Holographic gratings can also be made from computer-generated interference patterns. The patterns are written onto a chrome mask using an electron-beam machine. The patterns on the mask are then etched into a material, such as fused silica, using photolithographic masking and etching techniques. "Computer-generated gratings have really just reached maturity within the last two years," says Michael Feldman, of Digital Optics Corp. (Charlotte, NC). "They are very flexible and easy to mass-produce."

The least expensive (and most common) objectives, employed on a majority of laboratory microscopes, are the achromatic objectives. These objectives are corrected for axial chromatic aberration in two wavelengths (blue and red; about 486 and 656 nanometers, respectively), which are brought into a single common focal point. Furthermore, achromatic objectives are corrected for spherical aberration in the color green (546 nanometers; see Table 1). The limited correction of achromatic objectives can lead to substantial artifacts when specimens are examined and imaged with color microscopy and photomicrography. If focus is chosen in the green region of the spectrum, images will have a reddish-magenta halo (often termed residual color). Achromatic objectives yield their best results with light passed through a green filter (often an interference filter) and using black and white film when these objectives are employed for photomicrography. The lack of correction for flatness of field further hampers achromat objectives. In the past few years, most manufacturers have begun providing flat-field corrections for achromat objectives and have given these corrected objectives the name of plan achromats.

Kaiser Optical Systems Inc. (KOSI; Ann Arbor, MI), has developed an alternative to the classical or surface-relief holographic grating--the volume transmission holographic grating (see photo at top of this page; also Laser Focus World, Oct. 1995, p. 95). The grating is created in the traditional manner by recording interference patterns generated by two mutually coherent laser beams. After the pattern is defined in the photosensitive material, coated on glass, and the film developed, a top layer of glass is added, creating a totally transparent grating assembly. Light strikes the grating on one side and diffracts out through the other.

To remedy this, many high-performance apochromat dry objectives are fitted with correction collars, which allow adjustment to correct for spherical aberration by correcting for variations in cover glass thickness (see Figure 5). Optical correction for spherical aberration is produced by rotating the collar, which causes two of the lens element groups in the objective to move either closer together or farther apart. The objective on the left in Figure 5 has had the correction collar adjusted for a cover glass thickness of 0.20 mm by bringing the adjustable lens elements very close together. In contrast, the objective on the right in Figure 5 has the adjustable lens elements separated by a rather large distance to compensate for very thin cover glasses (0.13 mm). A majority of the correction collar objectives designed for upright transmitted light microscopy have an adjustment range for cover glass thickness variations between 0.10 and 0.23 millimeters. Many of the specialized phase contrast objectives designed for observing tissue culture specimens with an inverted microscope have an even broader compensation range of 0 to 2 millimeters. This allows specimens to be viewed through the bottom of most culture vessels, which often have dramatic thickness fluctuations in this size range. Uncovered specimens, such as blood smears, can also be observed with correction collar objectives when the adjustment is set to 0 to account for the lack of a cover glass.

"The grooves are similar to the indentations made by a plow in soil," says John Hoose of Richardson Grating Laboratory (Rochester, NY), except that they are much closer together. Anywhere from one to 10,000 fine parallel lines per millimeter can be engraved. Light waves diffracted from these lines interfere, and all wavelengths but one are canceled in any particular direction through destructive interference. The depth of the groove changes the wavelength of the light wave being diffracted.

Since the invention of the replication technique, diffraction gratings have replaced prisms in many commercial spectrometers. A prism will bend short wavelengths more than longer ones (see Laser Focus World, Jan. 1997, p. 101). Prisms that transmit visible light absorb most UV and infrared wavelengths, whereas reflection gratings can be suitably coated for high reflectivity in wide spectral regions. Gratings are considered superior to prisms in many applications. Seeking to combine the best of both, Richardson Grating Laboratory has fabricated a "grism," a part-grating, part-prism optical element useful in spectrometers that require in-line presentation of the spectrum, as in astronomy. The light diffracted by the grating is bent back in line by the refracting effect of the prism. The dispersion of the grism is not linear, because the dispersive effects of the prism and grating are superimposed.

Light traveling along the fiber core impinges on the grating, and each area of different refractive index scatters a small portion of the beam. If the wavelength of the signal is twice the distance between the periodic refractive elements (typically <1 µm), then the signals scattered back down the fiber core will add constructively to give a large reflection. The wavelength at which the reflection occurs is the Bragg wavelength. A Bragg grating can operate at precise wavelengths that can be accurately preset and maintained, says Keith Brundin at 3M Specialty Optical Fibers (West Haven, CT).

Properly designed oil immersion objective lenses also correct for chromatic defects that are introduced by the first two lens elements, while introducing a minimum amount of spherical aberration. The fact that the light cone is partially converged before entering the first lens element aids in the control of spherical aberration. It should be noted that employing an oil immersion objective without the application oil between the coverslip and first lens element results in defective images. This due to refraction that occurs at the surface of the front lens, which introduces spherical aberration that cannot be corrected by subsequent lens components within the objective.

What is grating constant

Major microscope manufacturers offer a wide range of objective designs, which feature excellent optical characteristics under a wide spectrum of illumination conditions and provide various degrees of correction for the primary optical aberrations. The objective illustrated in Figure 1 is a 60x oil immersion apochromat, which contains 15 optical elements that are cemented together into three groups of lens doublets, a lens triplet group, and three individual internal single-element lenses. The objective also has a hemispherical front lens and a meniscus second lens, which work synchronously to assist in capturing light rays at high numerical aperture with a minimum of spherical aberration. As is the case with most oil immersion objectives, the apochromat illustrated in Figure 1 is equipped with a spring-loaded retractable nosecone assembly that protects the front lens elements and the specimen from collision damage. Internal lens elements are carefully oriented and tightly packed into a tubular brass housing that is encapsulated by the objective barrel. Specific objective parameters such as numerical aperture, magnification, optical tube length, degree of aberration correction, and other important characteristics are imprinted or engraved on the external portion of the barrel. Although the objective featured in Figure 1 is designed to operate utilizing oil as the imaging medium between the objective front lens and specimen, other objectives have front lens elements that allow them to be use either in air or immersed in water, glycerin, or a other specialized hydrocarbon-based oils.

A Raman band may vary in shift (position), narrow or broaden (width), or vary in intensity (height). These changes can reveal compressive/tensile stresses in ...

Order of diffractionexample

"It also has a high efficiency," says Arns. "Depending on the configuration, the grating can produce 90% efficiency in the first order. If the thickness or the frequency of the grating is high enough, higher orders that otherwise might be propagated are extinguished." Another advantage, says Arns, is that the element can be handled and cleaned in the same fashion as a high-quality cemented lens because the grating is sandwiched between two layers of glass. Also, because the Bragg-type grating is a transmission device, optical elements and instruments can be brought close to it, resulting in a compact design.

The highest level of correction (and expense) is found in apochromatic objectives, illustrated in Figures 2 and 3. Apochromats represent the most highly corrected microscope lenses currently available, and their high price reflects the sophisticated design and careful assembly required in their manufacture. In Figure 3, we compare lens elements in a series of apochromatic objectives ranging from 10x to 100x in magnification. The lower power apochromat objectives (10x and 20x) have a longer working distance and the overall objective length is shorter than in higher power (40x and 100x) apochromat objectives. Apochromats are corrected chromatically for three colors (red, green, and blue), almost eliminating chromatic aberration, and are corrected spherically for either two or three wavelengths (see Table 1). Apochromatic objectives are the best choice for color photomicrography in white light. Because of their high level of correction, apochromat objectives usually have, for a given magnification, higher numerical apertures than do achromats or fluorites. Many of the newer high-performance fluorite and apochromat objectives are corrected for four (dark blue, blue, green, and red) or more colors chromatically and four colors spherically.

High numerical aperture dry objectives lacking a correction collar often produce images that are inferior to those of lower numerical aperture objectives where cover glass thickness is of less concern. For this reason, it is often prudent to choose a lower magnification (and numerical aperture) objective in order to obtain superior contrast without the accompanying artifacts introduced by cover glass fluctuations. As an example, a 40x objective having a numerical aperture of 0.65 may be able to produce better images with sharper contrast and clarity than a 60x-0.85 numerical aperture objective, even though the resolving power of the higher magnification objective is theoretically greater.

Diffractiongrating formula

Uncorrected field curvature is the most severe optical aberration that occurs in fluorite (semi-apochromat) and apochromat objectives, and it was tolerated as an unavoidable artifact for many years. During routine use, the viewfield would have to be continuously refocused between the center and the edges to capture all specimen details. The introduction of flat-field (plan) correction to objectives perfected their use for photomicrography and video microscopy, and today these corrections are standard in both general use and high-performance objectives. Correction for field curvature adds a considerable number of lens elements to the objective as illustrated in Figure 4 with a simple achromat. The uncorrected achromat on the left in Figure 4 contains two lens doublets, in addition to a simple thin-lens front element. In contrast, the corrected plan achromat on the right in Figure 4 contains three lens doublets, a central lens triplet group, and a meniscus lens positioned behind the hemispherical front lens. Plan correction, in this instance, has led to the addition of six lens elements bundled into more sophisticated lens groupings, which dramatically increases the optical complexity of the objective. The significant increase in lens elements for plan correction also occurs with fluorite and apochromat objectives, frequently resulting in an extremely tight fit of lens elements (see Figure 1) within the internal objective sleeve. In general, plan objectives corrected for field curvature sacrifice a considerable amount of free working distance, and many of the high-magnification versions have a concave front lens, which can be extremely difficult to clean and maintain.

In 1882, Henry A. Rowland invented the process of ruling, or scratching parallel notches into metal deposited onto the surface of a flat, clear glass plate—a method that produced gratings of exceptionally high quality. Modern ruled gratings can be either reflective or transmissive and are fabricated with a single diamond point that burnishes grooves on flat or concave surfaces.

The objective is the most difficult component of an optical microscope to design and assemble, and is the first element that light encounters as it proceeds from the specimen to the image plane. Objectives derive their name from the fact that they are, by proximity, the closest component to the object (specimen) being imaged.

The author wishes to thank John Hoose of Richardson Grating Laboratory (Rochester, NY) for his help in preparing this article.

Digital holography optically generates a hologram, which is then recorded on a CCD camera, and an image is reconstructed using digital techniques.

What isorder of diffractionin Bragg's law

Diffraction gratings are fundamental optical elements that have a precise pattern of grooves superimposed on them. These minute, periodic structures diffract, or disperse, incident light in such a way that the individual wavelengths making up the incident light can be differentiated. Gratings are indispensable in helping physicists determine the structure of atoms or helping astronomers calculate the chemical composition of stars and the rotation of galaxies. Applications are expanding; one of the fastest growing areas for gratings—laser pulse compression—didn’t even exist until a few years ago.

The concept of diffraction gratings is simple, yet elegant. For more than one hundred years, they have been used in dispersive optical systems. Applications for gratings are expanding as the fabrication technology grows. Fields as diverse as telecommunications, astronomy, microlithography, lasers, and metal analysis are driving these changes.

Modern objectives, made up of many glass elements, have reached a high state of quality and performance, with the extent of correction for aberrations and flatness of field determining the usefulness and cost of an objective. Construction techniques and materials used to manufacture objectives have greatly improved over the course of the past 100 years. Today, objectives are designed with the assistance of Computer-Aided-Design (CAD) systems using advanced rare-element glass formulations of uniform composition and quality having highly specific refractive indices. The enhanced performance that is demonstrated using these advanced techniques has allowed manufacturers to produce objectives that are very low in dispersion and corrected for most of the common optical artifacts such as coma, astigmatism, geometrical distortion, field curvature, spherical and chromatic aberration. Not only are microscope objectives now corrected for more aberrations over wider fields, but image flare has been dramatically reduced with a substantial increase in light transmission, yielding images that are remarkably bright, sharp, and crisp.

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The next higher level of correction and cost is found in objectives called fluorites or semi-apochromats (illustrated by center objective in Figure 2), named for the mineral fluorite, which was originally used in their construction. Figure 2 depicts the three major classes of objectives: The achromats with the least amount of correction, as discussed above; the fluorites (or semi-apochromats) that have additional spherical corrections; and, the apochromats that are the most highly corrected objectives available. The objective positioned on the far left in Figure 2 is a 10x achromat, which contains two internal lens doublets and a front lens element. Illustrated in the center of Figure 2 is a 10x fluorite objective having several lens groups including two doublets and a triplet, in addition to a hemispherical front lens and a secondary meniscus lens. On the right in Figure 2 is a 10x apochromat objective that also contains multiple lens groups and single elements. Although similar in construction to fluorite objectives, the lenses have different thicknesses and curvatures and are arranged in a configuration that is unique to apochromat objectives.

Objectives that use water and/or glycerin as an imaging medium are also available for applications with living cells in culture or sections of tissue immersed in physiological saline solution. Plan apochromat water immersion lenses are equipped with correction collars and numerical apertures up to 1.2, slightly less than their oil immersion counterparts. These objectives allow microscopists to focus through up to 200 microns of aqueous media and still retain excellent optical correction. The downside is that high numerical aperture water immersion lenses often cost many thousands of dollars and the image can still degrade when the objective is focused deeply through refractile tissue or cell parts. For more details on water, glycerin, and oil immersion objectives, visit the Molecular Expressions Microscopy Primer.

Secondorder diffraction

Fiber Bragg gratings, another recent development in grating applications, are made within a fiberoptic cable. Fiber gratings are fabricated by exposing the core of a single-mode fiber, 8 to 10 µm thick, to a periodic pattern of intense ultraviolet light. This pattern is created when a 248- or 193-nm laser passes through a special diffractive phase mask. When a fiber is placed in the intense UV light pattern of the mask, a permanent modulation of the index of refraction is generated in the fiber core. This photo-generated index modulation acts as a grating.

World-class Nikon objectives, including renowned CFI60 infinity optics, deliver brilliant images of breathtaking sharpness and clarity, from ultra-low to the highest magnifications.

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Investigate how internal lens elements in a high numerical aperture dry objective may be adjusted to correct for fluctuations in coverslip thickness.

The intermediate image in an infinity-corrected system appears at the reference focal length (formerly, the optical tube length) behind the tube lens in the optical pathway. This length varies between 160 and 250 millimeters, depending upon design constraints imposed by the manufacturer. The magnification of an infinity-corrected objective is calculated by dividing the reference focal length by the focal length of the objective lens.

In most biological and petrographic applications, a cover glass is utilized in mounting the specimen, both to protect the integrity of the specimen and to provide a clear window for observation. The cover glass acts to converge the light cones originating from each point in the specimen, but also introduces chromatic and spherical aberration (and consequent loss of contrast) that must be corrected by the objective. The degree to which light rays are converged is determined by the refractive index, dispersion, and thickness of the cover glass. Although the refractive index should be relatively constant within a batch of cover glasses, the thickness can vary between 0.13 and 0.22 millimeters. Another concern is the aqueous solvent or excess mounting medium that lies between the specimen and cover glass in wet or thickly mounted preparations. For example, in physiological saline whose refractive index is significantly different from that of the coverslip, the objective must focus through a layer of water only a few microns thick, leading to significant aberrations and a deviation of the point spread function that is no longer symmetrical above and below the focal plane. These factors add to the effective variations in refractive index and thickness of the coverslip and are very difficult for the microscopist to control.

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Microscope objectives are perhaps the most important components of an optical microscope because they are responsible for primary image formation and play a central role in determining the quality of images that the microscope is capable of producing. Objectives are also instrumental in determining the magnification of a particular specimen and the resolution under which fine specimen detail can be observed in the microscope.

Order of diffractionformula

Commercial surface-relief gratings are produced using an epoxy casting replication process developed in the mid-1900s. The process involves pouring a liquid into a mold, allowing the liquid to harden, and then removing the hardened material from the mold without damaging either. The replication process yields a grating that is an optically identical copy of the original. The two basic types of grating masters are ruled and interference.

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Light incident on a diffraction grating is dispersed away from the grating surface at an angle dependent on its wavelength, allowing a grating to be used to select a narrow spectral band from a much wider band. This ability of a grating is particularly useful for laser tuning, especially in the visible region of the spectrum. Two primary configurations for selecting a narrow wavelength are Littrow and Littman. In the Littrow configuration, the wavelength of interest diffracts at exactly the same angle as the light incident on the grating. Littrow tuning is done either with fine-pitch first-order gratings (typically 1800 or 2400 grooves/mm, either ruled or holographic) or a coarser grating used in higher orders. The alternative approach is to use the grating in a fixed grazing incidence mode together with a rotating reflecting mirror.

Fluorite objectives are produced from advanced glass formulations that contain materials such as fluorspar or newer synthetic substitutes. These new formulations allow for greatly improved correction of optical aberration. Similar to the achromats, the fluorite objectives are also corrected chromatically for red and blue light. In addition, the fluorites are also corrected spherically for two or three colors instead of a single color, as are achromats. The superior correction of fluorite objectives compared to achromats enables these objectives to be made with a higher numerical aperture, resulting in brighter images. Fluorite objectives also have better resolving power than achromats and provide a higher degree of contrast, making them better suited than achromats for color photomicrography in white light.

Firstorder diffractionFormula

Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

Grating applicationsLight incident on a diffraction grating is dispersed away from the grating surface at an angle dependent on its wavelength, allowing a grating to be used to select a narrow spectral band from a much wider band. This ability of a grating is particularly useful for laser tuning, especially in the visible region of the spectrum. Two primary configurations for selecting a narrow wavelength are Littrow and Littman. In the Littrow configuration, the wavelength of interest diffracts at exactly the same angle as the light incident on the grating. Littrow tuning is done either with fine-pitch first-order gratings (typically 1800 or 2400 grooves/mm, either ruled or holographic) or a coarser grating used in higher orders. The alternative approach is to use the grating in a fixed grazing incidence mode together with a rotating reflecting mirror. Pairs of diffraction gratings can also be used to compress or stretch a laser pulse. When a spectrally broad laser pulse is incident on a diffraction grating, the various wavelengths that make up the pulse will diffract from the grating at angles determined by those wavelengths. If the pulse is chirped so that the frequency changes linearly during the length of the pulse, then diffraction will spread the pulse out across the second grating. When the light diffracts from the second grating, which is oriented parallel to the first grating, the different parts of the pulse will diffract at angles that yield a pulse whose parts are synchronized. This increases the peak power while the total energy remains the same. Pulse compression uses two gratings with the same groove frequency and efficiencies peaked for the polarization and wavelength of the laser. If the gratings are arranged in a nonparallel arrangement, a pulse can be stretched. Pulse stretching uses two identical gratings, allowing lower peak power to be transmitted through the laser system and increasing the amount of stored energy that can be extracted. Since the invention of the replication technique, diffraction gratings have replaced prisms in many commercial spectrometers. A prism will bend short wavelengths more than longer ones (see Laser Focus World, Jan. 1997, p. 101). Prisms that transmit visible light absorb most UV and infrared wavelengths, whereas reflection gratings can be suitably coated for high reflectivity in wide spectral regions. Gratings are considered superior to prisms in many applications. Seeking to combine the best of both, Richardson Grating Laboratory has fabricated a "grism," a part-grating, part-prism optical element useful in spectrometers that require in-line presentation of the spectrum, as in astronomy. The light diffracted by the grating is bent back in line by the refracting effect of the prism. The dispersion of the grism is not linear, because the dispersive effects of the prism and grating are superimposed.New fabrication techniquesKaiser Optical Systems Inc. (KOSI; Ann Arbor, MI), has developed an alternative to the classical or surface-relief holographic grating--the volume transmission holographic grating (see photo at top of this page; also Laser Focus World, Oct. 1995, p. 95). The grating is created in the traditional manner by recording interference patterns generated by two mutually coherent laser beams. After the pattern is defined in the photosensitive material, coated on glass, and the film developed, a top layer of glass is added, creating a totally transparent grating assembly. Light strikes the grating on one side and diffracts out through the other.An advantage of a transmission volume grating is its relative insensitivity to angle, says James Arns of KOSI. A Bragg-type structure follows the classical grating equation concerning image position but with the added ability to adjust the intensity profile over a range of wavelengths. To describe the capability, Arns compares a Venetian blind to lines painted on a window. When the blind is positioned with the slats horizontal, it diffracts light in the same way as the painted lines or a surface-relief grating. When the slats are angled, the element of depth is added to how the light is diffracted. Because of this added dimension, the grating efficiency can be adjusted over the wavelength bandwidth to favor one side or the other. Also, the low sensitivity to incidence angle means the grating can be angularly tuned without influencing the image position."It also has a high efficiency," says Arns. "Depending on the configuration, the grating can produce 90% efficiency in the first order. If the thickness or the frequency of the grating is high enough, higher orders that otherwise might be propagated are extinguished." Another advantage, says Arns, is that the element can be handled and cleaned in the same fashion as a high-quality cemented lens because the grating is sandwiched between two layers of glass. Also, because the Bragg-type grating is a transmission device, optical elements and instruments can be brought close to it, resulting in a compact design.Holoplexing, a technique devised by KOSI in which two gratings are placed together in the same structure to cover multiple spectral ranges at one time, is useful for imaging on charge-coupled-device (CCD) cameras for broadband applications. Holographic transmission gratings are also used in Raman spectroscopy and for pulse compression in ultrafast lasers.Holographic gratings can also be made from computer-generated interference patterns. The patterns are written onto a chrome mask using an electron-beam machine. The patterns on the mask are then etched into a material, such as fused silica, using photolithographic masking and etching techniques. "Computer-generated gratings have really just reached maturity within the last two years," says Michael Feldman, of Digital Optics Corp. (Charlotte, NC). "They are very flexible and easy to mass-produce." Their versatility offers many advantages. "Ruled and holographic gratings are limited to relatively simple structures by the fabrication methods that are used," says W. Hudson Welch, also of Digital Optics. "The flexibility provided by computer-generated gratings allows the creation of essentially arbitrary grating patterns."Fiber gratingsFiber Bragg gratings, another recent development in grating applications, are made within a fiberoptic cable. Fiber gratings are fabricated by exposing the core of a single-mode fiber, 8 to 10 µm thick, to a periodic pattern of intense ultraviolet light. This pattern is created when a 248- or 193-nm laser passes through a special diffractive phase mask. When a fiber is placed in the intense UV light pattern of the mask, a permanent modulation of the index of refraction is generated in the fiber core. This photo-generated index modulation acts as a grating. Light traveling along the fiber core impinges on the grating, and each area of different refractive index scatters a small portion of the beam. If the wavelength of the signal is twice the distance between the periodic refractive elements (typically <1 µm), then the signals scattered back down the fiber core will add constructively to give a large reflection. The wavelength at which the reflection occurs is the Bragg wavelength. A Bragg grating can operate at precise wavelengths that can be accurately preset and maintained, says Keith Brundin at 3M Specialty Optical Fibers (West Haven, CT).There are also long-period fiber gratings that have index modulations with periods of hundreds of microns (see Laser Focus World, June 1996, p. 293). Instead of producing a reflected signal, these gratings create a phase-matching, or Bragg, condition that couples a forward-traveling signal into forward-traveling cladding modes. The signals coupled into the cladding are absorbed by the coating, creating a loss. Long-period gratings thus act as wavelength-selective absorption filters and are used in wavelength-division-multiplexing networks and in gain-shaping filters for rare-earth-doped fiber amplifiers. Fiber Bragg gratings have been commercially available only since 1995. They are becoming increasingly popular in telecommunications and the laser industry for such applications as external reflectors for stabilizing semiconductor lasers (see Fig. 4) and single- frequency fiber lasers.

An advantage of a transmission volume grating is its relative insensitivity to angle, says James Arns of KOSI. A Bragg-type structure follows the classical grating equation concerning image position but with the added ability to adjust the intensity profile over a range of wavelengths. To describe the capability, Arns compares a Venetian blind to lines painted on a window. When the blind is positioned with the slats horizontal, it diffracts light in the same way as the painted lines or a surface-relief grating. When the slats are angled, the element of depth is added to how the light is diffracted. Because of this added dimension, the grating efficiency can be adjusted over the wavelength bandwidth to favor one side or the other. Also, the low sensitivity to incidence angle means the grating can be angularly tuned without influencing the image position.

The advantages of oil immersion objectives are severely compromised if the wrong immersion fluid is utilized. Microscope manufacturers produce objectives with tight tolerances to refractive index and dispersion, which require matching values in the liquid placed between the cover glass and objective front lens. It is advisable to employ only the oil intended by the objective manufacturer, and to not mix immersion oils between manufacturers to avoid unpleasant artifacts such as crystallization or phase separation.

Objective numerical aperture can be dramatically increased by designing the objective to be used with an immersion medium, such as oil, glycerin, or water. By using an immersion medium with a refractive index similar to that of the glass coverslip, image degradation due to thickness variations of the cover glass are practically eliminated whereby rays of wide obliquity no longer undergo refraction and are more readily grasped by the objective. Typical immersion oils have a refractive index of 1.51 and a dispersion similar to that of glass coverslips. Light rays passing through the specimen encounter a homogeneous medium between the coverslip and immersion oil and are not refracted as they enter the lens, but only as they leave its upper surface. It follows that if the specimen is placed at the aplanatic point of the first objective lens, imaging by this portion of the lens system is totally free of spherical aberration.

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The imaging medium between the objective front lens and the specimen coverslip is also very important with respect to correction for spherical aberration and coma in the design of lens elements for objectives. Lower power objectives have relatively low numerical apertures and are designed to be used dry with only air as the imaging medium between the objective front lens and the cover glass. The maximum theoretical numerical aperture obtainable with air is 1.0, however in practice it is virtually impossible to produce a dry objective with a numerical aperture above 0.95. The effect of cover glass thickness variation is negligible for dry objectives having numerical apertures less than 0.4, but such deviation becomes significant at numerical apertures exceeding 0.65, where fluctuations as small as 0.01 millimeter can introduce spherical aberration. This poses problems with high-power apochromats, which must use very short working distances in air and contain sensitive corrections for spherical aberration that tend to make it difficult to obtain sharp images.

If the gratings are arranged in a nonparallel arrangement, a pulse can be stretched. Pulse stretching uses two identical gratings, allowing lower peak power to be transmitted through the laser system and increasing the amount of stored energy that can be extracted.

All three types of objectives suffer from pronounced field curvature and project images that are curved rather than flat, an artifact that increases in severity with higher magnification. To overcome this inherent condition arising from curved lens surfaces, optical designers have produced flat-field corrected objectives, which yield images that are in common focus throughout the viewfield. Objectives that have flat-field correction and low distortion are called plan achromats, plan fluorites, or plan apochromats, depending upon their degree of residual aberration. Such correction, although expensive, is quite valuable in digital imaging and conventional photomicrography.

Their versatility offers many advantages. "Ruled and holographic gratings are limited to relatively simple structures by the fabrication methods that are used," says W. Hudson Welch, also of Digital Optics. "The flexibility provided by computer-generated gratings allows the creation of essentially arbitrary grating patterns."

Secondorder diffractionformula

Joseph Fraunhofer first used diffraction gratings in 1819 to observe the spectrum of the sun. Earliest devices were multiple-slit assemblies, consisting of a grid of fine wire or thread wound about and extending between two parallel screws, which served as spacers. A wavefront that passed through the system was confronted by alternate opaque and transparent regions, so that it underwent a modulation in amplitude.

Holoplexing, a technique devised by KOSI in which two gratings are placed together in the same structure to cover multiple spectral ranges at one time, is useful for imaging on charge-coupled-device (CCD) cameras for broadband applications. Holographic transmission gratings are also used in Raman spectroscopy and for pulse compression in ultrafast lasers.

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The general design of a practical oil immersion objective includes a hemispherical front lens element, followed by a positive meniscus lens and a doublet lens group. Presented in Figure 6 are the aplanatic refractions that occur at the first two lens elements in a typical apochromatic oil immersion objective. The specimen is sandwiched between the microscope slide and cover glass at point P, the aplanatic point of the hemispherical lens element. Light rays refracted at the rear of the hemispherical lens appear to proceed from point P(1), which is also the center of curvature for the first surface of the meniscus lens. The refracted light rays enter the meniscus lens along the radius of its first surface and experience no refraction at that surface. At the rear surface of the meniscus lens, light rays are refracted aplanatically, so they appear to diverge from point P(2). Refraction of the light rays at the surfaces of subsequent lens groups in the objective complete the convergence of light rays originating from point P, thus forming the intermediate image.

There are also long-period fiber gratings that have index modulations with periods of hundreds of microns (see Laser Focus World, June 1996, p. 293). Instead of producing a reflected signal, these gratings create a phase-matching, or Bragg, condition that couples a forward-traveling signal into forward-traveling cladding modes. The signals coupled into the cladding are absorbed by the coating, creating a loss. Long-period gratings thus act as wavelength-selective absorption filters and are used in wavelength-division-multiplexing networks and in gain-shaping filters for rare-earth-doped fiber amplifiers.

The standard thickness for cover glasses is 0.17 millimeters, which is designated as a number 1½ cover glass. Unfortunately, not all 1½ cover glasses are manufactured to this close tolerance (they range from 0.16 to 0.19 millimeters) and many specimens have media between them and the cover glass. Compensation for cover glass thickness can be accomplished by adjusting the mechanical tube length of the microscope, or (as previously discussed) by the utilization of specialized correction collars that change the spacing between critical elements inside the objective barrel. The correction collar is utilized to adjust for these subtle differences to ensure the optimum objective performance. Proper utilization of objective lenses with correction collars demands that the microscopist is experienced and alert enough to reset the collar using appropriate image criteria. In most cases, focus may shift and the image may wander during adjustment of the correction collar. Use the steps listed below to make small incremental adjustments to an objective's correction collar while observing changes in the specimen image.

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Older objectives generally have lower numerical apertures, and are subject to an aberration termed chromatic difference of magnification that requires correction by the use of specially designed compensating oculars or eyepieces. This type of correction was prevalent during the reign of fixed tube length microscopes, but is not necessary with modern infinity-corrected objectives and microscopes. In recent years, modern microscope objectives have their correction for chromatic difference of magnification either built into the objectives themselves (Olympus and Nikon) or corrected in the tube lens (Leica and Zeiss).

pellicle* The living, proteinaceous, layered structure which surrounds the cells in many types of protozoa. It is immediately below the cell membrane [1] ...

Pairs of diffraction gratings can also be used to compress or stretch a laser pulse. When a spectrally broad laser pulse is incident on a diffraction grating, the various wavelengths that make up the pulse will diffract from the grating at angles determined by those wavelengths. If the pulse is chirped so that the frequency changes linearly during the length of the pulse, then diffraction will spread the pulse out across the second grating. When the light diffracts from the second grating, which is oriented parallel to the first grating, the different parts of the pulse will diffract at angles that yield a pulse whose parts are synchronized. This increases the peak power while the total energy remains the same. Pulse compression uses two gratings with the same groove frequency and efficiencies peaked for the polarization and wavelength of the laser.