Microscopy

Introduction: Light microscopy is the corner stone of microbiology for it is through the microscope that most scientists first become acquainted with microorganisms. Microbiologists use a variety of microscopes, each with specific advantages and limitations. This exercise presents the principles of microscopy. A microscope is an optical instrument consisting of one or more lenses in order to magnify images of minute objects. Microscopes are of two categories.

a. Light Microscope: Magnification is obtained by a system of optical lenses using light waves. It includes (i) Bright field (ii) Dark field (iii) Fluorescence (iv) Phase contrast and (v) UV Microscope.

b. Electron Microscope: A system of electromagnetic lenses and a short beam of electrons are used to obtain magnification. It is of two types: (I) Transmission electron microscope (TEM) (ii) Scanning electron microscope (SEM).

Light microscopes can be broadly grouped into two categories. (a) Simple microscope: It consists of only one bi-convex lens along with a stage to keep the specimen. (b) Compound microscope: It employs two separate lens systems namely, (i) objective and (ii) ocular (eye piece).

Parts of Compound Microscope:

The compound student microscope is a bright field microscope. It consists of mechanical and optical parts.

1. Mechanical parts: These are secondary but are necessary for working of a microscope. A ‘Base’, which is horsehoe, shaped supports the entire framework for all parts. From the base, a ‘Pillar’ arises. At the top of the pillar through an ‘Inclination Joint’ arm or limb is attached. At the top of the pillar, a stage with a central circular opening called ‘Stage aperture’ is fixed, with a stage clip to fix the microscopic slide. Beneath the stage, there is one stage called ‘sub stage’ which carries the condenser. At the top of the arm, a hollow cylindrical tube of standard diameter is attached in-line with the stage aperture, called ‘body tube’. The body tube moves up and down by two separate arrangements called ‘coarse adjustment’ worked with pinion head and ‘fine adjustment’ worked with micrometer head. At the bottom of the body tube an arrangement called ‘revolving nose-piece’ is present for screwing different objectives. At the top of the body tube eye- piece is fixed.

2. Optical parts: It includes mirror, condenser, objective and ocular lenses. All the optical parts should be kept in perfect optical axis.

a. Objectives : Usually 3 types of magnifying lenses (i) Low power objective (10x) (ii) High dry objective (45x) and (iii) Oil immersion objective (100x)

b. Eye-piece : Mostly have standard dimensions and made with different power lenses. (5x, 10x, 15x, 20x). A compound microscope with a single eyepiece is said to be monocular, and one with two eyepieces is said to be binocular.

c. Condenser : Condenses the light waves into a pencil shaped cone there by preventing the escape of light waves. Also raising or lowering the condenser can control light intensity. To the condenser, iris diaphragm is attached which helps in regulating the light.

d. Mirror : It is mounted on a frame and attached to the pillar in a manner that it can be focused in three different directions. The mirror is made of a lens with one plane surface and another concave surface. Plane surface is used, when the microscope is with a condenser.

How specimen is magnified in compound microscope

Procedure for using microscope:

Rotate the nose-piece and bring the low power objective [10X] in line with the eye piece i.e. in optical axis
Adjust the concave mirror, so that the brightly illuminated microscopic field is seen through the objective.
View through the ocular(s) open and close the iris diaphragm by the lever to get an optimum illumination.
Keep the slide with specimen side up over the hole in the centre of the stage.
With the coarse adjustment bring the low power objective close to the slide and slowly raise it, until the specimen comes into focus.
To get a clear image focus with the fine adjustment.
Bring the specimen of our interest to the centre of the field with the stage adjustments.
Rotate the nosepiece and bring the high power [45x] in working position.
If the microscope is of parfocal type the objective comes into focus and adjust with fine adjustment to get a clear image.
If the microscope is not parfocal, lower the objective close to the specimen and slowly raise it with coarse adjustment until the specimen comes into focus.
Adjust with the fine adjustment to get a clear image.

Microscopy - Principles

The light is the primary source on which magnification is based in light microscopes. The magnification is obtained by a system of optical lenses using light waves. Magnification refers the number of times a specimen is appeared to be larger than its original size.

Basic units for microscope

1 meter = 1000 millimeter

1 millimeter = 1000 micrometer (mm) = 10^-6 meter

1 micrometer = 1000 nanometer (nm) = 10^-9meter

1 Angstrom (1 A) = 10^-10 meter

1 nanometer = 10 Angstrom

Relative size of the microorganisms and their visibility: Man can see about 0.5 mm sized object whereas the light microscopes can be used to visualize up to 1 mm and EM (electron microscopes) can be used to view 1 nm objects.

Quality of an image

The microscopic images should have four basic quality parameters, through which the microscopes can be graded.

Focus: It refers whether the image is well defined or blurry (out of focus). The focus can be adjusted through course and fine adjustment knobs of the microscope which will adjust the focal length to get clear image. The thickness of specimen, slide and coverslip also decide the focus of the image. (Thin specimens will have good focus).
Brightness: It refers how light or the dark the image is. Brightness of the image is depends on the illumination system and can be adjusted by changing the voltage of the lamp and by condenser diaphragm.
Contrast: It refers how best the specimen is differentiated from the background or the adjacent area of microscopic field. More the contrast will give good images. It depends on the brightness of illumination and colour of the specimen. The contrast can be achieved by adjusting illumination and diaphragm and by adding colour to the specimen. The phase contrast microscopes are designed in such a way that the contrast can be achieved with out colouring the specimen.
Resolution: It refers the ability to distinguish two objects close to each other. The resolution depends on the resolving power, which refers minimum distance between the two objects which can be distinguishable.

How to improve resolution in light microscopes?

The total magnification of compound microscope is the product of the magnifications of objective lens and eyepiece. Magnification of about 1500x is the upper limit of compound microscopes. This limit is set because of the resolution.

Resolution refers the ability of microscopes to distinguish two objects close to each other, it depends on resolving power, which refers the minimum distance. Ex : Man has the resolving power of 0.2 mm (meaning that he can distinguish two objects with a distance of 0.2 mm close to each other) If he want to see beyond the limit of his resolving power, further magnification is necessary.

Resolving power = lambda (micrometer)/n(sin(theta), where, lambda is the wave length of light source and n (sin theta ) is the numerical aperture (NA). (The resolving power (RP) should be of SMALLER value for good quality microscope)

For compound microscopes, resolving power is lambda/2NA.

The resolving power of an microscope can be improved either by reducing the wave length of light or by increasing the n(sin q) value.

a. Reducing the wave length: The following are the wavelength of some colours: blue – 400 nm; red – 700 nm; green – 550 nm used as filtered to improve the resolution of images. If we use violet light, we can get high resolution (meaning lowest resolving power). But, blue light (approx 450 nm) is sensitive to human being, which is commonly used in microscopes. The other lights will be removed from the light source using filters.

b. Increasing NA (n sin theta): Numerical aperture measures how much light cone spreads out between condenser & specimen. More spread of light gives less resolving power means better resolution. The numerical aperture depends on the objective lens of the microscope. There are two types of objective lenses are available in any compound microscope.

a. Dry objective lens – which can view the specimen without any fluid. Air is the medium between the objective lens and the specimen. b. Oil-immersion objective lens – which can view the specimen in the presence of immersion oil. This oil is the medium between lens and the specimen. The oil immersion objective lens has more NA than dry objective lens.

Microscopy with Oil Immersion

Principle: When light passes from a material of one refractive index to material of another, as from glass to air or from air to glass, it bends. The refractive index of air is 1.0, which is less than that glass slide (1.56). So, when light passes from glass (dense medium) to air (lighter medium), the rays get refracted, which led to loss of resolution of image. Light of different wavelengths bends at different angles, so that as objects are magnified the images become less and less distinct. This loss of resolution becomes very apparent at magnifications of above 400x or so. Even at 400x the images of very small objects are badly distorted. Placing a drop of oil (Cedar wood oil) with the same refractive index (1.51) as glass between the cover slip and objective lens eliminates two refractive surfaces and considerably enhances resolution, so that magnifications of 1000x or greater can be achieved.

The limit of resolution: The limit of resolution refers the smallest distance by which two objects can be separated and still be distinguishable or visible as two separate objects.

How to improve contrast in light microscopes?

Contrast refers the ability to differentiate the image from the background. There are two ways to increase the contrast as (1) Staining (colouring) the colour-less bacterial cells so as to differentiate from the background in the microscopic field; (2) Use of special light microscopes which will increase the contrast without straining. Obviously, the former method will allow to view the dead cells (cells will be killed during staining), while the microscopy methods will be used to see live cells without staining.

Staining: Dyes are used to stain cells and increase their contrast so that they can be more easily seen in the bright-field microscope. Dyes are the organic compounds, and each class of dye has an affinity for specific cellular materials. Many dyes used in microbiology are positively charged and hence, they are called basic dyes. Examples of basic dyes include methylene blue, crystal violet and safranin. Basic dyes bind strongly to negatively charged cell components, such as nucleic acids and acidic polysaccharides. Because cell surfaces tend to be negatively charged, these dyes also combine with high affinity to the surfaces of cells, and hence are very useful general-purpose stains. The dyes with negatively charged are called acidic dyes. Example of acidic dyes are nigrosine, picric acid, eosin, acid fuschin. As most of the cell wall outer membrane and capsules are negatively charged, this dye will repel and form a halo zone. Since, the background will be stained, and thus increased the contrast.

Special microscopes to increase the contrast: Staining, although a widely used procedure in light microscopy, kills cells and can distort their features. Two forms of light microscopy improve image contrast without the use of stain, and thus do not kill cells. These are phase-contrast microscopy and dark-field microscopy. The phase-contrast microscope in particular is widely used in teaching and research for the observation of wet-mount (living) preparations.

A. Phase contrast microscope: Phase-contrast microscopy is based on the principle that cells differ in refractive index (a factor by which light is slowed as it passes through a material) from their surroundings. Light passing through a cell thus differs in phase from light passing through the surrounding liquid. This subtle difference is amplified by a device in the objective lens of the phase-contrast microscope called the phase ring, resulting in a dark image on a light background. The ring consists of a phase plate that amplifies the minute variation in phase. The development of phase-contrast microscopy stimulated other innovations in microscopy, such as fluorescence and confocal microscopy, and greatly increased use of the light microscope in microbiology.

B. Dark field microscope: The dark-field microscope is a light microscope in which light reaches the specimen from the sides only. The only light that reaches the lens is that scattered by the specimen, and thus the specimen appears light on a dark background. Resolution by dark-field microscopy is somewhat better than by light microscopy, and objects can often be resolved by dark-field that cannot be resolved by bright-field or even phase-contrast microscopes. Dark-field microscopy is also an excellent way to observe microbial motility, as bundles of flagella (the structures responsible for swimming motility) are often resolvable with this technique.

C. Fluorescence Microscopy: Fluorescence microscopes are used to visualize specimens that fluoresce—that is, emit light of one color following absorption of light of another color. Cells fluoresce either because they contain naturally fluorescent substances such as chlorophyll or other fluorescing components, a phenomenon called auto-fluorescence or because the cells have been stained with a fluorescent dye. DAPI (4,6-diamidino-2-phenylindole) is a widely used fluorescent dye, staining cells bright blue because it complexes with the cell’s DNA. DAPI can be used to visualize cells in various habitats, such as soil, water, food, or a clinical specimen. Fluorescence microscopy using DAPI or related stains is therefore widely used in clinical diagnostic microbiology and also in microbial ecology for enumerating bacteria in a natural environment.

Three-dimensional imaging in light microscopy

All the above microscopes are light microscopes and the images observed through these are two-dimensional only. In order to visualize the images in three-dimensional way, certain forms of light microscopes were developed. These include (1) Differential interface contrast microscope; (2) Atomic force microscope and (3) Confocal scanning laser microscope.

A. Differential interference contrast (DIC) microscopy: It is a form of light microscopy that employs a polarizer in the condenser to produce polarized light (light in a single plane). The polarized light then passes through a prism that generates two distinct beams. These beams traverse the specimen and enter the objective lens where they are recombined into one. Because the two beams pass through different substances with slightly different refractive indices, the combined beams are not totally in phase but instead create an interference effect. This effect visibly enhances subtle differences in cell structure. Thus, by DIC microscopy, cellular structures such as the nucleus of eukaryotic cells or endospores, vacuoles, and granules of bacterial cells, appear more three-dimensional. DIC microscopy is typically used for observing unstained cells because it can reveal internal cell structures that are nearly invisible by the bright-field technique.

Atomic Force Microscopy: This microscope is useful for three-dimensional imaging of biological structures. In atomic force microscopy, a tiny stylus is positioned extremely close to the specimen such that weak repulsive forces are established between the probe on the stylus and atoms on the surface of the specimen. During scanning, the stylus surveys the specimen surface, continually recording any deviations from a flat surface. The pattern that is generated is processed by a series of detectors that feed the digital information into a computer, which then outputs an image. Although the images obtained from an AFM appear similar to those from the scanning electron microscope, the AFM has the advantage that the specimen does not have to be treated with fixatives or coatings. The AFM thus allows living specimens to be viewed, something that is generally not possible with electron microscopes.

Confocal scanning laser microscope (CSLM): It is a computerized microscope that couples a laser source to a fluorescent microscope. This generates a three-dimensional image and allows the viewer to profile several planes of focus in the specimen. The laser beam is precisely adjusted such that only a particular layer within a specimen is in perfect focus at one time. By precisely illuminating only a single plane of focus, the CSLM eliminates stray light from other focal planes. Thus, when observing a relatively thick specimen such as a microbial biofilm, not only are cells on the surface of the biofilm apparent, as would be the case with conventional light microscopy, but cells in the various layers can also be observed by adjusting the plane of focus of the laser beam. Using CSLM it has been possible to improve on the 0.2 micrometer resolution of the compound light microscope to a limit of about 0.1 micrometer.

Cells in CSLM preparations are typically stained with fluorescent dyes to make them more distinct. Alternatively, false-color images of unstained preparations can be generated such that different layers in the specimen are assigned different colors. The CLSM comes equipped with computer software that assembles digital images for subsequent image processing. Thus, images obtained from different layers can be digitally overlaid to reconstruct a three-dimensional image of the entire specimen. CSLM has found widespread use in microbial ecology, especially for identifying populations of cells in a microbial habitat or for resolving the different components of a structured microbial habitat, such as a biofilm (Figure 2.8a). CSLM is particularly useful anywhere thick specimens are assessed for microbial content with depth.

Electron Microscopy

Electron microscopes use electrons instead of visible light (photons) to image cells and cell structures. Electromagnets function as lenses in the electron microscope, and the whole system operates in a vacuum. Electron microscopes are fitted with cameras to allow a photograph, called an electron micrograph, to be taken.

Transmission Electron Microscopy (TEM): TEM is used to examine cells and cell structure at very high magnification and resolution. The resolving power of a TEM is much greater than that of the light microscope, even enabling one to view structures at the molecular level. This is because the wavelength of electrons is much shorter than the wavelength of visible light, and wavelength affects resolution. For example, whereas the resolving power of a high-quality light microscope is about 0.2 micrometer, the resolving power of a high-quality TEM is about 0.2 nanometer. With such powerful resolution, even individual protein and nucleic acid molecules can be visualized in the transmission electron microscope. Unlike visible light, however, electron beams do not penetrate very well; even a single cell is too thick to reveal its internal contents directly by TEM. Consequently, special techniques of thin sectioning are needed to prepare specimens before observing them. A single bacterial cell, for instance, is cut into many, very thin (20–60 nm) slices, which are then examined individually by TEM. To obtain sufficient contrast, the preparations are treated with stains such as osmic acid, or permanganate, uranium, lanthanum, or lead salts. Because these substances are composed of atoms of high atomic weight, they scatter electrons well and thus improve contrast.

Scanning Electron Microscopy: If only the external features of an organism are to be observed, thin sections are unnecessary. Intact cells or cell components can be observed directly by TEM with a technique called negative staining. Alternatively, one can image the specimen using a scanning electron microscope (SEM). In scanning electron microscopy, the specimen is coated with a thin film of a heavy metal, such as gold. An electron beam then scans back and forth across the specimen. Electrons scattered from the metal coating are collected and activate a viewing screen to produce an image. In the SEM, even fairly large specimens can be observed, and the depth of field (the portion of the image that remains in sharp focus) is extremely good. A wide range of magnifications can be obtained with the SEM, from as low as 15x up to about 100,000x, but only the surface of an object is typically visualized.

Electron micrographs taken by either TEM or SEM are black and- white images. Often times, false color is added to these images to boost their artistic appearance by manipulating the micrographs with a computer. But false color does not improve resolution of the micrograph or the scientific information it yields; resolution is set by the magnification used to take the original micrograph.

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