For many years, objective lenses designed for biological applications from most manufacturers all conformed to an international standard of parfocal distance. Thus, a majority of objectives had a parfocal distance of 45.0 millimeters and were considered interchangeable. With the migration to infinity-corrected tube lengths, a new set of design criteria emerged to correct for aberrations in the objective and tube lenses. Coupled to an increased demand for greater flexibility to accommodate the need for ever-greater working distances with higher numerical apertures and field sizes, interchangeability between objective lenses from different manufacturers disappeared. This transition is exemplified by the modern Nikon CFI-60 optical system that features "Chrome Free" objectives, tube lenses, and eyepieces. Each component in the CFI-60 system is separately corrected without one being utilized to achieve correction for another. The tube length is set to infinity (parallel light path) using a tube lens, and the parfocal distance has been increased to 60 millimeters. Even the objective mounting thread size has been altered from 20.32 to 25 millimeters to meet new requirements of the optical system.

High powerobjective microscope function

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Stage clipsmicroscope function

Achromatic lenses are used to diminish chromatic and spherical aberrations which are the loss of color and focus that can happen when light wavelengths refract in direct light. These aberrations can be controlled by using an objective lens that contains both a convex and concave lens inside. Mounting these two different types of lenses to each other can bring wavelengths of red and blue light closer together, which puts them in the same focus and cancels out chromatic aberration. Another type of lens used to correct for both color and spherical aberration is the plan (or planar) lens. These produce a flatter field and can also give you a much larger working distance. However, they can be more expensive than achromatic lenses, so choosing between the two depends largely on how much power you need in your objective lens, and whether or not you need to adjust for field curvature, which only plan lenses can do. Achromatic lenses and plan lenses both come in dozens of magnifications and types, accommodating a wide variety of microscopy needs.

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Presented in Figure 1 is a cut-away diagram of a microscope objective being illuminated by a simple two-lens Abbe condenser. Light passing through the condenser is organized into a cone of illumination that emanates onto the specimen and is then transmitted into the objective front lens element as a reversed cone. The size and shape of the illumination cone is a function of the combined numerical apertures of the objective and condenser. The objective angular aperture is denoted by the Greek letter θ and will be discussed in detail below.

Specialized microscopes, such as metallurgical microscopes, require their own specific metallurgical objective lenses. These devices are most often used to examine structural detail of ceramics, metals and other non-living materials. Another common microscope objective accessory is a Barlow lens. These can be added to the bottom of an objective lens to either increase or decrease its working distance, field of view or magnification. Since they can be interchanged between lenses, they are a cost-effective way to change the power and magnification of lenses you already own. Lastly, if all these lenses are starting to seem overwhelming, remember one quick trick for determining magnification at a glance: look at the band of color near the bottom of your objective lens. While the magnification number is usually written right on the lens, you can also quickly determine its strength by the color ring. Red indicates 5x magnification, while yellow means 10x, light blue means 40x and white can mean 100-250x.

Infinity objective lenses did not become common until the 1980s but have since carved out a permanent spot in the microscope objective market. Previously, all microscopes had a standard tube length–the distance from the eyepiece to the objective lens was always 160 mm. Once microscope manufacturers began developing microscopes with varying tube lengths, lens manufacturers had to catch up with the changing technology. New tube lengths meant that microscopy equipment developers needed to adjust for these changes in their accessories, including objective lenses. Infinity optical systems use multiple sets of lenses within the lens house to correct a wide range of tube lengths–typically from 160-200 mm. This enables the lenses to be more versatile between microscopes of varying tube lengths.

Function ofcondenser inmicroscope

The last, but perhaps most important, factor in determining the resolution of an objective is the angular aperture, which has a practical upper limit of about 72 degrees (with a sine value of 0.95). When combined with refractive index, the product:

The focal length of a lens system is defined as the distance from the lens center to a point where parallel rays are focused on the optical axis (often termed the principal focal point). An imaginary plane perpendicular to the principal focal point is called the focal plane of the lens system. Every lens has two principal focal points for light entering each side, one in front and one at the rear. By convention, the objective focal plane that is nearer to the front lens element is known as the front focal plane and the focal plane located behind the objective is termed the rear focal plane (see Figure 4). The actual position of the rear focal plane varies with objective construction, but is generally situated somewhere inside the objective barrel for high magnification objectives. Objectives of lower magnification often have a rear focal plane that is exterior to the barrel, located in the thread area or within the microscope nosepiece.

The field diameter in an optical microscope is expressed by the field-of-view number or simply field number, which is the diameter of the viewfield expressed in millimeters and measured at the intermediate image plane. The field diameter in the object (specimen) plane becomes the field number divided by the magnification of the objective. Although the field number is often limited by the magnification and diameter of the ocular (eyepiece) field diaphragm, there is clearly a limit that is also imposed by the design of the objective. In early microscope objectives, the maximum usable field diameter was limited to about 18 millimeters (or considerably less for high magnification eyepieces), but modern plan apochromats and other specialized flat-field objectives often have a usable field that can range between 22 and 28 millimeters or more when combined with wide-field eyepieces. Unfortunately, the maximum useful field number is not generally engraved on the objective barrel and is also not commonly listed in microscope catalogs.

Obtaining high-contrast images of transparent specimens is difficult, especially when your specimen is alive and moving on a slide. Phase-contrast lenses allow you to observe microorganisms without having to fix and stain them. When your specimens are kept alive, a variety of biological functions can be examined and analyzed in real-time. Phase plates at the top of the objective lens diffract light, allowing these specialized lenses to tap into tiny changes in wavelength amplitude, which appears to the viewer as starker contrast on the slide. This makes the specimen much easier to view and observe.

Objective lensmagnification

Ocularlens microscope function

The axial range through which an objective can be focused without any appreciable change in image sharpness is referred to as the depth of field. This value varies radically from low to high numerical aperture objectives, usually decreasing with increasing numerical aperture (see Table 2 and Figure 2). At high numerical apertures, the depth of field is determined primarily by wave optics, while at lower numerical apertures, the geometrical optical "circle of confusion" dominates. The total depth of field is given by the sum of the wave and geometrical optical depths of field as:

The clearance distance between the closest surface of the cover glass and the objective front lens is termed the working distance. In situations where the specimen is designed to be imaged without a cover glass, the working distance is measured at the actual surface of the specimen. Generally, working distance decreases in a series of matched objectives as the magnification and numerical aperture increase (see Table 1). Objectives intended to view specimens with air as the imaging medium should have working distances as long as possible, provided that numerical aperture requirements are satisfied. Immersion objectives, on the other hand, should have shallower working distances in order to contain the immersion liquid between the front lens and the specimen. Many objectives designed with close working distances have a spring-loaded retraction stopper that allows the front lens assembly to be retracted by pushing it into the objective body and twisting to lock it into place. Such an accessory is convenient when the objective is rotated in the nosepiece so it will not drag immersion oil across the surface of a clean slide. Twisting the retraction stopper in the opposite direction releases the lens assembly for use. In some applications (see below), a long free working distance is indispensable, and special objectives are designed for such use despite the difficulty involved in achieving large numerical apertures and the necessary degree of optical correction.

Illustrated in Figure 3 is a schematic drawing of light waves reflecting and/or passing through a lens element coated with two antireflection layers. The incident wave strikes the first layer (Layer A in Figure 3) at an angle, resulting in part of the light being reflected (R(o)) and part being transmitted through the first layer. Upon encountering the second antireflection layer (Layer B), another portion of the light is reflected at the same angle and interferes with light reflected from the first layer. Some of the remaining light waves continue on to the glass surface where they are again both reflected and transmitted. Light reflected from the glass surface interferes (both constructively and destructively) with light reflected from the antireflection layers. The refractive indices of the antireflection layers vary from that of the glass and the surrounding medium (air). As the light waves pass through the antireflection layers and glass surface, a majority of the light (depending upon the incident angle--usual normal to the lens in optical microscopy) is ultimately transmitted through the glass and focused to form an image.

Just as the brightness of illumination in a microscope is governed by the square of the working numerical aperture of the condenser, the brightness of an image produced by the objective is determined by the square of its numerical aperture. In addition, objective magnification also plays a role in determining image brightness, which is inversely proportional to the square of the lateral magnification. The square of the numerical aperture/magnification ratio expresses the light-gathering power of the objective when utilized with transmitted illumination. Because high numerical aperture objectives are often better corrected for aberration, they also collect more light and produce a brighter, more corrected image that is highly resolved. It should be noted that image brightness decreases rapidly as the magnification increases. In cases where the light level is a limiting factor, choose an objective with the highest numerical aperture, but having the lowest magnification factor capable of producing adequate resolution.

Table 1 lists working distance and numerical aperture as a function of magnification for the four most common classes of objectives: achromats, plan achromats, plan fluorites, and plan apochromats. Note that dry objectives all have a numerical aperture value of less than 1.0 and only objectives designed for liquid immersion media have a numerical aperture that exceeds this value.

Function ofstage inmicroscope

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

where R is the separation distance, λ is the illumination wavelength, n is the imaging medium refractive index, and θ is one-half of the objective angular aperture. In examining the equation, it becomes apparent that resolution is directly proportional to the illumination wavelength. The human eye responds to the wavelength region between 400 and 700 nanometers, which represents the visible light spectrum that is utilized for a majority of microscope observations. Resolution is also dependent upon the refractive index of the imaging medium and the objective angular aperture. Objectives are designed to image specimens either with air or a medium of higher refractive index between the front lens and the specimen. The field of view is often quite limited, and the front lens element of the objective is placed close to the specimen with which it must lie in optical contact. A gain in resolution by a factor of approximately 1.5 is attained when immersion oil is substituted for air as the imaging medium.

The objective lens is the most important optical component of the microscope. It’s the part that sits in closest proximity to the specimen being examined, gathering light to produce optimal images for observation and analysis. This lens creates the first magnification by spreading out the light’s rays to make the object appear considerably larger by the time it meets your field of view at the other end of the eyepiece. Such a critical piece of equipment doesn’t come in a one-size-fits-all package. Below, we will discuss some of the different types of microscope objective lenses and the unique roles they play in microscopy.

Resolution for a diffraction-limited optical microscope can be described as the minimum detectable distance between two closely spaced specimen points:

Typesof objectivelenses

Magnesium fluoride is one of many materials utilized in thin-layer optical antireflection coatings, but most microscope manufacturers now produce their own proprietary formulations. The general result is a dramatic improvement in contrast and transmission of visible wavelengths with a concurrent destructive interference in harmonically-related frequencies lying outside the transmission band. These specialized coatings can be easily damaged by mis-handling and the microscopist should be aware of this vulnerability. Multilayer antireflection coatings have a slightly greenish tint, as opposed to the purplish tint of single-layer coatings, an observation that can be employed to distinguish between coatings. The surface layer of antireflection coatings used on internal lenses is often much softer than corresponding coatings designed to protect external lens surfaces. Great care should be taken when cleaning optical surfaces that have been coated with thin films, especially if the microscope has been disassembled and the internal lens elements are subject to scrutiny.

When a manufacturer's set of matched objectives, e.g. all achromatic objectives of various magnifications (a single subset of the objectives listed in Table 1), are mounted on the nosepiece, they are usually designed to project an image to approximately the same plane in the body tube. Thus, changing objectives by rotating the nosepiece usually requires only minimal use of the fine adjustment knob to re-establish sharp focus. Such a set of objectives is described as being parfocal, a useful convenience and safety feature. Matched sets of objectives are also designed to be parcentric, so that a specimen centered in the field of view for one objective remains centered when the nosepiece is rotated to bring another objective into use.

There is a wealth of information inscribed on the objective barrel. Briefly, each objective has inscribed on it the magnification (e.g. 10x, 20x or 40x etc.); the tube length for which the objective was designed to give its finest images (usually 160 millimeters or the Greek infinity symbol); and the thickness of cover glass protecting the specimen, which was assumed to have a constant value by the designer in correcting for spherical aberration (usually 0.17 millimeters). If the objective is designed to operate with a drop of oil between it and the specimen, the objective will be engraved OIL or OEL or HI (homogeneous immersion). In cases where these latter designations are not engraved on the objective, the objective is meant to be used dry, with air between the lowest part of the objective and the specimen. Objectives also always carry the engraving for the numerical aperture (NA) value. This may vary from 0.04 for low power objectives to 1.3 or 1.4 for high power oil-immersion apochromatic objectives. If the objective carries no designation of higher correction, one can usually assume it is an achromatic objective. More highly corrected objectives have inscriptions such as apochromat or apo, plan, FL, fluor, etc. Older objectives often have the focal length (lens-to-image distance) engraved on the barrel, which is a measure of the magnification. In modern microscopes, the objective is designed for a particular optical tube length, so including both the focal length and magnification on the barrel becomes somewhat redundant.

One of the most significant advances in objective design during recent years is the improvement in antireflection coating technology, which helps to reduce unwanted reflections that occur when light passes through a lens system. Each uncoated air-glass interface can reflect between four and five percent of an incident light beam normal to the surface, resulting in a transmission value of 95-96 percent at normal incidence. Application of a quarter-wavelength thick antireflection coating having the appropriate refractive index can increase this value by three to four percent. Nikon's more recent CFI Plan Apochromat Lambda Series of objective lenses utilize their proprietary Nano Crystal Coat technology, which consists of several layers of ultra-low refractive index nano-sized crystals. As objectives become more sophisticated with an ever-increasing number of lens elements, the need to eliminate internal reflections grows correspondingly. Some modern objective lenses with a high degree of correction can contain as many as 15 lens elements having many air-glass interfaces. If the lenses were uncoated, the reflection losses of axial rays alone would drop transmittance values to around 50 percent. The single-layer lens coatings once utilized to reduce glare and improve transmission have now been supplanted by multilayer coatings that produce transmission values exceeding 99.9 percent in the visible spectral range.

is known as the numerical aperture (abbreviated NA), and provides a convenient indicator of the resolution for any particular objective. Numerical aperture is generally the most important design criteria (other than magnification) to consider when selecting a microscope objective. Values range from 0.1 for very low magnification objectives (1x to 4x) to as much as 1.6 for high-performance objectives utilizing specialized immersion oils. As numerical aperture values increase for a series of objectives of the same magnification, we generally observe a greater light-gathering ability and increase in resolution. The microscopist should carefully choose the numerical aperture of an objective to match the magnification produced in the final image. Under the best circumstances, detail that is just resolved should be enlarged sufficiently to be viewed with comfort, but not to the point that empty magnification hampers observation of fine specimen detail.

During assembly of the objective, lenses are first strategically spaced and lap-seated into cell mounts, then packaged into a central sleeve cylinder that is mounted internally within the objective barrel. Individual lenses are seated against a brass shoulder mount with the lens spinning in a precise lathe chuck, followed by burnishing with a thin rim of metal that locks the lens (or lens group) into place. Spherical aberration is corrected by selecting the optimum set of spacers to fit between the lower two lens mounts (the hemispherical and meniscus lens). The objective is parfocalized by translating the entire lens cluster upward or downward within the sleeve with locking nuts so that objectives housed on a multiple nosepiece can be interchanged without losing focus. Adjustment for coma is accomplished with three centering screws that can optimize the position of internal lens groups with respect to the optical axis of the objective.

What isobjective lensinmicroscope

where λ is the wavelength of illumination, n is the refractive index of the imaging medium, NA is the objective numerical aperture, M is the objective lateral magnification, and e is the smallest distance that can be resolved by a detector that is placed in the image plane of the objective. Notice that the diffraction-limited depth of field (the first term on the right-hand side of the equation) shrinks inversely with the square of the numerical aperture, while the lateral limit of resolution is reduced with the first power of the numerical aperture. The result is that axial resolution and the thickness of optical sections are affected by the system numerical aperture much more than is the lateral resolution of the microscope (see Table 2).

There are hundreds of unique objective lenses to choose from, but once you have a greater understanding of the most common types, you can make a more informed decision regarding which lens is right for you. Whether you are a hobbyist or whether you require the use of a microscope in your day-to-day research, it’s important to gain an understanding of the strengths and weaknesses across the spectrum of objective lenses. Once you know exactly what you’re looking for, you’ll be well on your way to obtaining the best results and having an optimal viewing experience.

Three critical design characteristics of the objective set the ultimate resolution limit of the microscope. These include the wavelength of light used to illuminate the specimen, the angular aperture of the light cone captured by the objective, and the refractive index in the object space between the objective front lens and the specimen.

As light rays pass through an objective, they are restricted by the rear aperture or exit pupil of the objective, as illustrated in Figure 4. The diameter of this aperture varies between 12 millimeters for low magnification objectives down to around 5 millimeters for the highest power apochromatic objectives. Aperture size is extremely critical for epi-illumination applications that rely on the objective to act as both an imaging system and condenser, where the exit pupil also becomes an entrance pupil. The image of the light source must completely fill the objective rear aperture to produce even illumination across the viewfield. If the light source image is smaller than the aperture, the viewfield will experience vignetting from uneven illumination. On the other hand, if the light source image is larger than the rear aperture, some light does not enter the objective and the intensity of illumination is reduced.