FUNDAMENTALS OF MICROBIOLOGY

Lesson 4. LIGHT AND ELECTRON MICROSCOPY

Module 2. Microscopy

Lesson 4

LIGHT AND ELECTRON MICROSCOPY

4.1 Introduction

On the basis of source of illumination and types of lenses used, microscopes can be broadly categorized into (i) light microscopes and (ii) electron microscopes. Light or optical microscopes are characterized by light as a source of illumination and optical lenses as magnifying objects while in electron microscopes, these functions are performed by electrons and electromagnetic lenses respectively.

4.2 Light Microscopy

Microscopes using light can be categorized into following types: • Bright-field microscop

• Darkfield microscope
• Phase-contrast microscope
• Fluorescence microscopes

All these applications employ compound microscope, which essentially involve image formation by the action of two or more lenses (Fig. 4.1).

Fig. 4.1 Compound microscope

4.2.1 Bright-field microscopy

The bright-field light microscope is an instrument that magnifies images using two-lens systems. Initial magnification occurs in the objective lens. Most microscopes have at least three objective lenses on a rotating base, and each lens may be rotated into alignment with the eyepiece or ocular lens in which the final magnification occurs. The objective lenses are identified as the low-power, high-dry, and oil immersion objectives. Each objective is also designated by other terms. These terms give either the linear magnification or the focal length. The latter is about equal to or greater than the working distance between the specimen, when in focus, and the tip of the objective lens. For example, the low-power objective is also called the 10×, or 16 millimeter (mm), objective; the high-dry is called the 40×, or 4 mm, objective; and the oil immersion is called the 90×, 100×, or 1.8 mm objective. As the magnification increases, the size of the lens at the tip of the objective becomes progressively smaller and admits less light. This is one of the reasons that changes in position of the substage condenser and iris diaphragm are required when using different objectives if the specimens viewed are to be seen distinctly. The condenser focuses the light on a small area above the stage, and the iris diaphragm controls the amount of light that enters the condenser. When the oil immersion lens is used, immersion oil fills the space between the objective and the specimen. Because immersion oil has the same refractive index as glass, the loss of light is minimized. The eyepiece, or ocular, at the top of the tube magnifies the image formed by the objective lens. As a result, the total magnification seen by the observer is obtained by multiplying the magnification of the objective lens by the magnification of the ocular, or eyepiece. For example, when using the 10X ocular and the 40X objective, total magnification is 10 × 40 = 400 times.

4.2.2 Darkfield microscopy

The compound microscope may be fitted with a darkfield condenser (Fig. 4.2) that has a numerical aperture (resolving power) greater than the objective. The condenser also contains a darkfield stop. The compound microscope now becomes a darkfield microscope. Light passing through the specimen is diffracted and enters the objective lens, whereas undiffracted light does not, resulting in a bright image against a dark background. Since light objects against a dark background are seen more clearly by the eye than the reverse, darkfield microscopy is useful in observing unstained living microorganisms, microorganisms that are difficult to stain, and spirochetes, which are poorly defined by bright-field microscopy.

Fig. 4.2 Darkfield microscopy

A central obstruction blocks the central cone. The sample is only illuminated by the marginal rays. These marginal rays must be at angles too large for the objective lens to collect. Only light scattered by the object is collected by the lens.

4.2.3 Phase contrast microscopy

This is first microscopic method which allowed visualization of live cells in action. The Nobel prize in physics was awarded to Frits Zernike in 1953 for its discovery. Certain transparent, colorless living microorganisms and their internal organelles are often impossible to see by ordinary bright or darkfield microscopy because they do not absorb, reflect, refract, or diffract sufficient light to contrast with the surrounding environment or the rest of the microorganism. Microorganisms and their organelles are only visible when they absorb, reflect, refract, or diffract more light than their environment. The phase contrast microscope permits the observation of otherwise invisible living, unstained microorganisms. It enhances contrast in transparent and colorless objects by influencing the optical path of light. It uses the fact that light passing through the specimen travels slower than the undisturbed light beam, i.e. its phase is shifted. In figure 4.3, let S be light passing through medium surrounding sample and D light interacting with specimen. S and D typically interfere to yield P, which is what we can usually detect. P will be phase shifted compared to S, but our eyes cannot detect phase shifts. Phase contrast microscopy effectively converts this phase shift into an intensity difference we can detect.

Fig. 4.3 Phase contrast formation

In the phase contrast microscope, the condenser has an annular diaphragm, which produces a hollow cone of light; the objective has a glass disk (the phase plate) with a thin film of transparent material deposited on it, which accentuates phase changes produced in the specimen. This phase change is observed in the specimen as a difference in light intensity. Phase plates may either retard (positive phase plate) the diffracted light relative to the undiffracted light, producing dark phase contrast microscopy, or advance (negative phase plate) the undiffracted light relative to the directed light, producing bright phase contrast microscopy.

4.2.4 Fluorescence microscopy

A fluorescence microscope is much the same as a conventional light microscope with added features to enhance its capabilities.

• The conventional microscope uses visible light (400-700 nanometers) to illuminate and produce a magnified image of a sample.
• A fluorescence microscope, on the other hand, uses a much higher intensity light source which excites a fluorescent species in a sample of interest. This fluorescent species in turn emits a lower energy light of a longer wavelength that produces the magnified image instead of the original light source.

Fluorescence microscopy is based on the principle of removal of incident illumination by selective absorption, whereas light that has been absorbed by the specimen and re-emitted at an altered wavelength is transmitted. The light source must produce a light beam of appropriate wavelength. An excitation filter removes wavelengths that are not effective in exciting the fluorochrome used. The light fluoresced by the specimen is transmitted through a filter that removes the incident wavelength from the beam of light. As a result, only light that has been produced by specimen fluorescence contributes to the intensity of the image being viewed (Figure 4.4).

Fig. 4.4 Principle of fluorescence microscopy

4.3 Electron Microscopy

Electron microscopes were developed in order to overcome the limitations of light microscopes, which are constrained by the physics of light to 500x or 1000x magnification and a resolution of 0.2 µm

In the early 1930s this theoretical limit had been reached and there was a scientific desire to see the fine details of the interior structures of organic cells (nucleus, mitochondria, etc.). This required 10,000x plus magnification which was just not possible using Light Microscopes. Electro microscopes based on their construction and application are of two types viz. transmission and scanning electron microscopes. The Transmission Electron Microscope (TEM) was the first type of EM to be developed and is patterned exactly on the Light Transmission Microscope except that a focused beam of electrons is used instead of light to ‘see through’ the specimen. It was developed by Max Knoll and Ernst Ruska in Germany in 1931. The first Scanning Electron Microscope (SEM) debuted in 1942 with the first commercial instruments around 1965. Its late development was due to the electronics involved in ‘scanning’ the beam of electrons across the sample. Electron Microscopes (EMs) function exactly as their optical counterparts except that they use a focused beam of electrons instead of light to ‘image’ the specimen and gain information as to its structure and composition.

4.4 Scanning Electron Microscope

SEM is patterned after reflecting light microscopes and yield similar information.

• Topography: The surface features of an object or ‘how it looks’, its texture; detectable features limited to a few manometers.

• Morphology: The shape, size and arrangement of the particles making up the object that are lying on the surface of the sample or have been exposed by grinding or chemical etching; detectable features limited to a few manometers.

• Composition: The elements and compounds the sample is composed of and their relative ratios, in areas ~1 µm in diameter.

• Crystallographic Information: The arrangement of atoms in the specimen and their degree of order; only useful on single-crystal particles >20 µm

A detailed explanation of how a typical SEM functions follows: (Fig. 4.6)

• The ‘Virtual Source’ at the top represents the electron gun, producing a stream of monochromatic electrons.

• The stream is condensed by the first condenser lens (usually controlled by the ‘coarse probe current knob’). This lens is used to both form the beam and limit the amount of current in the beam. It works in conjunction with the condenser aperture to eliminate the high-angle electrons from the beam.

• The beam is then constricted by the condenser aperture (usually not user selectable), eliminating some high-angle electrons.

• The second condenser lens forms the electrons into a thin, tight, coherent beam and is usually controlled by the ‘fine probe current knob’.

• A user selectable objective aperture further eliminates high-angle electrons from the beam.

• A set of coils then ‘scan’ or ‘sweep’ the beam in a grid fashion (like a television), dwelling on points for a period of time determined by the scan speed (usually in the microsecond range).

• The final lens, the objective, focuses the scanning beam onto the part of the specimen desired.

• When the beam strikes the sample (and dwells for a few microseconds) interactions occur inside the sample and are detected with various instruments.

• Before the beam moves to its next dwell point these instruments count the number of interactions and display a pixel on a CRT whose intensity is determined by this number (the more reactions the brighter the pixel).

• This process is repeated until the grid scan is finished and then repeated, the entire pattern can be scanned 30 times per second

Fig. 4.6 (A) Diagrammatic representation of pathway of electrons in SEM, (B) SEM
available at Electron Microscopy Centre, NDRI, Karnal

4.5 Transmission Electron Microscope

TEM are patterned after Transmission Light Microscopes and will yield similar information.

• Morphology: The size, shape and arrangement of the particles which make up the specimen as well as their relationship to each other on the scale of atomic diameters.

• Crystallographic Information: The arrangement of atoms in the specimen and their degree of order, detection of atomic-scale defects in areas a few nanometers in diameter

TEM work the same way except that they shine a beam of electrons (like the light) through the specimen (like the slide). Whatever part is transmitted is projected onto a phosphor screen for the user to see. A more technical explanation of a typical TEM working is as follows: (Fig. 4.7)

• The ‘Virtual Source’ at the top represents the electron gun, producing a stream of monochromatic electrons.

• This stream is focused to a small, thin, coherent beam by the use of condenser lenses 1 and 2. The first lens (usually controlled by the ‘spot size knob’) largely determines the lens (usually controlled by the ‘intensity or brightness knob’ actually changes the size of the spot on the sample; changing it from a wide dispersed spot to a pinpoint beam.

• The beam is restricted by the condenser aperture (usually user selectable), knocking out high angle electrons (those far from the optic axis, the dotted line down the center).

• The beam strikes the specimen and parts of it are transmitted.

• This transmitted portion is focused by the objective lens into an image.

• Optional objective and selected area metal apertures can restrict the beam; the objective aperture enhancing contrast by blocking out high-angle diffracted electrons, the selected area aperture enabling the user to examine the periodic diffraction of electrons by ordered arrangements of atoms in the sample.

• The image is passed down the column through the intermediate and projector lenses, being enlarged all the way.

• The image strikes the phosphor image screen and light is generated, allowing the user to see the image. The darker areas of the image represent those areas of the sample that fewer electrons were transmitted through (they are thicker or denser). The lighter areas of the image represent those areas of the sample that more electrons were transmitted through (these are thinner or less dense).

Fig. 4.7 (A) Diagrammatic representation of pathway of electrons in TEM, (B): EM
available at Electron Microscopy Centre, NDRI, Karnal