variations. The external medium can also be replaced with another having either a higher or lower refractive index to generate changes in specimen image contrast.
In fact, the effect on image contrast of refractive index variations in the surrounding medium forms the basis of the technique known as immersion refractometry.
Presented in Figure 7 are several semi-transparent specimens imaged with both positive and negative phase contrast optical systems. Figures 7 a and 7 b illustrate a ctenoid fish scale in positive phase contrast Figure 7 a and negative phase contrast Figure 7 b at relatively high magnification x.
These scales are commonly found in the majority of bony fishes referred to as the Teleostei. The anterior or front part of each scale is usually tucked behind the rear portion of the preceding scale.
As the fish grows, so do the scales, resulting in a pattern of concentric growth "rings" that increase in number with the scale size and appear similar to those found in the cross section of tree trunks. In some cases, ctenoid scale growth patterns are utilized to estimate the age of a fish. The growth rings appear dark surrounded by lighter gray halo regions in positive phase contrast Figure 7 a , but are rendered much lighter and surrounded by dark troughs with negative phase contrast Figure 7 b.
A culture of living Chinese hamster ovary cells appears transparent in brightfield illumination mode when bathed in growth medium, and the cells have a refractive index that is very close to the nutrient saline buffer. In positive phase contrast Figure 7 c , internal cellular details, including the nucleus and organelles, can be readily visualized.
However, when examined under negative phase contrast illumination, the cellular outlines become difficult to distinguish, and internal details are largely obscured with the exception of high-refractive index organelles that become very bright Figure 7 d.
Finally, human erythrocytes appear as dark gray oblate ellipsoids featuring a doughnut contour and bright centers in positive phase contrast Figure 7 e , but the same cells are bright with dark centers Figure 7 f and stand out sharply from the background in negative phase contrast.
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. Although these patterns occur as a natural result of the phase contrast optical system, they are often referred to as phase artifacts or image distortions.
In all forms of positive phase contrast, bright phase halos usually surround the boundaries between large specimen features and the medium.
Identical halos appear darker than the specimen with negative phase contrast optical systems. These effects are further accentuated by optical path difference fluctuations, which can turn bright halos dark in positive phase contrast, and dark halos bright in negative phase contrast. Halos occur in phase contrast microscopy because the circular phase-retarding and neutral density ring located in the objective phase plate also transmits a small degree of diffracted light from the specimen it is not restricted to passing surround waves alone.
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 difference in width between the phase plate ring and surround wavefront is usually around 25 to 40 percent, but is necessary due to restrictions and requirements of the optical design. Because of the spatial location of the circular phase-altering ring in the objective diffraction plane, only those wavefronts corresponding to low spatial frequencies diffracted by the specimen pass through the annulus of the phase plate.
Thus, the diffracted specimen waves passing through the phase plate remain degrees a quarter-wavelength out of phase relative to the zeroth-order undeviated or surround light. The resulting phase contrast halo artifact is due to attenuation of the low spatial frequency information diffracted by the specimen through a very shallow angle with respect to the zeroth-order surround wavefronts.
In effect, the absence of destructive interference between low spatial frequency wavefronts diffracted by the specimen and undeviated light waves produces a localized contrast reversal manifested by the halo surrounding the specimen.
In order to create a sharp edge in the image, all of the spatial frequencies diffracted by the specimen must be represented in the final image. 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.
Phase contrast halos are especially prominent and noticeable around large, low spatial frequency objects such as nuclei, diatoms, and entire cells. Another contributing factor to the halo artifact is the redistribution of light energy at the image plane, from regions where it is destructive to regions where it is constructive. Large, high contrast halos can produce confusing images for specimens generating large optical path differences, such as erythrocytes, molds, protozoa, yeast cells, and bacteria.
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.
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