MICROSCOPY / MICROSCOPES

Microscopes are magnifying piece of instruments that are used for studying microorganisms (including bacteria, fungi, algae, viruses, and protozoa) which are too small to be seen by a naked eye. Since microorganisms are invisible to the human eyes, nothing was known about them until the 1600s when the works of Antony Von Leeuwenhoek and Robert Hooke brought them into limelight through their innovative development and use of the microscope to view and make clear the microbial world. The scientific technique of preparing microorganisms and specimens/samples of clinical and non-clinical origin for viewing under the microscope is generally referred to as microscopy.

Microscopy is the technical field of magnifying smaller forms of living things which cannot be seen by the normal human eyes using the microscopes. The field of microbiology flourished following the discovery and application of microscopes which made it possible for scientists (especially the microbiologists) to see the very small forms of living organisms (microorganisms) that are ubiquitous in our environment. The microscopes therefore, are invaluable tools for both a microbiologist and the medical laboratory because of its exceptional ability to produce the true imaging of microorganisms which cannot be seen with an unaided eye.

Microscopes enable the human eyes to observe smaller forms of life (i.e. the microbial world) in a more magnified, clear and enlarged form using a combination of lenses. The field of Microbiology as an applied science developed at a very faster pace because of the availability of the microscope (which made clear the microbial world that the naked eyes could not see) and the ability to isolate and grow pure cultures of microorganisms (which are necessary for the proper understanding of the form and development of microorganisms). The discovery and outright development of the microscopes with key microscopy innovations transformed the fields of microbiology and biology and, the application of microscopy is still a very essential technique in both the physical and life sciences.

BRIEF HISTORY OF THE ADVENT OF THE MICROSCOPE

Before the advent of microscopes and its actual application in viewing and making clear the microbial world which our eyes are not competent enough to see, mankind in time past have been using shiny stones and glasses to view objects; and they discovered that the objects become enlarged when viewed under such objects they termed “magnifying objects or magnifiers”. Top among those that experimented on the use of such glasses in the first century (100 AD) to view objects were the Romans who discovered that viewing objects using the magnifiers made them to appear larger. They named these magnifying objects “lenses” because their shapes resemble that of the lentil bean seeds.

The term “lens” was not adopted and used until the end of the 13th century when the eye glasses (also known as spectacles) was developed by an Italian called Salvino D’Armate who invented the world’s first wearable eye glasses. During this period, other spectacle/glass makers continued experimenting on different glasses (or lenses) and they discovered magnifying glasses (with about 6x-10x magnification) which were only used as at the time to observe small/tiny insects such as fleas. These magnifying glasses were the earlier microscopes, and they were simply small tubes made of a plate at one end for the object and, a lens at the other end for magnification of the object being viewed.

The first compound microscope (i.e. a microscope that uses two or more lenses) was actually invented in 1590 by Hans Janssen and his son, Zacharias Janssen who were both Dutch spectacle/eye glass makers and, who co-experimented on the earlier lenses used previously for viewing objects (e.g. tiny insects). The Janssen’s microscopes were able to provide a much larger and enlarged image of objects than the previously discovered magnifying glasses (simple microscopes). Their experiments and breakthrough in this aspect gave impetus to Galileo (the father of modern physics and astronomy) who developed the telescope with a much better focusing device in 1609. Galileo’s work helped describe the working principles of lenses and light rays and, this helped to improve the microscopes and even the telescope that he discovered.

In 1660, Robert Hooke (1635-1703), an English Physicist observed some biological materials with the best compound microscopes as at the time, and he discovered small pores in them which he termed “cells”. Hooke’s findings were published in a journal known as the “Micrographia”- which showed detailed drawings of his discoveries. The first real microscope was actually discovered and invented in the late 17th century (precisely 1676) by Anthony Von Leeuwenhoek (1632-1723), a Dutch draper (clothing and dry goods dealer) from Holland who developed one of the finest simple microscopes (with a magnification of about 270x) as at the time.

Anthony Von Leeuwenhoek is regarded as the father of microscopy and the field of microbiology because of his many biological discoveries that was made possible with his new improved microscope which enabled him to see some living things that was never discovered as at the time. He was the first person to see and describe bacteria, blood cells, yeast cells and other tiny forms of life. Leeuwenhoek’s work together with that of Hooke’s helped to further study in to the field of microbiology and other biological sciences. Carl Zeiss and Ernst Abbe pioneered the development of immersion lenses in 1883. Both helped to improve the basic understanding about the optical principles and quality of the microscope. The first transmission electron microscope was constructed by Ernst Ruska in 1933.

Very little was made in the development of the microscope until the late 19th century that ushered in great innovations in the field of microscopy which lead to the development of very fine optical lenses and equipment, thus making the use of microscope a very important tool for scientists as at the time even till date. Top among the scientists that improved the technical workings of the microscope was the American scientist; Charles A. Spencer (1813-1881) who was America’s first microscope maker and, who was famously noted for his development of achromatic objectives with great aperture angle. Joseph Jackson showed in 1830 that several weak lenses used together at certain distances gave good magnification without blurring the image of the object. In 1872, Ernst Abbe developed a mathematical formular called the “Abbe Sine Equation” which made possible the maximum resolutions of microscope lenses. The phase contrast microscope was invented in 1936 by Frits Zernike while the scanning microscope was invented by Gerd Binnig and Heinrich Rohrer in 1981.

RESOLUTION AND MAGNIFICATION: THE OPTICS BEHIND THE MICROSCOPE

Resolution is the ability of a microscope’s lens to distinguish between (two) small objects that are close together as distinct and separate. It is the ability of a lens system to allow an observer (the microbiologist) to see fine details of different small objects even when they are close together. The resolution (resolving power) of a microscope is dependent on the quality of the objective lens which must produce a clear image and not necessarily an enlarged image of an object being viewed. The greater the resolving power of the objective lenses of microscope, the clearer the image produced. The resolution range of the normal human eyes is not sufficient enough (or competent) to view, enlarge and produce a clear image of objects (or small forms of living things).

Thus; with the aid of a microscope whose objective lenses have a good resolving power, two closely spaced elements of an object or specimen can be properly made clear and distinguished for better interpretation by the microscopist (i.e. a scientist that uses the microscope for research). The resolving power of a microscope is both a function of the wavelength of light used and the quality of the objective lenses of the microscope. The measure of the light gathering ability of a microscopes objective lens is known as the numerical aperture (NA). Notably, lenses with higher magnification usually have higher numerical apertures.

The resolving power of a microscope is given by the following formular:

Resolving power (RP) = 0.5λ / Numerical aperture

Where λ = wavelength of light used

0.5 = the diameter of the smallest object that can be resolved by any lens

Numerical aperture = n sin θ

The resolving power of a microscope is greatest when blue light is used to illuminate an object or a specimen, and this works in line with an objective lens that has a very high numerical aperture. The reason for the preference of blue light over white light in providing a good resolution of a specimen is because blue light has a shorter wavelength than the red or white light. This has lead many manufacturers of the light microscope to fit in blue light filters over the condenser lens of microscopes in order to improve its resolving power.

Oil immersion lenses or objectives are lenses of microscopes (usually designated as 100x) on which immersion oil is used; and the 100x objectives are mainly used for viewing stained slides. The purpose of immersion oil is to increase the light gathering ability (NA) of a lens. It does this by allowing rays emerging from an object or specimen at angles to be collected and viewed. These rays would have been lost to the objective lens, but the immersion oil helps to collect them for a better and clear image of the specimen. The resolving power of a microscope’s objective lens can be significantly increased through a proper specimen illumination during viewing.

MAGNIFICATION

Magnification is the enlargement of a specimen or an object seen through a microscope. It is a function performed solely by two parts of a microscope i.e. the eye piece (ocular) lenses and the objective lenses. The final image of a specimen under a microscope is seen by the ocular lens after being received from the objective lens that first magnifies the image to produce the true image. The total magnification of an image is therefore mathematically expressed as follows:

Magnification (m) = magnification of the objective lens X magnification of the eye piece                                             lens.

The magnification of a microscope can be increased without limitation because it is not dependent on physical properties of light waves, but this is not the case for resolving power which is highly dependent on the type of light used. Therefore what the image of an object or specimen seen by a microscopist through a microscope is determined by the resolution and not the magnification of the microscope. Microscopes are fitted with an artificial source of light (usually from a tungsten lamp) in order to ensure proper magnification and resolution of images through effective illumination since the illumination from daylight cannot be controlled. A microscope is normally fitted with about 3 or 4 objective lenses having magnifying power that includes: 4x, 10x, 40x and 100x.

Higher magnifications of a specimen’s image are usually possible with sophisticated microscopes such as the transmission electron microscopes that provide enough resolution for very high magnifications (usually above 100,000x). The light microscopes and other compound microscopes are usually limited in terms of their magnification as they have an upper limit of magnification (which is usually in the range of 1500x) beyond which an image becomes blurred or unclear. The resolution of the objective lens does not improve above this upper limit of magnification of the bright field microscopes; instead the image becomes more and more distorted.

During viewing under the microscope, the objective lenses can be switched or changed from one magnifying power to another (e.g. from 10x to 40x) and, the image of the object or specimen is expected to still remain in focus while this is being done. This property of the microscope is known as parfocality (i.e. the microscope is said to be parfocal). It should be noted that the resolution and magnification of a microscope are normally imprinted on the body of the microscope. The distance between the front surface of an objective lens and the surface of the specimen (when the objective lens is in sharp focus) or the surface of the cover glass (if used) is known as the working distance of the microscope’s objective lens.

FUNCTIONS OF THE PARTS OF A MICROSCOPE

The microscope has various parts that perform specific function; and it is important that scientists and students acquaint themselves of these components and their functions in order to make optimum use of the equipment (Figure 1). An understanding of the different parts of the microscope is vital for the proper use of the apparatus in order to avoid its damage. The main parts of the microscope and their functions shall be discussed in this section.

Figure 1. Bright filed microscope

  • Ocular lens: The ocular lens which can also be called the eye piece lens is used to enlarge the primary image produced by the objective lens.
  • The fine adjustment knob: The coarse adjustment knob is used to obtain a fine and sharp image of a specimen after focusing with the coarse adjustment knob. It is used to bring the object on the stage into perfect focus.
  • The coarse adjustment knob: The coarse adjustment knob is used to focus specimens on the mechanical stage, and it brings the specimen into general focus. It is first used to attain an approximate focus of the object prior to using the fine adjustment knob.
  • The stage: The stage of a bright-field microscope is usually a mechanical one (i.e. it is movable and adjustable) and, it is helps to hold a slide in place for proper focusing while allowing movement to the left or right, and up or down.
  • The objective lens: The objective lens which can also be called the nose piece lens helps to magnify the specimen to produce its real image first before sending it to the ocular lens.
  • The nose piece: The nose piece helps to hold the objective lenses together.
  • The diaphragm: The diaphragm is a lever-like device fitted into the diaphragm and, which is used to control the amount of light reaching the specimen on the stage and the entire lens system of the microscope.
  • The base: The base supports the entire weight of the microscope. When carrying the microscope, one hand is placed underneath the base and while the other holds the arm (or limb).
  • The condenser: The condenser is fitted directly under the stage and, it helps to collect and focus light rays from the illuminator upwards and onto the specimen or object on the stage. It is located between the light source and the stage.
  • The illuminator: The illuminator is the source of light to the microscope.
  • Light control knob: It is used to adjust and control the intensity of the light source (i.e. the illuminator).
  • Stage clip: The stage clip is used to hold glass slide in position so that they can be moved around during viewing.
  • The arm (handle): This is the point where the microscope is held during its movement from one place to another. It can also be called the limb.
  • Stage adjuster: The stage adjuster is used to control the movement of the mechanical stage to and fro (i.e. side by side to the left or right position).

TYPES OF MICROSCOPES

There abound several numbers of microscopes that can be used by a microscopist to view specimens or samples and microorganisms in the laboratory. The choice of the microscope to be used is usually dependent on the task to be performed by the user and, on the type of specimen or microorganism to be investigated. Normally, all the different types of microscope is geared towards serving the same purpose which is to magnify and make clear, small forms of life (i.e. microorganisms) which the normal human eyes cannot be seen. Nevertheless, microscopes still vary in their technicality, choice, and function. Experience on the part of the user of a microscope is very important to getting a better result from their usage. This section describes the different types of microscopes used in the microbiology laboratory.

  1. Compound light microscope

The compound light microscope makes use of visible light to illuminate the cell structure of microorganisms or specimens viewed with it. Compound light microscopes still remain one of the most versatile and available magnifying piece of equipment found in most academic and medical institutions. They are usually fitted with a number of objective lenses and other supplementary lenses which help to magnify the images of specimens. In the compound light microscopes, the primary image of a specimen is formed first by the objective lens and then, it is enlarged by the ocular lens to produce the virtual or actual image. There are about four (4) different types of compound light microscopes that are widely available today.

  1. The bright field microscope

The bright field microscope is the most commonly used microscope in the microbiology laboratory (both in the education and medical profession) for teaching purposes and in observations that are not too detailed. It is usually fitted with two objective lenses and two eye lenses that works together to produce an enlarged and clear image of an object or specimen being viewed with it. The bright field binocular microscope (Figure 1) is limited in its usage in that it cannot observe living cells.

It can only be used to view non-living (or dead) cells and stained specimens or microorganisms. The bright field microscope forms a dark image of a specimen against a brighter background. Microorganisms must first be stained and fixed thereafter before in order to increase contrast and create variations in colour between the cell structures of organisms being viewed under the bright field microscope. Staining helps to improve contrast between the specimen and its surrounding environment when using the bright field microscope. This measure makes it easy to view cells or image of specimens whenever the bright field microscope is used.

  1. The phase contrast microscope

The phase contrast microscope can be used to view living cells of microorganisms unlike the bright field microscope that lacks this ability. It allows microorganisms to be viewed directly without being stained (Note: staining distorts cell features and kills the organism eventually). Phase contrast microscope is widely used for studying eukaryotic cells and, the motility, shape, and cellular components (e.g. spores) of prokaryotic cells. The working principle of the phase contrast microscope is based on the rationale that microbial cells differ in their refractive index; and this variation in cellular components allows some amount of the light passing through the cell to be refracted (or bent) and be magnified, thus making it possible for this microscope to make clear the image of unpigmented living cells.

In phase contrast microscopy, a dark image is formed on a light background. Phase contrast microscopy is widely used in biomedical research because of its unique ability to observe wet preparations of living microorganisms. The differential interference contrast microscope is a compound light microscope that is similar and works with the same principle of the phase contrast microscope. It can be used to observe the endospores, cell walls, vacuoles and other vital cell components of prokaryotic cells and even the nuclei of eukaryotic cells. Differential interference contrast microscope together with other newly developed microscopes such as the confocal scanning laser microscope and the atomic force microscope all detect and produce the images of living microorganisms or specimen being viewed under it in three dimensions.      

  1. The dark field microscope

The dark field microscope is also used to observe living unstained cells and microorganisms. It is a light microscope that is fitted with a modified condenser which allows the light reaching the specimen or cell being viewed to come only from one side. The specimen or cell is not illuminated directly, thus light comes from only one side; and this allows the light to be scattered or diffused by the specimen. This makes the specimen or cell to appear light against a dark background. In dark field microscopy, only refracted light is used to form an image. Dark field microscopy allows living cells to be observed easily and more clearly than when the bright field microscope is used. Cell structures and features such as motility can be observed with this type of microscopy.

  1. The fluorescence microscope

The fluorescence microscope (Figure 2) is used to observe living cells or microorganisms that fluoresces (i.e. organisms that emits coloured lights when light of a different colour is shown on it). Some microorganisms naturally contains substances that emits light in them while others (e.g. M. tuberculosis, E. coli)  can emit light because they have been stained with a fluorescing or fluorescent dye. Other microscopes (including the bright field, dark field and the phase contrast microscope) produce the image of a specimen or a cell by passing light through the specimen being viewed. But this is not the case for the fluorescence microscopy which is based on the light emitted by an organism. In fluorescence microscopy, specimens are exposed to ultraviolet (UV) light and, the image is formed from the light emitted by the specimen or that coated on it. Fluorescence microscope has applications in the detection of antigen-antibody reactions, clinical diagnostics and microbial ecology.

Figure 2. Fluorescence microscope

  1. Electron microscope

The electron microscope makes use of beams of electrons instead of a beam of light (as is the case in other types of microscopes previously described) to form or produce the image of living microorganisms. In electron microscopy, electromagnets instead of lenses are used to focus specimens or living organisms. Electron microscopes are large and expensive and, difficult to use unlike the other types of microscopes. They are usually fitted with internal cameras that produce the photographs of specimens or living cells being viewed with them. Electron microscopes (whose components are enclosed in a tube that allows a complete vacuum) use electromagnetic lenses to form images from electrons that have passed through a thin section of the specimen or microbial cell. The electron microscopy has a very high resolution because electrons are known to have a short wavelength and, wavelength affects the resolving power of microscopes.

Internal structures of both prokaryotic and eukaryotic cells together with viral cells and other microbial cells can be comfortably visualized by the use of electron microscopy. An electron microscope has a resolving power that is much greater than the light microscopes – with a practical resolution that is about 1,000 times better than that of the light microscope. Electron microscopy allows microorganisms to be viewed at the molecular level that is not possible with the use of light microscope. It can magnify images of a specimen or object thousands of times smaller than a wavelength of light; thus, electron microscopy is useful for three-dimensional imaging of living cells or specimens. Special training and techniques are required for specimen preparations prior to the usage of the electron microscope for viewing. Electron microscope is cumbersome and cannot be moved from one place to another as is applicable for other types of microscopes; and the electron microscope is usually coupled in a separate room where it can be used for microscopical analysis. Transmission electron microscope (TEM) and scanning electron microscope (SEM) are examples of electron microscope.

A. Transmission electron microscope (TEM)

The transmission electron microscope allow intact cells of microorganisms and their constituent cell structures to be observed directly by the use of a technique called negative staining (which reveals special structures such as capsules that surrounds a microbial cell). In the use of TEM, thin sections of microorganisms must be prepared in order to allow for the proper viewing and imaging of internal structures of the even the smallest cell. Negative staining allows easy study of the structure of viruses with the use of TEM. TEM is basically used to study the internal surfaces or features of cells and viruses.

B. Scanning electron microscopy (SEM)

The scanning electron microscope is used for the studying of the three-dimensional imaging of microorganisms and, it can also be used to examine the external surfaces of living cells unlike TEM which can be used to view internal surfaces or structures of living cells. SEM allows the external features of microorganisms (e.g. cell wall) to be observed even without the preparation of a thin section of the specimen or cell. In SEM, the specimen or microorganisms is usually covered with a thin film of a heavy metal (e.g. gold or silver) which scatters electrons. The specimen is allowed to go through an electron beam scanning and, the scattered electrons emitted from the metal used for the coating or covering of the specimen are then collected. The collected electrons activate a viewing screen which allows the image produced to be seen. A photograph of the image produced can also be obtained because SEM is fitted with an internal camera for this purpose. SEM is basically used to study the external surfaces or features of microorganisms in an excellent 3-dimensional pattern. Both TEM and SEM are used extensively in many current microbiological and biomedical researches.

  1. Confocal microscopy

Confocal microscope is used in confocal microscopy. Confocal microscopy can also be called confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM). This type of microscopy is an optical imaging technique for increasing optical resolution and contrast of a specimen by means of using a spatial pinhole to block out-of-focus light in image formation. Confocal microscope (Figure 3) has an added advantage over fluorescence microscope which can also perform similar operation like the confocal microscope. Some of the many advantages of confocal microscope include shallow depth of field, elimination of out-of-focus glare, and the ability to collect serial optical sections from thick specimens. Living or fixed cells and tissues can be viewed or observed using confocal microscopy after proper staining of samples with fluorescence dyes or probes. And with the use of confocal microscopy, the three dimensional structure of the sample can be obtained. Confocal microscopy also allows the live viewing of specimens, making it possible to observe cells in their natural milieu.

Figure 3. Confocal microscope

CARE OF THE MICROSCOPE

Due to the critical role of microscope in microbiological and other biomedical researchers, it is very important that microscopes (which allow us to see microorganisms distinctly) are cautiously used and preserved in order to ensure their continued usage and value during laboratory undertakings. Microscopes (whether electron or compound light) are required for the visualization of microorganisms and, without a magnifying instrument like it, it would have been impossible for us to see clearly and understand the major workings of the microbial world. Thus it is very important that all users of the microscopes ensure that they take proper care of them by keeping to some of the following guidelines as they use these all important equipment in the microbiology laboratory:

  1. The lens system of microscopes should be properly cleaned using the appropriate lens tissue and cleaning solvent prior to and after usage in order to ensure a greater efficiency when in use.
  2. The microscope should be carried at the base and arm parts and carefully moved from one place to another in order to avoid dropping.
  3. Cover slips should always be used when viewing wet preparations (i.e. unstained specimens).
  4. When viewing a stained preparation, immersion oil should always be used with the oil immersion objective (100x) in order to achieve a high resolution. The oil immersion objective must make contact with the immersion oil and not the slide.
  5. No part of the microscope should be dismounted or removed without the permission of instructor or scientist in charge of it.
  6. The adjustment knobs and stage adjusters should be carefully used in order to avoid smashing the slide carrying the specimen to be examined.
  7. The bench on which the microscope will be placed must be a balanced bench and, it should be free from all obstructions during its usage.
  8. Both eyes and not one should be used to look through the microscope during the observation of a microscope or specimen.
  9. Cleaning of the microscope should be only done using authorized cleaning materials, and the manufacturers instruction should be kept during such procedures.
  10. Ocular lenses and objective lenses that are not in use should be carefully kept away in tight containers.
  11. Proper light illumination and good stage adjustment should be observed in order to get optimal visualization and focus.
  12. Always ensure that the microscope is covered with its dust cover after use in order to prevent dust from settling on it.
  13. Disconnect the microscope from the source of electricity after use.

REFERENCES

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Glick B.R and Pasternak J.J (2003). Molecular Biotechnology: Principles and Applications of Recombinant DNA. ASM Press, Washington DC, USA.

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Prescott L.M., Harley J.P and Klein D.A (2005). Microbiology. 6th ed. McGraw Hill Publishers, USA.

Willey J.M, Sherwood L.M and Woolverton C.J (2008). Harley and Klein’s Microbiology. 7th ed. McGraw-Hill Higher Education, USA.

 

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