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Objective lenses are roughly classified basically according to the intended purpose, microscopy method, magnification, and performance (aberration correction). Classification according to the concept of aberration correction among those items is a characteristic way of classification of microscope objectives.

A variety of microscopy methods have been developed for optical microscopes according to intended purposes. The dedicated objective lenses to each microscopy method have been developed and are classified according to such a method. For example, "reflected darkfield objective (a circular-zone light path is applied to the periphery of an inner lens)", "Differential Interference Contrast (DIC) objective (the combination of optical properties with a DIC( Nomarski）prism is optimized by reducing lens distortions)", "fluorescence objective (the transmittance in the near-ultraviolet region is improved)", "polarization objective (lens distortions are drastically reduced)", and "phase difference objective (a phase plate is built in) are available.

High powerobjective microscopefunction

Geoffrey Rolls is a Histology Consultant with decades of experience in the field. He is a former Senior Lecturer in histopathology in the Department of Laboratory Medicine, RMIT University in Melbourne, Australia.

Of these options, only whole-mounts and sections preserve the structural relationships between individual cells and extracellular components. Smears and squash preparations provide detail about individual cells and relative cell numbers, but structural relationships are lost. The preparation of sections is the most technically complicated of these methods as it requires specialized equipment and considerable expertise. The microscopic examination of sections by a pathologist forms the cornerstone of cancer diagnosis. Although the methodology for preparing sections from both animal and plant material is similar, the following description relates to animal (human) tissues.

The purposes of optical microscopes are broadly classified into two; "biological-use" and "industrial-use". Using this classification method, objective lenses are classified into "biological-use" objectives and "industrial-use" objectives. A common specimen in a biological use is fixed in place on the slide glass, sealing it with the cover glass from top. Since a biological-use objective lens is used for observation through this cover glass, optical design is performed in consideration of the cover glass thickness (commonly 0.17mm). Meanwhile, in an industrial use a specimen such as a metallography specimen, semiconductor wafer, and an electronic component is usually observed with nothing covered on it. An industrial-use objective lens is optically designed so as to be optimal for observation without any cover glass between the lens end and a specimen.

What are the3objectivelenseson a microscope

Specimens received for histological examination may come from a number of different sources. They range from very large specimens or whole organs to tiny fragments of tissue. For example, the following are some of the specimen types commonly received in a histopathology lab.

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2. Alternatively, specimens can be infiltrated with a liquid agent that can subsequently be converted into a solid that has appropriate physical properties that will allow thin sections to be cut from it. Various agents can be used for infiltrating and supporting specimens, including epoxy and methacrylate resins, but paraffin wax-based histological waxes are the most popular for routine light microscopy. This produces so-called “paraffin sections”. These sections are usually prepared with a “rotary” microtome. “Rotary” describes the cutting action of the instrument. In all histopathology laboratories, paraffin sections are routinely prepared from almost every specimen and used in diagnosis.

After processing, the specimens are placed in an embedding center where they are removed from their cassettes and placed in wax-filled molds. At this stage, specimens are carefully orientated because this will determine the plane through which the section will be cut and ultimately may decide whether an abnormal area will be visible under the microscope. The cassette in which the tissue has been processed carries the specimen identification details, and it is now placed on top of the mold and is attached by adding further wax. The specimen “block” is now allowed to solidify on a cold surface, and when set, the mold is removed. The cassette, now filled with wax and forming part of the block, provides a stable base for clamping in the microtome. The block containing the specimen is now ready for section cutting.

Axial chromatic aberration correction is divided into three levels of achromat, semiapochromat (fluorite), and apochromat according to the degree of correction. The objective lineup is divided into the popular class to high class with a gradual difference in price. An objective lens for which axial chromatic aberration correction for two colors of C ray (red: 656,3nm) and F ray (blue: 486.1nm) has been made is known as Achromat or achromatic objective. In the case of Achromat, a ray except for the above two colors (generally violet g-ray: 435.8nm) comes into focus on a plane away from the focal plane. This g ray is called a secondary spectrum. An objective lens for which chromatic aberration up to this secondary spectrum has satisfactorily been corrected is known as Apochromat or apochromatic objective. In other words, Apochromat is an objective for which the axial chromatic aberration of three colors (C, F, and g rays) has been corrected. The following figure shows the difference in chromatic aberration correction between Achromat and Apochromat by using the wavefront aberration. This figure proves that Apochromat is corrected for chromatic aberration in wider wavelength range than Achromat is.

Objective lens microscopefunction

Most fresh tissue is very delicate, easily distorted, and damaged. Thus, it is impossible to prepare thin sections (slices) from it unless it is supported in some way whilst it is being cut. Usually, the specimen also needs to be preserved or “fixed” before sections are prepared. Broadly, there are two strategies that can be employed to provide this support.

High powerobjective lens

There are many reasons to examine human cells and tissues under the microscope. Medical and biological research is underpinned by knowledge of the normal structure and function of cells and tissues and the organs and structures that they make up. In the normal healthy state, the cells and other tissue elements are arranged in regular, recognizable patterns. Changes induced by a wide range of chemical and physical influences are reflected by alterations in the structure at a microscopic level, and many diseases are characterized by typical structural and chemical abnormalities that differ from the normal state. Identifying these changes and linking them to particular diseases is the basis of histopathology and cytopathology, important specializations of modern medicine. Microscopy plays an important part in haematology (the study of blood), microbiology (the study of microorganisms including parasites and viruses), and more broadly in the areas of biology, zoology, and botany. In all these disciplines, specimens are examined under a microscope.

Objective lensmagnification

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.

Objective lensfunction

An optical microscope is used with multiple objectives attached to a part called revolving nosepiece. Commonly, multiple combined objectives with a different magnification are attached to this revolving nosepiece so as to smoothly change magnification from low to high only by revolving the nosepiece. Consequently, a common combination lineup is comprised from among objectives of low magnification (5x, 10x), intermediate magnification (20x, 50x), and high magnification (100x). To obtain a high resolving power particularly at high magnification among these objectives, an immersion objective for observation with a dedicated liquid with a high refractive index such as immersion oil or water charged between the lens end and a specimen is available. Ultra low magnification (1.25x, 2.5x) and ultra high magnification (150x) objectives are also available for the special use.

Where large batches of specimens are processed for paraffin section preparation, automated instruments called “tissue processors” are used. These instruments allow the specimens to be infiltrated with a sequence of different solvents finishing in molten paraffin wax. The specimens are in an aqueous environment to start with (water-based) and must be passed through multiple changes of dehydrating and clearing solvents (typically ethanol and xylene) before they can be placed in molten wax (which is hydrophobic and immiscible with water). The duration and step details of the “processing schedule” chosen for a particular batch of specimens will depend on the nature and size of the specimens. Schedules can be as short as one hour for small specimens or as long as twelve hours or more for large specimens. In many labs, the bulk of processing is carried out overnight. At present, there is considerable pressure on laboratories to use processors capable of rapid processing in an effort to improve workflow and reduce turnaround times.

Fixation is a crucial step in preparing specimens for microscopic examination. Its objective is to prevent decay and preserve cells and tissues in a “life-like” state. It does this by stopping enzyme activity, killing microorganisms, and hardening the specimen while maintaining sufficient molecular structure to enable appropriate staining methods to be applied (including those involving antigen-antibody reactions and those depending on preserving DNA and RNA). The sooner fixation is initiated following the separation of a specimen from its blood supply, the better the result will be. The most popular fixing agent is formaldehyde, usually in the form of a phosphate-buffered solution (often referred to as “formalin”). Ideally, specimens should be fixed by immersion in formalin for six to twelve hours before they are processed.

1. The tissue can be rapidly frozen and kept frozen while sections are cut using a cryostat microtome (a microtome in a freezing chamber). These are called “frozen sections”. Frozen sections can be prepared very quickly and are therefore used when an intra-operative diagnosis is required to guide a surgical procedure or where any type of interference with the chemical makeup of the cells is to be avoided (as in some histochemical investigations).

The following paragraphs describe the major steps in preparing paraffin sections. These steps generally dictate the layout and workflow in large, specialist histopathology laboratories where hundreds of specimens are handled every day.

Types ofobjectivelenses

Photography or image pickup with a video camera has been common in microscopy and thus a clear, sharp image over the entire field of view is increasingly required. Consequently, Plan objective lenses corrected satisfactorily for field curvature aberration are being used as the mainstream. To correct for field curvature aberration, optical design is performed so that Petzval sum becomes 0. However, this aberration correction is more difficult especially for higher-magnification objectives. (This correction is difficult to be compatible with other aberration corrections) An objective lens in which such correction is made features in general powerful concave optical components in the front-end lens group and powerful concave ones in the back-end group.

Meanwhile, an objective lens for which the degree of chromatic aberration correction to the secondary spectrum (g ray) is set to medium between Achromat and Apochromat is known as Semiapochromat (or Flulorite).

Apart from a few natural pigments such as melanin, the cells and other elements making up most specimens are colorless. In order to reveal structural detail using brightfield microscopy, some form of staining is required. The routine stain used universally as a starting point in providing essential structural information is the hematoxylin and eosin (H&E) stain. With this method, cell nuclei are stained blue, and cytoplasm and many extra-cellular components in shades of pink. In histopathology, many conditions can be diagnosed by examining an H&E alone. However, sometimes additional information is required to provide a full differential diagnosis, and this requires furthermore specialized staining techniques. These may be “special stains” using dyes or metallic impregnations to define particular structures or microorganisms, or immuno-histochemical methods (IHC) involving the location of diagnostically useful proteins using labeled antibodies. Molecular methods such as in-situ hybridisation (ISH) may also be required to detect specific DNA or RNA sequences. 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.

Specimens are usually received in fixative (preservative) but sometimes arrive fresh and must be immediately fixed. Before specimens are accepted by a laboratory, the identification (labeling) and accompanying documentation will be carefully checked, all details recorded, and “specimen tracking” commenced. It is vital that patient or research specimens are properly identified, and the risk of inaccuracies minimized.

Grossing, often referred to as “cut-up”, involves a careful examination and description of the specimen that will include the appearance, the number of pieces, and their dimensions. Larger specimens may require further dissection to produce representative pieces from appropriate areas. For example, multiple samples may be taken from the excision margins of a tumour to ensure that the tumour has been completely removed. In the case of small specimens, the entire specimen may be processed. The tissues selected for processing will be placed in cassettes (small perforated baskets), and batches will be loaded onto a tissue processor for processing through to wax.

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What are the objective lens on a microscopeexplain

Sections are now “floated out” on the surface of warm water in a flotation bath to flatten them and then picked up onto microscope slides. After thorough drying, they are ready for staining.

In the optical design of microscope objectives, commonly the larger is an N.A. and the higher is a magnification, the more difficult to correct the axial chromatic aberration of a secondary spectrum. In addition to axis chromatic aberration, various aberrations and sine condition must be sufficiently corrected and therefore the correction of the secondary spectrum is far more difficult to be implemented. As the result, a higher-magnification apochromatic objective requires more pieces of lenses for aberration correction. Some objectives consist of more than 15 pieces of lenses. To correct the secondary spectrum satisfactorily, it is effective to use "anomalous dispersion glass" with less chromatic dispersion up to the secondary spectrum for the powerful convex lens among constituting lenses. The typical material of this anomalous dispersion glass is fluorite (CaF2) and has been adopted for apochromatic objectives since a long time ago, irrespective of imperfection in workability. Recently, optical glass with a property very close to the anomalous dispersion of fluorite has been developed and is being used as the mainstream in place of fluorite.

Sections are cut on a precision instrument called a “microtome” using extremely fine steel blades. Paraffin sections are usually cut at a thickness of 3 - 5µm, ensuring that only a single layer of cells makes up the section (a red blood cell has a diameter of about 7µm). One of the advantages of paraffin wax as an embedding agent is that as sections are cut, they will stick together edge-to-edge, forming a “ribbon” of sections. This makes handling easier.

An objective lens is the most important optical unit that determines the basic performance/function of an optical microscope To provide an optical performance/function optimal for various needs and applications (i.e. the most important performance/function for an optical microscope), a wide variety of objective lenses are available according to the purpose.

There are many different forms of microscopy, but the one most commonly employed is “brightfield” microscopy where the specimen is illuminated with a beam of light that passes through it (as opposed to a beam of electrons as in electron microscopy). The general requirements for a specimen to be successfully examined using brightfield microscopy are: