The Microscope The first compound microscope was constructed in Holland (by Hans and Zacharias Janssen) over

400 years ago (1590). A compound microscope refers to a tube with a lens on either end, one of which is placed close to the object, and hence is called the objective, and the other close to the eye (ocular). Antioni Van Leeuwenhoek, using a simple strong magnifier and specimen holder, in 1673 discovered microorganisms in water at a magnification of about 275X. Giovanni Battista Amici, among his other improvements to microscopy, improved the image quality and brightness by filling the space between the objective and cover glass with water, the first water immersion objective. Amici's work was advanced by Carl Zeiss, a master instrument maker, and professor of physics Ernst Abbe. Abbe improved the Amici immersion system by using a suitable oil that possessed the same refractive index as glass. This is the oil immersion system commonly used today. Zeiss and Abbe, working with a glass chemist, formulated lenses that were color corrected--the first apochromatic objectives. Early in the twentieth century, Professor Khler introduced the method of microscope illumination which is used today throughout the world. Khler provides the highest intensity of even illumination 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 obtained without revealing fine structures. How does a microscope magnify? The closer an object 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 is considered 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 object occupies 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 magnifier must be used. The very strong magnifiers are called simple microscopes. They are capable of magnifications in the range of 250X. The modern microscope magnifies through two separate lens systems, the eyepiece and the objective, and is, therefore, called a compound microscope. Microscopic magnification is brought about in the same manner, i.e., the total magnification is equal to the product of the magnification of the objective and the magnification of the eyepiece. Resolving power is the ability of a lens to enable the observer to see fine details in a specimen. Thus, a 40X microscope may be superior to a 200X microscope is the resolving power of the 40X microscope is better. The better the resolving power, the closer two small objects can and still be distinguished as two objects. Thus, a lens system with a resolving power of 2.5 m has poorer resolving power than a lens with a resolving power of 1.0 m. The practical resolving power of the microscope is limited by that of the human eye. From a distance of 25 cm, which is approximately the distance of the optical tube in a microscope, the human eye can distinguish two small objects that are 0.1 mm apart. Therefore, the practical limit of resolving power for optical microscopes 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 a specimen into a point in the image. We call failure to do so an aberration. Chromatic

Pl P 421 Lab 1 aberration occurs because the focal length of a simple lens varies noticeably with wavelength. Blue rays are shorter in wavelength and focus closer to the lens than green or red rays. The single lens is unable to bring all colors to a common focus, resulting in a slightly different sized image for each wavelength at slightly different focal points. Chromatic aberration makes lines look like colored bands. Achromatism was achieved through the combination of two or three lenses of different optical properties cemented together to form a doublet or triplet, respectively.

specimen

specimen spherical aberration

chromatic aberration

Spherical aberration occurs when light rays passing through the central and outer portions 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. Spherical aberration causes fuzzy images by focusing some rays of light closer to the lens and some rays further from the lens. You may recall that the problem with the Hubble Space Telescope was due to miscalculation of about 1 mm in orienting the lenses that led to spherical aberration. Ernst Abbe derived an equation for calculating approximate resolving power. Thus, the quality of lenses could be compared numerically. Abbe's equation may be stated 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 allows them to be seen as two structures. According to the equation, resolving power is better in green light than in red light: 400 nm---------490nm----------560nm---------590nm---------630nm---------700nm blue green yellow orange red While blue light will theoretically provide better resolving power, the visual acuity of the human eye is greatest in green light. Thus, green filters are generally used. The variable other than wavelength of light that determines resolving power is called 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 off at an angle. The finer the details in the specimen, the greater the angle of bending. The rays that are bent by the specimen are the image-forming rays. The more of these that can be gathered and focused by the objective, the better the image. Numerical aperture is an expression of the ability of an objective to collect these angled, image-forming rays of light. The larger the N.A., the greater the ability of the objective to collect the rays and hence, the better its resolving power. The theoretical maximum N.A. possible for dry objectives is 1.0. The actual highest numerical aperture

Pl P 421 Lab 1 for a dry objective is about 0.95. The numerical aperture for any objective is engraved on the objective itself. The only way to have an objective of N.A. equal to or greater than 1.0 is to place a liquid medium of higher refractive index between the lens and the specimen slide. Air has a refractive index of 1.0, oil of 1.51, and water of 1.333. When immersion oil is used, image-forming rays of light are bent less than they would be in air and thus more of them enter the objective; hence, resolution is increased. Some imageforming rays of light are lost due to reflection where air meets the surface of the glass coverslip. Immersion oil is used that has a refractive index equal to that of the glass used in coverslips (1.515). Immersion oil is convenient because it gets neither sticky nor dry. However, it is critical to carefully clean the objective after using oil, with lens paper only, to prevent a build-up of oil on the objective; despite the previous statement, immersion oil dries to some extent when left for long periods on the objective. Only oil immersion lenses are designed to withstand immersion in oil. Generally, that is the 100X lens, although it is possible to get oil immersion lenses for lower power objectives. An oil immersion lens will 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 nearly 100% of the image-forming kind. It does this by focusing light into the shape of an inverted cone that has its apex at the specimen and its base at the objective. The type of condenser on a microscope and the cone of light it produces determines the different types of illumination available to the microscopist for specific specimen characteristics. We have available brightfield, phase contrast and Nomarski differential interference. Khler Illumination In Khler illumination, a central fraction of the lamp condenser may be focused in the plane of the specimen. In the simplest arrangement, the light source (lamp filament) is focused 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 the aperture of the substage condenser in use. The required distance changes with magnification and some other variables. In practice, since the lamp distance is rarely changed, the image or the source is usually kept large enough to fill the whole aperture of the condenser. The substage condenser is focused to bring the image of the lamp condenser into the plane of the specimen. The steps for setting up Khler illumination are 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 routine work. Ordinarily, you can leave the condenser's height as it is once you have set it, but for critical work, it too should be readjusted whenever you change objectives. 3

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Phase-Contrast Microscopy Many of the fungi produce hyaline structures. Unstained, hyaline structures often are often difficult to examine with the bright field microscope. Sometimes a person who is observing a transparent, unstained specimen will deliberately close the condenser's iris to exaggerate contrast. This closing will reduce resolving power, but because of the increased contrast, the observer may be able to see structures that would otherwise be invisible. The phase-contrast microscope was designed to exaggerate contrast without sacrificing resolving power. The theoretical basis for this microscope was published in 1935 by Frits Zernike, and Carl Zeiss Inc. built a prototype phase-contrast microscope in 1936 based on Zernike's principles. The phase-contrast microscope contributed so much to science that Zernike received the Nobel Prize for his contribution to it. The phase-contrast method involves a direct and deviated beam. In a phase contrast system the phase of the central beam is changed by one quarter of a wavelength at the rear focal plane of the objective. This converts the imaging conditions of transparent 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 through a medium of different refractive index, one will be speeded up or slowed down relative to the other. The two, when recombined, may be out of phase. If so, interference occurs and the recombined beam is not as intense as the original. This canceling will create darkened places in the image that the eye can detect. In phase-contrast, an annular ring retards a wave by 1/4 wavelength; light waves that are delayed by 1/2 wavelength will cancel (i.e., destructively interfere with) undelayed light from the same source. Interference-Contrast Microscopy The 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 phasecontrast. Interference takes place between two types of beams, not between two image components as in the phase-contrast systems. The Nomarski interference system consists of 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 the objectives is a second Wollaston prism and an analyzer. When the light passes through the polarizer and enters the first Wollaston prism, it is split into two plane-polarized components. Two separate beams, the object beam and the reference bream, are focused in 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 pass through a relatively clear area of the slide in order to minimize the imaging of unwanted detail, which would intrude on the subject detail. Therefore, a specimen slide prepared for observation with DIC microscopy should consist of a small amount of tissue, wellspread 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 a specimen. By focusing up and down the effect is that of sectioning through the specimen. Other Types of Microscopy Fluorescence microscopy is becoming more widely used by both mycologists and plant pathologists. The principle is simple: some materials will emit light of a longer 4

Pl P 421 Lab 1 wavelength when excited by short wavelengths of radiation. This phenomenon is called fluorescence. Ultraviolet radiation and blue light are often used as exciting radiation to produce visible light of longer wavelengths. If a substance does fluoresce, the effect is called autofluorescence. Some materials do not fluoresce by themselves but can be impregnated with chemicals, such as dyes that will fluoresce. Dyes of this type are called fluorochromes; the effect in the original material is called secondary fluorescence. Fluorochromes are absorbed by cell organelles or bind to specific residues inside or on the cell. Fluorochromes have been used for many years in medicine, cell biology and industry. Mycologists have also made extensive use of fluorochromes, albeit their potential as a tool for probing fungal cell components and studying fungal differentiation and growth has not been fully realized by many researchers.