7. Once you have brought the specimen into sharp focus with a low-powered lens, preparation may be made for visualizing the spec-imen under oil immersion. Place a drop of oil on the slide directly over the area to be viewed. Rotate the nosepiece until the oil-immersion objective locks into position. Care should be taken not to allow the high-power objective to touch the drop of oil.The slide is observed from the side as the objective is rotated slowly into position. This will ensure that the objective will be properly immersed in the oil. The fine-adjustment knob is readjusted to bring the image into sharp focus.

Above the stage and attached to the arm of the microscope is the body tube. This structure houses the lens system that magnifies the specimen. The upper end of the tube contains the ocular or eyepiece lens. The lower portion consists of a movable nosepiece containing the objective lenses. Rotation of the nosepiece posi-tions objectives above the stage opening. The body tube may be raised or lowered with the aid of coarse-adjustment and fine-adjustment knobs that are located above or below the stage, depending on the type and make of the instrument.

You will be responsible for the proper care and use of microscopes. Since microscopes are expensive, you must observe the following regu-lations and procedures.

2. Rotate the scanning lens or the low power lens into position. While watching from the side to insure that the lens doesn't touch the specimen, turn the coarse focus knob to move the stage as close as it can get to the lens without touching the lens. (Always watch from the side whenever you move a specimen towards any objective lens to make sure the lens doesn't crash through the specimen and get damaged!)

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Between the light source and the condenser is the iris diaphragm, which can be opened and closed by means of a lever; thereby regulating the amount of light entering the condenser. Excessive illumination may actually obscure the specimen because of lack of contrast. The amount of light entering the microscope differs with each objec-tive lens used. A rule of thumb is that as the mag-nification of the lens increases, the distance between the objective lens and slide, called working distance, decreases, whereas the numerical aperture of the objective lens increases.

Due to Fresnel reflection, as light passes from air through an uncoated glass substrate approximately 4% of the light will be reflected at each interface. This results in a total transmission of only 92% of the incident light, which can be extremely detrimental in many applications (Figure 1). Excess reflected light reduces throughput and can lead to laser-induced damage in laser applications. Anti-reflection (AR) coatings are applied to optical surfaces to increase the throughput of a system and reduce hazards caused by reflections that travel backwards through the system and create ghost images. Back reflections also destabilize laser systems by allowing unwanted light to enter the laser cavity. AR coatings are especially important for systems containing multiple transmitting optical elements. Many low-light systems incorporate AR coated optics to allow for efficient use of light.

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Observation of microorganisms in an unstained state is possible with this microscope. Its optics include special objectives and a condenser that make visible cellular components that differ only slightly in their refractive indexes. As light is transmitted through a specimen with a refractive index different from that of the surrounding medium, a portion of the light is refracted (bent) due to slight varia-tions in density and thickness of the cellular components. The special optics convert the difference between transmitted light and refracted rays, resulting in a significant vari-ation in the intensity of light and thereby producing a discernible image of the struc-ture under study. The image appears dark against a light background.

5. Routinely adjust the light source by means of the light source transformer setting, and/or the iris diaphragm, for optimum illumination for each new slide and for each change in magnification.

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Effective illumination is required for efficient magnification and resolving power. Since the intensity of daylight is an uncontrolled variable, artificial light from a tungsten lamp is the most commonly used light source in microscopy. The light is passed through the con-denser located beneath the stage. The condenser contains two lenses that are necessary to produce a maximum numerical aperture. The height of the condenser can be adjusted with the con-denser knob. Always keep the condenser close to the stage, especially when using the oil-immersion objective.

(This passage was adapted from Microbiology: A Laboratory Manual, 5th edition, Cappuccino, J.S. and Sherman, N., Benjamin/Cummings Science Publishing.)

A fixed platform with an opening in the center allows for the passage of light from an illu-minating source below to the lens system above the stage. This platform provides a surface for the placement of a slide with its specimen over the central opening. In addition to the fixed stage, most microscopes have a mechanical stage that can be moved vertically or horizontally by means of adjustment controls. Less sophisticated micro-scopes have clips on the fixed stage, and the slide must be positioned manually over the central opening.

The most commonly used microscopes are equipped with a revolving nosepiece containing four objective lenses possessing different degrees of magnification. When these are combined with the magnification of the ocular lens, the total or overall linear magnification of the specimen is obtained.

Table 1 shows the reflectivity and guaranteed laser-induced damage threshold (LIDT) for Edmund Optics’ standard laser V-coats.

Edmund Optics offers all TECHSPEC® transmissive optics with a variety of anti-reflection (AR) coating options that vastly improve the efficiency of the optic by increasing transmission, enhancing contrast, and eliminating ghost images. Most AR coatings are also very durable, with resistance to both physical and environmental damage. For these reasons, the vast majority of transmissive optics include some form of anti-reflection coating. When specifying an AR coating to suit your specific application, you must first be fully aware of the full spectral range of your system. While an AR coating can significantly improve the performance of an optical system, using the coating at wavelengths outside the design wavelength range could potentially decrease the performance of the system.

8. During microscopic examination of microbial organisms, it is always necessary to observe several areas of the preparation. This is accomplished by scanning the slide with-out the application of additional immersion oil. This will require continuous, very fine adjustments by the slow, back-and-forth rotation of the fine adjustment knob only.

An external light source, such as a lamp, is placed in front of the mirror to direct the light upward into the lens system. The flat side of the mirror is used for artificial light, and the concave side for sunlight.

This component is found directly under the stage and contains two sets of lenses that collect and concentrate light passing upward from the light source into the lens sys-tems. The condenser is equipped with an iris diaphragm, a shutter controlled by a lever that is used to regulate the amount of light entering the lens system.

This microscope is used most frequently to visualize speci-mens that are chemically tagged with a fluorescent dye. The source of illumination is an ultraviolet (UV) light obtained from a high-pressure mercury lamp or hydrogen quartz lamp. The ocular lens is fitted with a filter that permits the longer ultraviolet wavelengths to pass, while the shorter wavelengths are blocked or eliminated. Ultraviolet radiations are absorbed by the fluorescent label and the energy is re-emitted in the form of a different wavelength in the visible light range. The fluorescent dyes absorb at wavelengths between 230 and 350 nanometers (nm) and emit orange, yellow, or greenish light. This microscope is used primarily for the detection of antigen-antibody reactions. Antibodies are conjugated with a fluorescent dye that becomes excited in the presence of ultraviolet light, and the fluorescent portion of the dye becomes visible against a black background.

Broadband anti-reflection (BBAR) coatings are designed to improve transmission over a much wider waveband. They are commonly used with broad-spectrum light sources and lasers with multiple-harmonic generation. BBAR coatings typically do not achieve reflectivity values quite as low as V-coats but are more versatile because of their wider transmission band. In addition to being applied to transmissive optical components including lenses and windows, AR coatings are also used on laser crystals and nonlinear crystals to minimize reflections, as Fresnel reflections occur where air and the crystal meet.1

VIS-NIR: Our visible/near-infrared broadband anti-reflection coating is specially optimized to yield maximum transmission (>99%) in the near-infrared.

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This instrument provides a revolutionary method of microscopy, with magnifications up to one million. This permits visualization of submicroscopic cel-lular particles as well as viral agents. In the electron microscope, the specimen is illu-minated by a beam of electrons rather than light, and the focusing is carried out by elec-tromagnets instead of a set of optics. These components are sealed in a tube in which a complete vacuum is established. Transmission electron microscopes require speci-mens that are thinly prepared, fixed, and dehydrated for the electron beam to pass freely through them. As the electrons pass through the specimen, images are formed by direct-ing the electrons onto photographic film, thus making internal cellular structures visi-ble. Scanning electron microscopes are used for visualizing surface characteristics rather than intracellular structures A narrow beam of electrons scans back and forth, producing a three-dimensional image as the electrons are reflected off the specimen's surface.

UV-AR and UV-VIS: Ultraviolet coatings are applied to our UV fused silica lenses and UV fused silica windows to increase their coating performance in the ultraviolet region.

This instrument contains two lens systems for magnifying specimens: the ocular lens in the eyepiece and the objective lens located in the nose-piece. The specimen is illuminated by a beam of tungsten light focused on it by a sub-stage lens called a condenser, and the result is that the specimen appears dark against a bright background. A major limitation of this system is the absence of contrast between the specimen and the surrounding medium, which makes it difficult to observe living cells. Therefore, most brightfield observations are performed on nonviable, stained preparations.

NIR I and NIR II: Our near-infrared I and near-infrared II broadband AR coatings offer exceptional performance in near-infrared wavelengths of common fiber optics, laser diode modules, and LED lights.

Telecom-NIR: Our telecom/near-infrared is a specialized broadband AR coating for popular telecommunications wavelengths from 1200 – 1600nm.

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Microbiology, the branch of science that has so vastly extended and expanded our knowledge of the living world, owes its existence to Antony van Leeuwenhoek. In 1673, with the aid of a crude microscope consisting of a biconcave lens enclosed in two metal plates, Leeuwenhoek introduced the world to the existence of microbial forms of life. Over the years, microscopes have evolved from the simple, single-lens instrument of Leeuwenhoek, with a magnification of 300, to the present-day electron microscopes capable of magnifications greater than 250,000. Microscopes are designated as either light microscopes or electron microscopes. The former use visible light or ultraviolet rays to illuminate specimens. They include brightfield, darkfield, phase-contrast, and fluorescent instruments. Fluorescent micro-scopes use ultraviolet radiations whose wavelengths are shorter than those of visible light and are not directly perceptible to the human eye. Electron microscopes use elec-tron beams instead of light rays, and magnets instead of lenses to observe submicro-scopic particles.

Microbiology is a science that studies living organisms that are too small to be seen with the naked eye. Needless to say, such a study must involve the use of a good compound microscope. Although there are many types and variations, they all fundamentally consist of a two-lens system, a variable but controllable light source, and mechanical adjustable parts for determining focal length between the lenses and specimen.

$\tfrac{\lambda}{4}$ MgF2: The simplest AR coating used is $ \tfrac{\lambda}{4} $ MgF2 centered at 550nm (with an index of refraction of 1.38 at 550nm). MgF2 coating is ideal for broadband use though it gives varied results depending upon the glass type involved.

AR coatings are designed so that the relative phase shift between the beam reflected at the upper and lower boundaries of a thin film is 180°. Destructive interference between the two reflected beams occurs, which cancels out both beams before they exit the surface (Figure 2). The optical thickness of the optical coating must be an odd integer multiple of $\tfrac{\lambda}{4}$, where $ \small{\lambda} $ is the design wavelength or wavelength being optimized for peak performance in order to achieve the desired path difference of $\tfrac{\lambda}{2}$ between the reflected beams. When achieved, this will lead to the cancellation of the beams. The index of refraction of a thin film $ \small{\left( n_f \right)} $ needed for complete cancelation of the reflected beams can be found by using the refractive indices of the incident medium $ \small{\left( n_0 \right)} $ and the substrate $ \small{\left( n_s \right)} $.

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Although magnification is important, you must be aware that unlimited enlargement is not possible by merely increasing the magnifying power of the lenses or by using additional lenses, because lenses are limited by a property called resolving power. By definition, resolving power is the ability of a lens to show two adjacent objects as discrete entities. When a lens cannot discriminate, that is, when the two objects appear as one, it has lost resolu-tion. Increased magnification will not rectify the loss, and will, in fact, blur the object. The resolv-ing power of a lens is dependent on the wave-length of light used and the numerical aperture, which is a characteristic of each lens and imprinted on each objective. The numerical aper-ture is defined as a function of the diameter of the objective lens in relation to its focal length. It is doubled by use of the substage condenser; which illuminates the object with rays of light that pass through the specimen obliquely as well as directly. Thus, resolving power is expressed mathematically, as follows:

However; as with magnification, resolving power also has limits. You might rationalize that merely decreasing the wavelength will automati-cally increase the resolving power of a lens. Such is not the case, because the visible portion of the electromagnetic spectrum is very narrow and borders on the very short wavelengths found in the ultraviolet portion of the spectrum.

On completion of the laboratory exercise, return the microscope to its cabinet in its original condition. The following steps are recommended:

The light source is positioned in the base of the instrument. Some microscopes are equipped with a built-in light source to pro-vide direct illumination. Others are provided with a mirror; one side flat and the other concave.

Because reflectivity increases rapidly as the wavelength of the source moves further away from the DWL, optical components with V-coats are meant for use at exactly or very close to the intended DWL of the coating. An interesting characteristic of V-coats is that the shape of their transmission curves is semi-periodic such that the reflectivity reaches a local minimum at harmonics of the DWL (e.g. $ \tfrac{\lambda_0}{2} $ or $ \tfrac{\lambda_0}{4} $) that are not as optimized for reflectivity as at the DWL. V-coats are usually comprised of only two coating layers. Simple V-coats can consist of a single layer with a thickness of a $ \tfrac{\lambda}{4} $, but more layers may be required to adjust the bandwidth or if a coating material with an appropriate index of refraction is not available. Multilayer coatings may also compensate for different angles of incidence, but are more complicated and tend to have larger bandwidths. If the thickness of the V-coat layers is incorrect, the reflectivity of the coating increases and the DWL changes. V-coats from Edmund Optics typically achieve minimum reflectivities significantly less than 0.25%, but all standard V-coats have specified reflectivities of <0.25% at the DWL. This allows for small shifts in the DWL from coating tolerances.

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While scientists have a variety of optical instruments with which to perform routine laboratory procedures and sophisticated research, the compound brightfield micro-scope is the "workhorse" and is commonly found in all biological laboratories. Although you should be familiar with the basic principles of microscopy, you probably have not been exposed to this diverse array of complex and expensive equipment. Therefore, only the compound brightfield microscope will be discussed in depth and used to examine specimens.

The relationship between wavelength and numerical aperture is valid only for increased resolving power when light rays are parallel. Therefore, the resolving power is dependent on another factor, the refractive index. This is the bending power of light passing through air from the glass slide to the objective lens. The refractive index of air is lower than that of glass, and as light rays pass from the glass slide into the air, they are bent or refracted so that they do not pass into the objective lens. This would cause a loss of light, which would reduce the numerical aperture and diminish the resolving power of the objective lens. Loss of refracted light can be compensated for by interposing mineral oil, which has the same refractive index as glass, between the slide and the objective lens. In this way, decreased light refraction occurs and more light rays enter directly into the objective lens, producing a vivid image with high resolution.

4. If this is the first specimen of the day, you should Kohler your microscope at this point (while it is in focus). Otherwise, if your microscope has already been Kohlered you won't need to do it again

Edmund Optics offers all TECHSPEC® lenses with an optional single-layer, dielectric anti-reflection (AR) coating to reduce surface reflections. In addition, custom single-layer, multi-layer, V, and 2V coatings are available for both our off-the-shelf and large volume custom orders. View Custom Optical Lens Coatings for information.

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This is similar to the ordinary light microscope; however, the condenser system is modified so that the specimen is not illuminated directly. The con-denser directs the light obliquely so that the light is deflected or scattered from the spec-imen, which then appears bright against a dark background. Living specimens may be observed more readily with darkfield than with brightfield microscopy.

VIS 0° and VIS 45°: VIS 0° (for 0° angle of incidence) and VIS 45° (for 45° angle of incidence) provide optimized transmission for 425 – 675nm, reducing average reflection to 0.4% and 0.75% respectively. VIS 0° AR coating is preferred over MgF2 for visible applications.

The instruments are housed in special cabinets and must be moved by users to their laboratory benches. The correct and only acceptable way to do this is to grip the microscope arm firmly with the right hand and the base with the left hand, and lift the instrument from the cabinet shelf. Carry it close to the body and gently place it on the laboratory bench. This will prevent collision with furniture or co-workers and will protect the instrument against damage.

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Enlargement or magnification of a specimen is the function of a two-lens system; the ocular lens is found in the eyepiece, and the objective lens is situated in a revolving nose-piece. These lenses are separated by the body tube. The objective lens is nearer the specimen and magnifies it, producing the real image that is projected up into the focal plane and then magnified by the ocular lens to produce the final image.

3. Now, while looking through the ocular lens, turn the coarse focus knob carefully, and slowly move the stage away from the lens until the specimen comes into vague focus. Then, use the fine focus knob to bring the specimen into sharp focus.

Based on this formula, the shorter the wave-length, the greater the resolving power of the lens. Thus, short wavelengths of the electromag-netic spectrum are better suited than longer wavelengths in terms of the numerical aperture.

3.Clean all lens svstems; the smallest bit of dust, oil, lint, or eyelash will decrease the efficiency ot the microscope. The ocular; scan-ning, low-power, and high-power lenses may be cleaned by wiping several times with acceptable lens tissue. Never use paper tow-eling or cloth on a lens surface. If the oil-immersion lens is gummy or tacky, a piece of lens paper moistened with methanol is used to wipe it clean. If the lens is very dirty it may be cleaned with xylol however the xylol cleansing procedure should be performed only by the instructor, and only if necessary. Consistent use of xylol may loosen the lens.

Anti-reflection V-coats are a type of AR coating designed to increase transmission over a very narrow waveband centered at a specified design wavelength (DWL). This coating type is called “V-coat” because the curve of the transmission versus wavelength forms a “V,” with a minimum at the DWL. V-coats are ideal for obtaining maximum transmission when using single-frequency, small linewidth lasers, or narrow full width-half max (FWHM) light sources.1 V-coats typically have a reflectivity of less than 0.25% at the DWL. However, the reflection curve for the coating locally has a nearly parabolic shape and the reflectivity is significantly higher at wavelengths besides the DWL (Figure 3).

6. Our microscopes are parfocal, which means that when one lens is in focus, other lenses will also have the same focal length and can be rotated into position without further major adjustment. In practice, however; usually a half-turn of the fine-adjustment knob in either direction is necessary for sharp focus.

To use the microscope efficiently and with minimal frustration, you should understand the basic principles of microscopy: magnification, resolution, numerical aperture, illumination, and focusing.