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The Microscope

The first compound microscope was constructed in Holland (by Hans andZacharias Janssen) over 400 years ago (1590). A compound microscope refers to a tubewith a lens on either end, one of which is placed close to the object, and hence is calledthe objective, and the other close to the eye (ocular). Antioni Van Leeuwenhoek, using asimple strong magnifier and specimen holder, in 1673 discovered microorganisms inwater at a magnification of about 275X. Giovanni Battista Amici, among his otherimprovements to microscopy, improved the image quality and brightness by filling thespace between the objective and cover glass with water, the first water immersionobjective. Amici's work was advanced by Carl Zeiss, a master instrument maker, andprofessor of physics Ernst Abbe. Abbe improved the Amici immersion system by usinga suitable oil that possessed the same refractive index as glass. This is the oil immersionsystem commonly used today. Zeiss and Abbe, working with a glass chemist, formulatedlenses that were color corrected--the first apochromatic objectives. Early in the twentiethcentury, ProfessorKhler introduced the method of microscope illumination which is

used today throughout the world. Khler provides the highest intensity of evenillumination from nonhomogenous sources.

What is the exact purpose of a microscope? It is to magnify, and also to resolve.Magnification itself is not sufficient. High orders of magnification can be obtainedwithout revealing fine structures. How does a microscope magnify? The closer anobject is brought to the eye the larger it becomes and the more detail we see. However,there is a limit, as close focusing of the human eye is limited. Normally 250 mm isconsidered to be normal viewing distance at which an object is seen at 1X magnification.With young people this distance is somewhat shorter and focusing is possible at 125 mm.This makes possible a 2X magnification, due to the fact that the image of the objectoccupies a larger area on the retina. In order to obtain a closer look at objects, it is

necessary to spread the image on the retina artificially. To spread this image, a magnifiermust be used. The very strong magnifiers are called simple microscopes. They arecapable of magnifications in the range of 250X. The modern microscope magnifiesthrough two separate lens systems, the eyepiece and the objective, and is, therefore,called a compound microscope. Microscopic magnification is brought about in the samemanner, i.e., the total magnification is equal to the product of the magnification of theobjective and the magnification of the eyepiece.

Resolving power is the ability of a lens to enable the observer to see fine detailsin a specimen. Thus, a 40X microscope may be superior to a 200X microscope is theresolving power of the 40X microscope is better. The better the resolving power, thecloser two small objects can and still be distinguished as two objects. Thus, a lenssystem with a resolving power of 2.5 m has poorer resolving power than a lens with aresolving power of 1.0 m. The practical resolving power of the microscope is limitedby that of the human eye. From a distance of 25 cm, which is approximately the distanceof the optical tube in a microscope, the human eye can distinguish two small objects thatare 0.1 mm apart. Therefore, the practical limit of resolving power for opticalmicroscopes that use visible light is about 0.2 m.

To form a clear image, a lens must focus each ray of light from a point in aspecimen into a point in the image. We call failure to do so an aberration. Chromatic

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aberration occurs because the focal length of a simple lens varies noticeably withwavelength. Blue rays are shorter in wavelength and focus closer to the lens than greenor red rays. The single lens is unable to bring all colors to a common focus, resulting ina slightly different sized image for each wavelength at slightly different focal points.Chromatic aberration makes lines look like colored bands. Achromatism was achievedthrough the combination of two or three lenses of different optical properties cementedtogether to form a doublet or triplet, respectively.

specimen specimen

chromatic aberration spherical aberration

Spherical aberration occurs when light rays passing through the central and outerportions of a lens are not brought to focus at the same distance from the lens. This

condition arises because light is refracted more at the edge of the lens. Sphericalaberration causes fuzzy images by focusing some rays of light closer to the lens and somerays further from the lens. You may recall that the problem with the Hubble SpaceTelescope was due to miscalculation of about 1 mm in orienting the lenses that led tospherical aberration.

Ernst Abbe derived an equation for calculating approximate resolving power.Thus, the quality of lenses could be compared numerically. Abbe's equation may bestated as:

resolving power = wavelength of light used/2 (numerical aperture of objective)(minimum distance)

This equation specifies the minimum distance between two minute structures that allowsthem to be seen as two structures. According to the equation, resolving power is better ingreen light than in red light:

400 nm---------490nm----------560nm---------590nm---------630nm---------700nmblue green yellow orange red

While blue light will theoretically provide better resolving power, the visual acuity of thehuman eye is greatest in green light. Thus, green filters are generally used.

The variable other than wavelength of light that determines resolving power iscalled numerical aperture (N.A.). When light strikes a specimen on a microscope

stage, some light passes straight through while some is bent by the specimen and goes offat an angle. The finer the details in the specimen, the greater the angle of bending. Therays that are bent by the specimen are the image-forming rays. The more of these thatcan be gathered and focused by the objective, the better the image.

Numerical aperture is an expression of the ability of an objective to collect theseangled, image-forming rays of light. The larger the N.A., the greater the ability of theobjective to collect the rays and hence, the better its resolving power. The theoreticalmaximum N.A. possible for dry objectives is 1.0. The actual highest numerical aperture

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for a dry objective is about 0.95. The numerical aperture for any objective is engravedon the objective itself. The only way to have an objective of N.A. equal to or greaterthan 1.0 is to place a liquid medium of higher refractive index between the lens and thespecimen slide. Air has a refractive index of 1.0, oil of 1.51, and water of 1.333. Whenimmersion oil is used, image-forming rays of light are bent less than they would be in airand thus more of them enter the objective; hence, resolution is increased. Some image-forming rays of light are lost due to reflection where air meets the surface of the glasscoverslip. Immersion oil is used that has a refractive index equal to that of the glass usedin coverslips (1.515).

Immersion oil is convenient because it gets neither sticky nor dry. However, it iscritical to carefully clean the objective after using oil, with lens paper only, to prevent abuild-up of oil on the objective; despite the previous statement, immersion oil dries tosome extent when left for long periods on the objective. Only oil immersion lenses aredesigned to withstand immersion in oil. Generally, that is the 100X lens, although it ispossible to get oil immersion lenses for lower power objectives. An oil immersion lenswill have some designation on it; look for the designation before immersing any lens.

A substage condenser is essential for maximizing the resolving power of the

objective. The condenser increases resolving power by providing light that is nearly100% of the image-forming kind. It does this by focusing light into the shape of aninverted cone that has its apex at the specimen and its base at the objective. The type ofcondenser on a microscope and the cone of light it produces determines the differenttypes of illumination available to the microscopist for specific specimen characteristics.We have available brightfield, phase contrast and Nomarski differential interference.

Khler IlluminationIn Khler illumination, a central fraction of the lamp condenser may be focused in theplane of the specimen. In the simplest arrangement, the light source (lamp filament) isfocused in the rear focal plane of the substage condenser. With a given lamp condenser

and source size, the distance of the lamp is such that the image of the source just fills theaperture of the substage condenser in use. The required distance changes withmagnification and some other variables. In practice, since the lamp distance is rarelychanged, the image or the source is usually kept large enough to fill the whole apertureof the condenser. The substage condenser is focused to bring the image of the lampcondenser into the plane of the specimen. The steps for setting up Khler illuminationare as follows:

1. Focus on a specimen using the 10X objective

2. Close down the condenser diaphragm so that the iris leaves are visible

3. Focus the substage condenser until the edges of the condenser iris are sharp

4. Open up the condenser iris so that it just fills the field

When you change objectives, you will need to adjust the condenser's iris even for routinework. Ordinarily, you can leave the condenser's height as it is once you have set it, but

forcritical work, it too should be readjusted whenever you change objectives.

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Phase-Contrast MicroscopyMany of the fungi produce hyaline structures. Unstained, hyaline structures often

are often difficult to examine with the bright field microscope. Sometimes a person whois observing a transparent, unstained specimen will deliberately close the condenser's iristo exaggerate contrast. This closing will reduce resolving power, but because of theincreased contrast, the observer may be able to see structures that would otherwise beinvisible.

The phase-contrast microscope was designed to exaggerate contrast withoutsacrificing resolving power. The theoretical basis for this microscope was published in1935 by Frits Zernike, and Carl Zeiss Inc. built a prototype phase-contrast microscope in1936 based on Zernike's principles. The phase-contrast microscope contributed so muchto science that Zernike received the Nobel Prize for his contribution to it.

The phase-contrast method involves a direct and deviated beam. In a phasecontrast system the phase of the central beam is changed by one quarter of a wavelengthat the rear focal plane of the objective. This converts the imaging conditions oftransparent objects into absorbing objects. When a ray of light from a single point is

split in two and each of the two rays is passed through the same transparent medium,they can be recombined without interference. But if each separated beam passes througha medium of different refractive index, one will be speeded up or slowed down relativeto the other. The two, when recombined, may be out of phase. If so, interference occursand the recombined beam is not as intense as the original. This canceling will createdarkened places in the image that the eye can detect. In phase-contrast, an annular ringretards a wave by 1/4 wavelength; light waves that are delayed by 1/2 wavelength willcancel (i.e., destructively interfere with) undelayed light from the same source.

Interference-Contrast MicroscopyThe difference between phase-contrast and interference-contrast is that it is the

optical system that produces the two interfering beams, not the specimen as in phase-contrast. Interference takes place between two types of beams, not between two imagecomponents as in the phase-contrast systems. The Nomarski interference system consistsof a polarizer at the light port of the microscope and Wollaston prisms in the condenser.One prism for each magnification is arranged in a turret mount. Situated above theobjectives is a second Wollaston prism and an analyzer. When the light passes throughthe polarizer and enters the first Wollaston prism, it is split into two plane-polarizedcomponents. Two separate beams, the object beam and the reference bream, are focusedin the plane of the specimen, with only the object beam passing through the specimen.The two beams are recombined below the eyepiece. The reference beam should passthrough a relatively clear area of the slide in order to minimize the imaging of unwanteddetail, which would intrude on the subject detail. Therefore, a specimen slide preparedfor observation with DIC microscopy should consist of a small amount of tissue, well-spread out on the slide. Normally, dark structures are not suitable for DIC microscopy.

Another advantage of DIC microscopy is that it permits optical sectioning of aspecimen. By focusing up and down the effect is that of sectioning through thespecimen.

Other Types of MicroscopyFluorescence microscopy is becoming more widely used by both mycologists and

plant pathologists. The principle is simple: some materials will emit light of a longer

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wavelength when excited by short wavelengths of radiation. This phenomenon is calledfluorescence. Ultraviolet radiation and blue light are often used as exciting radiation toproduce visible light of longer wavelengths. If a substance does fluoresce, the effect iscalled autofluorescence. Some materials do not fluoresce by themselves but can beimpregnated with chemicals, such as dyes that will fluoresce. Dyes of this type arecalled fluorochromes; the effect in the original material is called secondary fluorescence.Fluorochromes are absorbed by cell organelles or bind to specific residues inside or onthe cell. Fluorochromes have been used for many years in medicine, cell biology andindustry. Mycologists have also made extensive use of fluorochromes, albeit theirpotential as a tool for probing fungal cell components and studying fungal differentiationand growth has not been fully realized by many researchers.

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