Light Microscopes

Rather than the simple single lens instruments of van Leeuwenhoek (Section 10.2.1), today the compound light microscope is the conventional tool used (Figure 11.2). It routinely gives magnifications of up to 1000 power (x) by combining a 10x ocular (eyepiece) lens with an objective of up to 100 x. The total magnification is determined by multiplying the two together. Objectives of 4, 10, 20, and 40 x are also common, and oculars of 15 x are available.

It is resolution, however, rather than magnification itself, that is the primary limitation of observation with a light microscope. For the average human eye, as two points move closer together they would become one point—could no longer be resolved as separate— at a distance of about 75 mm. (Most prokaryotes, remember, are about 1 to 2 mm in size.) At 1000 x this would occur at a distance of 0.075 mm. However, visible light has wavelengths of about 450 to 720 nm (0.45 to 0.72 mm), which effectively limits resolution to about 0.2 mm. Any additional magnification does not lead to further resolution if visible light is used; the object simply looks grainy or fuzzy.

When the compound microscope is used for standard bright-field microscopy (Figure 11.3), the specimen, typically in water, is placed on a glass slide and a small glass coverslip is placed over it. The sample is then seen against a bright background. This is particularly useful in determining the color of cells, such as cyanobacteria and algae, and observing the yellow of sulfur inclusions. However, for most colorless

Ocular lens

Ocular lens

Objective lens

"^BPlatform

Condenser

Objective lens

Cover slip Sample

Glass slide

White light source

Figure 11.3 Bright-field microscopy: an original image viewed against a light or bright background.

microorganisms, there is little visible contrast between them and the water, and thus little detail is observable. This is a primary reason that so much effort was put into development of staining procedures (Section 10.4.4 and Figure 10.15) by early microscopists.

A disadvantage of traditional staining techniques is that they normally involve killing the cells. Thus, observations of motility and any other activity of the microorganisms is lost. One variation that is sometimes used is dark-field microscopy (Figure 11.4), in which objects are illuminated from the side. This makes them appear bright against a dark background and can improve visibility of some transparent or fine structures, such as the flagella of eukaryotes.

A major advance for observing live cultures was the development of phase-contrast microscopy. This specialized modification of the compound microscope makes small changes in refractive index visible, allowing improved observation of both the cell surface and internal structures (Figure 11.5). Frederik Zernike of the Netherlands was awarded a Nobel Prize in Physics in 1953 for this invention.

Another modification is the fluorescence microscope (Figure 11.6). Fluorescent compounds absorb light at one wavelength to become "excited" or "activated" and then emit the energy at another wavelength. A variety of fluorescent molecules have been developed that can penetrate or be taken up by cells. The specimen is then exposed to the specific activation wavelength of light, and through filters, the specific emission wavelength is observed.

The size of microorganisms can be measured using an ocular micrometer. This is a small "ruler" placed in one of the eyepieces, so that it appears superimposed on the magnified image. This scale is then calibrated at each magnification using a stage micrometer, a special glass slide with a precisely etched 1-mm scale broken down into tenths and hundredths of a millimeter (Figure 11.7). Usually, the ocular micrometer is designed to have markings at 10-p.m intervals at 100 x magnification, and at 1 mm for 1000 x, but this may depend on the specific microscope.

White light source

White light source

Condenser

Ocular lens

Objective lens

/Cover slip Sample Glass slide

Figure 11.4 Dark-field microscopy: a bright reflected image is viewed against a darkened or black background.

Ocular lens

Objective lens

Condenser

/Cover slip Sample Glass slide

Figure 11.4 Dark-field microscopy: a bright reflected image is viewed against a darkened or black background.

Figure 11.5 Phase-contrast microscopy. Object (a), with high refractive index (n), bends and retards the light more; after further retardation at the edges of the phase plate, the recombined light (retarded and unretarded) creates destructive interference, leading to a darker appearance. Object (b), with a low n, does not bend or retard the light, creating no interference, and thus appears bright. Thus, contrast was created between the two nearly transparent objects that was not otherwise visible. A special condenser (not shown) with a phase ring is also required.

Figure 11.5 Phase-contrast microscopy. Object (a), with high refractive index (n), bends and retards the light more; after further retardation at the edges of the phase plate, the recombined light (retarded and unretarded) creates destructive interference, leading to a darker appearance. Object (b), with a low n, does not bend or retard the light, creating no interference, and thus appears bright. Thus, contrast was created between the two nearly transparent objects that was not otherwise visible. A special condenser (not shown) with a phase ring is also required.

Example 11.1 At 100x (10x ocular and 10x objective) a ciliated protozoan is observed to have a length of 9.3 units on the ocular micrometer scale. If from a previous calibration it is known that at 100 x each micrometer unit is 10 mm, what is the length of the organism? What is the field diameter if it is observed to correspond to 161 units?

Answer 10 mm/unit x 9.3 units = 93-p.m-longciliate. 10 mm/unit x 161 units = 1610 mm = 1.61-mm field diameter.

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