Microscopy
essentially deals with the following three
(
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
.
Concepts
It is worthwhile to mentio
n here that before one looks into the different instruments related tomicroscopy 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 microscope
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, Phasecontrast
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 colourless
; 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 4.6.1.1.)
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 microorganisms
and
molecules in greater depth, such as : (a) Scanning Probe Microscope ; and (b) Scanning
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 microscopes
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.
Bright-Field Microscope
In actual, practice, the
‘ordinary microscope’ is usually refereed to as a bright-field microscope
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 isprovided 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.
[Adapted From : Tortora GJ
et. al. Microbiology an Introduction, The Benjamin/CummingsPublishing Co., Inc., New York, 5th edn, 1995].
(
a) Bright-field : Shows the path of light in the bright-field microscopy i.e., the specific kind of illumination
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.
Differential Interference Contrast (DIC) Microscope
The
differential interference contrast (DIC microscope bears a close resemblance to the phasecontrast
microscope
(Section 4.6.2.2.3.) 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 phasecontrast
microscope, due to addage of contrasting colours to the specific specimen.
Fluorescence Microscope
Interestingly, the various types of
microscopes discussed so far pertinently give rise to an ‘image’
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 luminescencebright 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 yellow
on being exposed to UV-light, gets strongly absorbed by
M. tuberculosis (a pathogenic ‘tuberculosis’
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 ofanthrax.
Methodology :
The various steps involved in the operational procedures of a fluorescence microscope
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 Infrared
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 fluorescent
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 remove
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 microorganisms
duly stained with either
fluorochromes e.g., acridine orange, and diamidino-2-phenylindole
(DAPI)
-a DNA specific stain or fluorochrome-labeled probes.
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 magnification
of objects. The final image is adequately viewed on a fluorescent screen or recorded on a photographic
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 1,00,000times 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 :
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 speciallyprepared ultrathin section of the investigative specimen, as illustrated
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 electrons
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.
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 surfaces
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.
duly depicts the diagramatic sketch fo a
Scanning Electron Microscope (SEM) that
vividly shows the
primary electrons sweeping across the particular investigative specimen togetherwith 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 microorganisms
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 accordingly
for record.
Notes :
(
i) The actual and exact number of secondary electrons that ultimately reach the ‘detector’ exclusively
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 ‘secondary
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 appearance.
Thus, one may obtain a realistic 3D image of the surface of the microorganism having a
visible intensive depth of focus.