Aside from specialized contrast-enhancing accessories, there are several locations in the microscope that enable the operator to adjust contrast. The most critical to the optical system are the field and condenser aperture diaphragm settings, but contrast can also be manipulated by varying electronic camera or traditional emulsion film gamma, altering the magnification for video detectors, processing images in real time, as well as specimen staining.
Because the human eye perceives an object by the contrast generated in its image, a certain degree of confusion can result unless there is prior knowledge of the optical events that occur to produce contrast in the image. Figure 1 illustrates a series of three digital images captured in transmitted light mode of the same viewfield containing a transparent, almost colorless Zygnema filamentous algae under differing contrast modes: brightfield, phase contrast, and differential interference contrast.
The three digital images appear quite dissimilar, and because of these variations, the microscopist might arrive at a different conclusion from independent examination of each viewfield. The algae filament illustrated in Figure 1 a was imaged through a microscope operating in brightfield mode with the condenser aperture reduced in size enough to render the edges visible and to expose some internal detail. Although the green chloroplasts can be distinguished within the ribs of the filament, the image generally suffers from an overall lack of contrast.
An identical viewfield of the filamentous algae captured with phase contrast optics is presented in Figure 1 b. Note the series of ribbed, ring-like structures that appear to be ordered in groups of two and the high-contrast spherical shapes that are revealed to be chloroplasts in Figure 1 a.
In general, the image displays dark regions surrounded by halos, which are a common artifact in phase contrast microscopy. Specimen shading introduced by the compensator results in one side of the image appearing dark while the other side is brighter, leading to the perception of a pseudo three-dimensional image. Each viewfield in Figure 1 provides a different specimen image and leads to slightly different interpretations, which can only be deciphered with knowledge about how the microscope created these images.
The absorption of light either naturally or mediated through the addition of synthetic dyes to produce colors or vary the brightness of a specimen has been the classical method of producing contrast in brightfield microscopy.
The term contrast refers to the ability of an individual specimen detail to be distinguished when compared to the background or other adjacent features. In effect, contrast is defined as the difference in light intensity between the specimen image and the adjacent background relative to the overall background intensity.
Specimen properties that produce changes in brightness, or color differences, arise from light absorption, reflection, spatial variation in refractive index, scattering, diffraction, birefringence, fluorescence and similar optical phenomena.
In general, contrast is measured by the relationship between the highest and lowest intensity in an image, and can be described by a simple formula:. From this equation, it is evident that specimen contrast refers to the relationship between the highest and lowest intensity in the image. If the specimen intensity is less darker than that of the background, contrast is referred to as being positive , while specimens that are lighter than the background display negative contrast.
When a specimen modifies the spectral distribution color of light passing through, it produces color contrast. This type of contrast is also produced by interference of white light in specimens with closely spaced periodic structures. The graph presented in Figure 2 illustrates the effect of background intensity on specimen contrast. When the background is a very dark gray value I b equals 0.
By lightening the background to a somewhat lighter gray I b equals 0. At still lighter background intensities I b is greater than or equal to about 0. In most situations, the background and image intensities are not discrete values, but vary over the entire viewfield, leading to fluctuations in contrast.
As a rule of thumb, transparent specimens in brightfield illumination mode display about 2 to 5 percent contrast, while phase and differential interference contrast images can have contrast levels between 15 and 20 percent, just slightly less than that observed with fixed and stained specimens in brightfield illumination about 25 percent.
Compared to darkfield and fluorescence microscopy average contrast levels of 60 and 75 percent, respectively , phase contrast and differential interference contrast illumination produce less overall contrast but still afford a similar degree of resolution.
Prior to the invention of optical contrast enhancement techniques, transmitted brightfield illumination was one of the most commonly utilized observation modes in optical microscopy, especially for fixed, stained specimens or other types of samples having high natural absorption of visible light. Collectively, specimens readily imaged with brightfield illumination are termed amplitude objects or specimens because the amplitude or intensity of the illuminating wavefronts is reduced when light passes through the specimen.
However, for many specimens in optical microscopy, especially unstained or living material, contrast is so poor that the specimen remains essentially invisible regardless of the ability of the objective to resolve or clearly separate details.
Often, for just such specimens, it is important not to alter them by killing or treatment with chemical dyes or fixatives. This necessity has led microscopists to experiment with contrast enhancing techniques for over a hundred years in an attempt to improve specimen visibility and to bring more detail to the image without altering the specimen itself. It is a common practice to reduce the condenser aperture diaphragm below the recommended size or to lower the substage condenser in order to increase specimen contrast.
Unfortunately, while these maneuvers will indeed increase contrast, they also seriously reduce resolution and sharpness. In brightfield illumination, both the light source usually a tungsten-halogen lamp and condenser are positioned to fill the objective front aperture with partially coherent wavefronts that are symmetrical with respect to the microscope optical axis.
Wavefronts that interact with stained regions of the specimen are reduced in amplitude, while those diffracted by the specimen produce prominent first-order diffraction side bands , which are degrees out of phase with light passing through the specimen. The diffracted side bands interfere at the image plane with the surround light waves during formation of the image, and the background appears bright, with absorbing structures in the specimen exhibiting a variety of colors and gray-level tones.
However, when the refractive index of an unstained specimen is similar to that of the surrounding medium typically referred to as the surround , the intensity of diffracted light waves is dramatically reduced. In addition, the relative phase of retardation imparted to diffracted wavefronts emanating from the specimen is shifted by only 90 degrees instead of the usual degrees observed with highly absorbing specimens.
Both of these effects combine to severely limit visibility of the specimen. As discussed above, specimens that alter the intensity of transmitted light are termed amplitude specimens and can be observed in the microscope as a consequence of their ability to absorb or otherwise affect the light intensity, which is proportional to the square of the light wave amplitude.
Other specimens that are naturally colored or artificially stained with chemical color dyes can also be clearly imaged in brightfield illumination. These stains or natural colors absorb some portion of the white light passing through and transmit or reflect other colors. Often, stains are combined to yield contrasting colors. For example, blue hematoxylin stain for cell nuclei is often combined with pink eosin that selectively stains cytoplasm.
It is a common practice to utilize stains on specimens that do not readily absorb light, thus rendering such images visible in the microscope. In contrast, transparent specimens that do not absorb light, but instead, produce a phase change to wavefronts passing through are termed phase objects or specimens.
These specimens are virtually invisible and very difficult to image because the human eye is insensitive to changes in the relative phase shifts between visible light waves. In addition, the eye is unable to detect orientational changes, such as polarizing effects, in the electric field vectors comprising electromagnetic radiation. Phase specimens are characterized by several criteria including their shape typically round or flat , the density of internal light scattering elements, thickness, and unique chemical or electrical structural properties collectively grouped as refractive index.
There are several types of staining media, each can be used for a different purpose. Commonly used stains and how they work are listed below. All these stains may be used on fixed, or non-living, cells and those that can be used on living cells are noted. After staining cells and preparing slides, they may be stored in the dark and possibly refrigerated to preserve the stained slide, and then observed with a microscope.
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Cell Structure And Its Functions 9. Reproduction In Animals Reaching The Age Of Adolescence However, sometimes additional information is required to provide a full differential diagnosis, and this requires furthermore specialized staining techniques. These methods can all be applied to paraffin sections, and in most cases, the slides produced are completely stable and can be kept for many years.
After staining, the sections are covered with a glass coverslip and are then sent to a pathologist who will view them under a microscope to make an appropriate diagnosis and prepare a report. The content, including webinars, training presentations and related materials is intended to provide general information regarding particular subjects of interest to health care professionals and is not intended to be, and should not be construed as, medical, regulatory or legal advice.
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