Microscopy : The Different Instruments

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Chapter: Pharmaceutical Microbiology : Identification of Microorganisms

Microscopy essentially deals with the following three cardinal goals, namely : (i) Examination of ‘objects’ via the field of a microscope, (ii) Technique of determining particle size distribution by making use of a microscope, and (iii) Investigation based on the application of a microscope e.g., optical microscopy, electron microscopy.

Microscopy : The Different Instruments


Microscopy essentially deals with the following three cardinal goals, namely :


(i) Examination of ‘objects’ via the field of a microscope,


(ii) Technique of determining particle size distribution by making use of a microscope, and


(iii) Investigation based on the application of a microscope e.g., optical microscopy, electron microscopy.




It is worthwhile to mention here that before one looks into the different instruments related to microscopy one may have to understand the various vital and important cocepts, such as :


Light microscopes usually make use of glass lenses so as to either bend or focus the emerging light rays thereby producing distinct enlarged images of tiny objects. A light micro-scope affords resolution which is precisly determined by two guiding factors, namely :

(a) Numerical aperture of the lens-system, and

(b) Wavelength of the light it uses :

However, the maximum acheivable resolution is approximately. 0.2 μm.


Light microscopes that are commonly employed are : the Bright field, Darkfield, Phase-contrast and Fluorescence microscopes. Interestingly, each different kind of these variants give rise to a distinctive image ; and, hence may be specially used to visualize altogether different prevailing aspects of the so called microbial morphology.


As a rather good segment of the microorganisms are found to be almost virtually colour-less ; and, therefore, they are not so easily visible in the Bright field Microscope directly which may be duly fixed and stained before any observation.


One may selectively make use of either simple or differential staining (see Section to spot and visualize such particular bacterial structures as : capsules, endospores and flagella.


The Transmission Electron Microscope accomplishes real fabulous resolution (approx. 0.5 nm) by employing direct electron beams having very short wave length in comparison to the visible light.


The Scanning Electron Microscope may used to observe the specific external features quite explicitely, that produces an image by meticulously scanning a fine electron beam onto the surface of specimens directly in comparison to the projection of electrons through them.


Advent of recent advances in research has introduced two altogether newer versions of microscopy thereby making a quantam jump in the improvement and ability to study the microor-ganisms and molecules in greater depth, such as : (a) Scanning Probe Microscope ; and (b) Scan-ning Laser Microscope.


Microscope Variants


Microbiology invariably deals with a host of microorganisms which are practically invisible with the unaided eye. This particular discipline essentially justifies the evolution of a variety of micro-scopes with crucial importance so that the scientists could carry out an elaborated and meaningful research.


The variants in microscopes are as stated under :

(a) Bright-field Microscope,

(b) Dark-field Microscope,

(c) Phase-contrast Microscope,

(d) Differential Interference Contrast (DIC) Microscope,

(e) Fluorescence Microscope, and

(f) Electron Microscope.


The aforesaid microscope variants shall now be treated individually and briefly in the sections that follows.


(a) Bright-Field Microscope


In actual, practice, the ‘ordinary microscope’ is usually refereed to as a bright-field micro-scope by virtue of the fact that it gives rise to a distinct dark image against a brighter background.


Description : Bright-field microscope essentially comprises of a strong metalic body with a base and an arm to which the various other components are duly attached as shown in Fig. 4.3 (a). It is provided with a ‘light source’ either an electric bulb (illuminator) or a plano-concave mirror strategi cally positioned at the base. Focusing is accomplished by two knobs, first, coarse adjustment knob, and secondly, fine adjustment knob which are duly located upon the arm in such a manner that it may move either the nosepiece or the stage so as to focus the image sharply.


In fact, the upper segment of the microscope rightly holds the body assembly to which is attached a nosepiece or eyepiece(s) or oculars. However, the relatively advanced microscopes do possess eyepieces meant for both the eyes, and are legitimately termed as binocular microscopes. Importantly, the body assembly comprises of a series of mirrors and prisms in order that the tubular structure very much holding the eyepiece could be tilted to afford viewing convenience. As many as 3 to 5 objectives having lenses of varying magnifying power that may be carefully rotated to such a position which helps in clear viewing of any objective help under the body assembly. In the right ideal perspective a microscope must be parfocal*.


In order to achieve high magnification (× 100) with markedly superb resolution, the lens should be of smaller size. Though it is very desired that the light travelling via the specific specimen as well as the medium to undergo refraction in a different manner, at the same time it is also preferred not to have any loss of light rays after they have gained passage via the stained specimen. Therefore, to preserve and maintain the direction of light rays at the maximum magnification, an immersion oil is duly placed just between the ‘glass slide’ and the ‘oil immersion objective lens’, as depicted in Fig. 4.3 (b). Interestingly, both ‘glass’ and ‘immersion oil’ do possess the same refractive index ; and, therefore, rendering the ‘oil’ as an integral part of the optics of the glass of the microscope. In fact, the ‘oil’ exerts more or less the identical effect as would have been accomplished by enhancing the diameter of the ‘objective’ ; and, therefore, it critically and significantly elevates the resolving power of the lenses. Thus, the condenser gives rise to a bright-field illumination.


(b) Dark-Field Microscope


A Dark-Field microscope is employed particularly for the examination of ‘living microbes’ which are either invisible in the ordinary light microscope i.e., cannot be properly stained by standard methods, or get distorted to a great extent after due staining that their characteristic features may not be identified satisfactorily. It essentially makes use of darkfield condenser comprising of an ‘opaque disc' instead of a normal condenser. In this particular instance the opaque disc blocks the passage of light completely which would have gained entry into the objective almost directly. Thus, the only light which is specifically reflected back the specimen under examination actually enters the objective lens precisly as illustread in Fig. 4.4(a) and (b).


The Dark-Field Microscopy has been successfully used in the following highly specific investi¬gative studies, such as :

(a) Examination of unstained bacteria suspended in an appropriate liquid,

(b) Studies related to the internal structure as observed in eukaryotic microbes, and

(c) Examination of highly specific and very thin spirochaetes e.g., Treponema pallidum - the causative agent of syphilis.


(c) Phase-Contrast Microscope


The development of the phase-contrast microscope came into being on account of the fact that generally the unpigmented living cells fail to show their presence vividly in the briglit-field micro¬scope as there exists practically no difference in contrast between the cells and water. Therefore, it became almost necessary to have the microorganisms first fixed and stained before carrying on with the observational procedures in order to enhance the much desired contrast as well as to produce distinct variations in colour composition between the prevailing cell structures.


Importantly, a phase-contrast microscope helps in the conversion of ensuing minimal differ¬ences both in the refractive index and cell density directly into appreciable detectable variations in the ‘light intensity’ ; and. therefore, ultimately serves as an excellent means and device to observe the living cells most conveniently, as shown in Fig. 4.5.


The actual behaviour of both undeviated and deviated or even undiffracted rays in the dark- phasc-contrast microscope is depicted in Fig. 4.6. As the light rays do have a tendency to cancel each other out significantly, the final observed image of the specific specimen under investigation shall ap¬pear as dark against a relatively brighter background.


The ensuing contrast-light pathways of bright-field, dark-field, and phase-contrast microscopes have been explicitely illustrated in the following Fig. 4.7.


(a) Bright-field : Shows the path of light in the bright-field microscopy i.e., the specific kind of illumi-nation produced by regular compound light microscopes.


(b) Dark-field : Depicts the path of light in the dark-field microscopy i.e., it makes use of a special condenser having an opaque disc which categorically discards all light rays in the very centre of the beam. Thus, the only light which ultimately reaches the specimen is always at an angle ; and thereby the only light rays duly reflected by the specimen (viz., gold rays) finally reaches the objective lens.


(c) Phase-contrast : Illustrates the path of light in the phase-contrast microscopy i.e., the light rays are mostly difracted altogether in a different manner ; and, therefore, do travel various pathways to reach the eye of the viewer. Thus, the diffracted light rays are duly indicated in gold ; whereas, the undiffracted light rays are duly shown in red.


(d) Differential Interference Contrast (DIC) Microscope


The differential interference contrast (DIC microscope bears a close resemblance to the phase-contrast microscope (Section wherein it specifically produces an image based upon the ensuing differences in two fundamental physical parameters, namely : (a) refractive indices ; and (b) thickness. In acutal practice, two distinct and prominent beams of plane-polarized light strategically held at right angles to each other are duly produced by means of prisms. Thus, in one of the particular set-ups, first the object beam happens to pass via the specimen ; and secondly the reference beam is made to pass via a clear zone in the slide. Ultimately, after having passed via the particular specimen, the two emerging beams are combined meticulously thereby causing actual interference with each other to give rise to the formation of an ‘image’.


Applications : DIC-microscope helps to determine :

(1) Live, unstained specimen appears usually as 3D highly coloured images.

(2) Clear and distinct visibility of such structures : cell walls, granules, vacuoles, eukaryotic nuclei, and endospores.


Note : The resolution of a DIC-Microscope is significantly higher in comparison to a standard phase-contrast microscope, due to addage of contrasting colours to the specific specimen.


(e) Fluorescence Microscope


Interestingly, the various types of microscopes discussed so far pertinently give rise to an ‘im-age’ from light which happens to pass via a specimen. It is, however, important to state here that the fluorescence microscopy exclusively based upon the inherent fluorescence characteristic feature of a ‘substance’ i.e., the ability of an object (substance) to emit light distinctly. One may put forward a plausible explanation for such an unique physical phenomenon due to the fact that ‘certain molecules’ do absorb radiant* energy thereby rendering them highly excited ; however, at a later convenient stage strategically release a reasonable proportion of their acquired trapped energy in the form of ‘light’ (an energy). It has been duly proved and established that any light given out by an excited molecule shall possess definitely a longer wavelength (i.e., having lower energy) in comparison to the radition absorbed initially’.


Salient Features : There are certain ‘salient features’ with regard to the fluorescence microscopy as stated under :


(1) Quite a few microbes do fluoresce naturally on being subjected to ‘special lighting’.


(2) Fluorochromes : In such an instance when the ‘specimen under investigation’ fails to fluoresce normally, it may be stained adequately with one of a group of fluorescent dyes termed as ‘fluorochromes’.


(3) Microorganisms upon staining with a fluorochrome when examined with the help of a fluorescence microscope in an UV or near-UV light source, they may be observed as luminescence bright objects against a distinct dark background.


Examples : A few typical examples are as follows :


(a) Mycobacterium tuberculosis : Auramine O (i.e., a fluorochrome) that usually glows yel-low on being exposed to UV-light, gets strongly absorbed by M. tuberculosis (a pathogenic ‘tuberculo-sis’ causing organism). Therefore, the dye when applied to a specific sample being investigated for this bacterium, its presence may be detected by the distinct visualization of bright yellow microbes against a dark background,


(b) Bacillus anthracis : Fluorescein isothiocyanate (FITC) (i.e., a fluorochrome) stains B. anthracis particularly and appears as ‘apple green’ distinctly. This organism is a causative agent of anthrax.

Fig. 4.8. illustrates the diagramatic sketch of the vital components and the underlying principles of operation of a Fluorescence Microscope.


Methodology : The various steps involved in the operational procedures of a fluorescence mi-croscope are as under :


(1) A particular specimen is exposed to UV-light or blue light or violet light thereby giving rise to the formation of an image of the ‘specified object’ along with the resulting fluorescence light.


(2) A highly intense beam is duly generated either by a Mercury Vapour Lamp (a) or any other appropriate source ; and the ensuing heat transfer is duly limited by a specially designed Infra-red Filter (b).


(3) Subsequently, the emerged light is made to pass through an Exciter Filter (c) which allows the specifc transmission of exclusively the desired wavelength.


(4) A Darkfield Condenser (d) critically affords a black background against which the fluo-rescent objects usually glow.


(5) Invariably the particular specimen is stained with fluorochrome (dye molecule) (e), which ultimately fluoresces brightly on being exposed to light of a particular wavelength ; whereas, there are certain organisms that are autofluorescing in nature.


(6) A Barrier Filter (f) is strategically positioned after the objective Lenses (g) helps to re-move any residual UV-light thereby causing two important functional advantages, namely :

(i) To protect the viewer’s eyes from getting damaged, and

(ii) To suitably eliminate the blue and violet light thereby minimising the image’s actual contrast.


Applications : The various useful applications of a Fluorescence Microscope are as enumerated under :


(1) It serves as an essential tool in both ‘microbial ecology’ and ‘medical microbiology’.


(2) Important microbial pathogens like : M. tuberculosis may be distinctly identified in two different modalities, for instance :

(i) particularly labeling the microorganisms with fluorescent antibodies employing highly specialized immunofluorescence techniques, and

(ii) specifically staining them (microbes) with fluorochromes.


(3) Ecological investigative studies is usually done by critically examining the specific micro-organisms duly stained with either fluorochromes e.g., acridine orange, and diamidino-2-phenylindole (DAPI)-a DNA specific stain or fluorochrome-labeled probes.


(f) Electron Microscope


An electron microscope refers to a microscope that makes use of streams of electrons duly deflected from their course either by an electrostatic or by an electromagnetic field for the magnifica-tion of objects. The final image is adequately viewed on a fluorescent screen or recorded on a photo-graphic plate. By virtue of the fact that an electron microscope exhibits greater resolution, the ensuing images may be magnified conveniently even upto the extent of 4,00,000 diameters.


It is, however, pertinent to mention here that objects that are smaller than 0.2 μm, for instance : internal structures of cells, and viruses should be examined, characterized and identified by the aid of an electron microscope.


Importantly, the electron microscope utilizes only a beam of electrons rather than a ray of light. The most acceptable, logical, and plausible explanation that an electron microscope affords much prominent and better resolution is solely on account of the fact that the ‘electrons’ do possess shorter wavelengths significantly. Besides, the wavelengths of electrons are approximately 1,00,000 times smaller in comparison to the wavelengths of visible light.


Interestingly, an electron microscope predominantly employs electromagnetic lenses, rather than the conventional glass lenses in other microscopes ; and ultimately, focused upon a ‘specimen’ a beam of electrons which is made to travel via a tube under vacuum (so as to eliminate any loss of energy due to friction collision etc.).


Types of Electron Microscopes :


The electron microscopes are of two different types, namely :


(a) Transmission Electron Microscope (TEM), and


(b) Scanning Electron Microscope (SEM).


These two types of electron microscopes shall be discussed briefly in the sections that follows :


(a) Transmission Electron Microscope (TEM)


The transmission electron microscope (TEM) specifically makes use of an extremely fine focused beam of electrons released precisely from an ‘electron gun’ that penetrates via a specially prepared ultrathin section of the investigative specimen, as illustrated in Fig. 4.9.


Methodology : The various operational steps and vital components of TEM are described below :


(1) The Electron Gun i.e., a pre-heated Tungsten Filament, usually serves as a beam of elec-trons which is subsequently focussed upon the desired ‘specimen’ by the help of Electromagnetic Condeser Lens.


(2) Because the electrons are unable to penetrate via a glass lens, the usage of doughnut-shaped electromagnets usually termed as Electromagnetic Objective Lenses are made so as to focus the beam properly.


(3) The entire length of the column comprising of the vairous lenses as well as specimen should be maintained under high vacuum*.


(4) The specimen causes the scattering of electrons that are eventually gaining an entry through it.


(5) The ‘electron beam’ thus emerged is adequately focused by the aid of Electromagnetic Projector Lenses strategically positioned which ultimately forms an enlarged and distinctly visible image of the ‘specimen’ upon a Fluorescent Screen (or Photographic Plate).


(6) Specifically the appearance of a relatively denser region in the ‘specimen’ helps to scatter much more electrons ; and, hence, may be viewed as darker zones in the image because only fewer electrons happen to touch that particular zone of the fluorescent screen (or photographic plate).


(7) Finally, in a particular contrast situation, the electron-transparent zones are definitely brighter always. The ‘screen’ may be removed and the image may be captured onto a ‘photographic plate’ to obtain a permanent impression as a record.


(b) Scanning Electron Microscope (SEM)


As it has been discussed under TEM that an image can be obtained from such radiation which has duly transmitted through a specimen. In a most recent technological advancement the Scanning Electron Microscope (SEM) has been developed whereby detailed in-depth examination of the sur-faces of various microorganisms can be accomplished with excellent ease and efficiency. In reality, the SEM markedly differs from several other electron microscopes wherein the image is duly obtained right from the electrons that are strategically emitted by the surface of an object in comparison to the transmitted electrons. Thus, there are quite a few SEMs which distinctly exhibit a resolution of 7 nm or even less.


Fig. 4.10. duly depicts the diagramatic sketch fo a Scanning Electron Microscope (SEM) that vividly shows the primary electrons sweeping across the particular investigative specimen together with the knock electrons emerging from its surface. In actual practice, these secondary electrons (or knock electrons) are meticulously picked up by a strategically positioned collector, duly amplified, and transmitted onto a viewing screen or a photographic plate (to have a permanent record/impression of the investigative specimen).


Methodology : The various steps involved in the operative sequential steps are as stated under :


(1) Specimen preparation : It is quite simple and not so cumbersome ; and even in certain cases one may use air-dried specimen for routine examination directly. In general, largely the microor-ganisms should first be fixed, dehydrated, and dried meticulously so as to preserve not only the so called ‘surface-structure’ of the specimen but also to prevent the ‘possible collapse of the cells’ when these are directly exposed to the SEM’s high vacuum. Before, carrying out the usual viewing activities, the dried samples are duly mounted and carefully coated with a very thin layer of metal sheet in order to check and prevent the buildup of an accumulated electrical charge onto the surface of the specimen and to provide a distinct better image.


(2) SEM helps in the scanning of a relatively narrow and tapered electron beam both back and forth onto the surface of the specimen. Thus, when the beam of electron happens to strike a specific zone of the specimen, the surface atoms critically discharge a small shower of electrons usually termed as ‘secondary electrons’, which are subsequently trapped and duly registered by a specially designed detector.


(3) The ‘secondary electrons’ after gaining entry into the ‘detector’ precisely strike a scintillator thereby enabling it to emit light flashes which is adequately converted into a stream of elctrical current by the aid of a photomultiplier tube. Finally, the emerging feeble electrical current is duly amplified.


(4) The resulting signal is carefully sent across to a strategically located cathode-ray tube, and forms a sharp image just like a television picture, that may be either viewed or photographed accord-ingly for record.


Notes :

(i) The actual and exact number of secondary electrons that ultimately reach the ‘detector’ exclu-sively depends upon the specific nature of the surface of the investigative specimen.

(ii) The ensuing ‘electron beam’ when strikes a raised surface area, a sizable large number ‘sec-ondary electrons’ gain due entry into the ‘detector’ ; whereas, fewer electrons do escape a depression in the surface of the specimen and then reach the detector. Therefore, the raised zones appear comparatively lighter on the screen, and the depressions are darker in appear-ance. Thus, one may obtain a realistic 3D image of the surface of the microorganism having a visible intensive depth of focus.


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