Introduction
to Microscope
Microscopy
is the science of using microscopes to observe objects that are too small to be
seen with the naked eye. It plays a vital role in a wide array of fields
including biology, medicine, materials science, and chemistry.
The
Microscope is an optical instrument consisting of a combination of lenses for
making enlarged or magnified images of minute objects. The term is derived from
the two Greek words ‘micro’ - small, and ‘scope’ - view. Since microorganisms are very small and
invisible to naked eyes, they must be magnified to be clearly seen. The use of
a microscope is absolutely indispensable to study microbiology.
Working
of lenses
When
a ray of light passes from one medium or material to another, refraction occurs,
the light ray will bend at the interface.
Refractive index is the measure of how a medium or material slows the
velocity of light. Glass has higher
refractive index and air have lower refractive index The direction and
magnitude of bending is determined by the refractive indexes of the two media or
materials forming the interface. When light passes from air into glass, it is
slowed and bent toward the normal, a line perpendicular to the surface and as
light leaves glass and returns to air, it accelerates and is bent away from the
normal. As shown in the below image, a
prism bends light because of the different refractive indices of glass and
air.
The Bending of Light by a Prism
(Prescott−Harley−Klein:Microbiology, Fifth Edition)
Lens
act like a collection of prisms operating as a unit. When parallel rays of
light strike the lens, a convex lens will focus these rays at a specific point
termed as the focal point (F). The distance between the centre of the lens and
the focal point is called the focal length (f).
Lens strength is related to focal length; a lens with a short focal length
will magnify an object more than a weaker lens having a longer focal length.
Lens functions like a collection of prisms
(Prescott−Harley−Klein:Microbiology,
Fifth Edition)
Magnification
and resolving power
Magnification
and resolving power are important concepts in microscopy and imaging. Magnification is the process of making an
object appear larger than its actual size. Magnification is expressed as the
ratio of the object's apparent size to its actual size. Resolving power or Resolution is the ability
to distinguish two objects that are close together. While higher magnification
can increase the amount of detail in an image, it doesn't necessarily improve
the ability to distinguish fine details. For example, two images may have the
same magnification, but one may have a higher resolution and be clearer.
The
minimum distance (d) between two objects that reveals them as separate entities
is given by the Abbe equation, in which lambda (λ) is the wavelength of light
used to illuminate the specimen and n sin θ is the numerical aperture (NA).
Numerical
aperture
The
measure for the resolving powers of a lens is the numerical aperture. It is n sin θ. The larger the numerical aperture, then the
greater is the resolving power of the lens. Theta is defined as 1/2 the angle
of the cone of light entering an objective. Light that strikes the microorganism
after passing through a condenser is cone- shaped. When this cone has a narrow
angle and tapers to a sharp point, it does not adequately separate images of
closely packed objects and thus the resolution will be low. If the cone of
light has a very wide angle and spreads out after passing through a specimen, closely
packed objects appear widely separated and thus the resolution will be high.
The
angle of the cone of light that enter a lens depends on the refractive index (n)
of the medium in which the lens works. The refractive index for air is 1.00.
Since sin θ cannot be greater than 1 (the maximum θ is 90° and sin 90° is
1.00), a lens working in air cannot have a numerical aperture greater than
1.00. In order to raise the numerical aperture above 1.00, to achieve higher
resolution, the refractive index is to be increased. This could be achieved by
using immersion oil, a colorless liquid having the same refractive index as
glass (about 1.515). If air is replaced with immersion oil, by adding a drop of
oil on the surface of glass slide containing the specimen, light rays that otherwise
may not enter the objective due to reflection and refraction will now enter the
lens. So, an increase in numerical aperture and thus
increase in resolution results.
Oil
immersion objective operating in air and with immersion oil (Prescott−Harley−Klein:Microbiology,
Fifth Edition)
Types of Microscopes
A
simple microscope consists merely of a single lens or magnifying glass held in
a frame, usually adjustable, and will have a stand for holding the object to be
viewed and a mirror for reflecting light. A compound microscope consists of two
sets of lenses, one known as an objective and the other as an eyepiece. Compound microscopes give more magnification
than simple microscope.
There
are different types of Microscopes. The major being a) Light Microscopes
(Optical Microscopes) and b) Electron Microscopes
A.
Light Microscope (Optical Microscope):
Light
microscopes use visible light and lenses to magnify objects. Magnification typically ranges from 40x to
1000x.
Compound
microscope and its parts (Fundamental Principles of Bacteriology : A.J. Salle)
Parts
of a Light Microscope:
Eyepiece
(ocular lens): The lens you look through, typically 10x magnification.
Objective
Lenses: These are multiple lenses that provide different magnifications
(usually 4x, 10x, 40x, 100x).
Stage:
The flat surface where the slide with the specimen is placed.
Condenser:
Focuses the light onto the specimen.
Diaphragm:
Controls the amount of light that passes through the sample.
Coarse
and Fine Focus Knobs: Adjust the focus of the image.
Illuminator:
A light source, usually a lamp, that illuminates the sample.
Types
of Light Microscopy:
Bright-Field
Microscopy: The most commonly used technique where light passes through the
specimen, and the image appears dark against a bright background.
Dark-Field
Microscopy: Light is directed at an angle to the sample, which makes the image
appear bright against a dark background.
Phase-Contrast
Microscopy: Enhances contrasts in transparent specimens and is useful for
studying live cells.
Fluorescence
Microscopy: Uses ultraviolet (UV) light to excite fluorophores attached to
specimen. When the fluorophores emit light, the specimen appears as brightly coloured
on a dark background.
B)
Electron Microscopes (EM)
Electron
microscopes use electrons instead of light to view specimens at much higher
magnifications and resolutions than light microscopes.
Transmission
Electron Microscope (TEM):
TEM
transmits a beam of electrons through a thin sample. The electrons interact
with the sample, and the resulting image is projected onto a screen or film and
can achieve magnifications up to 10 million times and capable of Resolution at
nanometer scale (less than 1 nm).
Scanning
Electron Microscope (SEM):
SEM
scans a focused electron beam over the surface of the sample. The electrons
interact with the surface and produce signals that are used to form an image. Can
achieve magnifications up to 1 million times and resolution of about 1-10
nanometers.
C.
Scanning Probe Microscopes (SPM)
Scanning
Probe Microscopes work by scanning a sharp probe across the surface of the
specimen. The most common type is the Atomic Force Microscope (AFM). This measures the force between the probe and
the surface and help for the construction of a high-resolution topographical
image.
D.
Other Advanced Microscopy Techniques
Confocal
Microscopy uses laser light to scan specimens and produce high-resolution 3D
images. Confocal microscopes capture optical sections of the sample, which can
then be digitally reconstructed to create a 3D image.
Multiphoton
Microscopy is a type of fluorescence microscopy that uses multiple photons to
excite fluorescent dyes.
Live-Cell
Microscopy is used to observe living cells in real-time, often with the help of
fluorescent dyes or proteins.
Super-Resolution
Microscopy includes techniques like STED (Stimulated Emission Depletion) and PALM
(Photo-Activated Localization Microscopy).
These provide resolutions of less than 100 nm.
The
Bright-Field Microscope
The
ordinary microscope is called a bright-field microscope because it forms a dark
image against a brighter background.
The
microscope consists of a sturdy metal body or stand composed of a base and an
arm to which the remaining parts are attached.
A
light source, either a mirror or an electric illuminator, is located in the
base.
Two
focusing knobs, the fine and coarse adjustment knobs, are located on the arm
and can move either the stage or the nosepiece to focus the image.
The
stage is positioned about halfway up the arm and holds microscope slides by
either simple slide clips or a mechanical stage clip. A mechanical stage allows
the operator to move a slide around smoothly during viewing by use of stage
control knobs.
The
substage condenser is mounted within or beneath the stage and focuses a cone of
light on the slide. Its position often is fixed in simpler microscopes but can
be adjusted vertically in advanced models.
The
curved upper part of the arm holds the body assembly, to which a nosepiece and
one or more eyepieces or oculars are attached. More advanced microscopes have
eyepieces for both eyes and are called binocular microscopes.
The
nosepiece holds three to five objectives with lenses of differing magnifying
power and can be rotated to position any objective beneath the body assembly.
Ideally a microscope should be parfocal—that is, the image should remain in
focus when objectives are changed.
The
objective lens forms an enlarged real image within the microscope, and the
eyepiece lens further magnifies this primary image. The total magnification is
calculated by multiplying the objective and eyepiece magnifications together.
For example, if a 45 X objective is used with a 10 X eyepiece, the overall
magnification of the specimen will be 450 X.
The resolution of a microscope depends upon the numerical aperture of its condenser as well as that of the objective.
The resolution of a light microscope can be calculated using the Abbe equation. The maximum theoretical resolving power of a microscope with an oil immersion objective (numerical aperture of 1.25) and blue-green light is approximately 0.2 µm.
So,
a bright-field microscope can distinguish between two dots which are 0.2 µm
apart, the size of a very small bacterium.
Generally,
a microscope is equipped with three or four objectives (4X, 10X, 40X, 100X). The working distance of an objective lens
is the distance between the front surface of the lens and the surface of the
specimen when it is in sharp focus. Objectives with large numerical apertures
and high resolving power have short working distances.
Normally, Microscopes come with 10 X eyepieces and have an upper limit of about 1,000 X with oil immersion (100 X objective). A 15 X eyepiece may be used to achieve a magnification of 1,500 X.
The
Dark-Field Microscope
The
principle of dark-field microscopy is based on scattering of light. Living,
unstained cells and organisms can be observed by enhancing the contrast by
illuminating them with light at an oblique angle. Light is directed from an angle, typically
using a special condenser to produce a hollow cone of light. This hollow cone of light is focused on the
specimen in such a way that only reflected and refracted rays enter the
objective lens and forms an image. Since
most of the direct light does not enter the objective lens, the background
remains dark, while the scattered light from the specimen appears bright. This
creates a high-contrast image. So, the
field surrounding a specimen appears black, while the object itself is brightly
illuminated.
This
microscopy is used to identify bacteria like Treponema, the causative
agent of syphilis. A microscope may be
converted to dark-field microscope by placing a dark-field stop or light stop
or central stop or central aperture underneath the condenser lens system.
The
central stop is a round piece of black metal, mounted into an attachment, a
slider or the disc of a turret condenser. The stop has to be aligned so that it
is exactly placed in the middle of the ray path.
Phase
Contrast Microscope
A
phase-contrast microscope converts slight differences in refractive index and
cell density into easily detectable variations in light intensity and enables to
observe living cells. It works by converting phase shifts (differences in the
speed of light as it passes through different parts of the specimen) into
variations in brightness, so otherwise invisible structures will become visible.
When light passes through cells, small phase shifts occur, and these small
phase shifts (which are invisible to the human eye) are converted into changes
in amplitude. These changes can be
observed as differences in image contrast in a phase-contrast microscope.
· The
condenser of a phase-contrast microscope has an annular stop (an opaque disk
with a thin transparent ring) which produces a hollow cone of light.
· As
this cone passes through a specimen, some light rays are bent due to variations in
density and refractive index within the specimen. These rays are retarded by about 1/4
wavelength.
· Now
the rays pass through phase-shift ring or the phase plate. The deviated rays pass through the plate and
undeviated light rays go through the ring in the phase plate (a special optical
disk located in the objective). Now the undeviated
light are advanced by 1/4 wavelength.
So,
the deviated and undeviated waves will be about 1/2 wavelength out of phase and Constructive
interference occurs, the waves combine in such a way that their crests (the
highest points) and troughs (the lowest points) align. This
alignment amplifies the overall wave, resulting in a wave with a larger
brightness. The undeviated light forms the bright background and the object
appears dark and well-defined.
Deviated and undeviated light rays in dark phase-contrast microscope (Prescott−Harley−Klein:Microbiology, Fifth Edition)
https://microbenotes.com/phase-contrast-microscopy/
Phase-contrast
microscopy is useful for studying microbial motility, shape of living cells, endospores
and inclusion bodies, etc.
Confocal
Microscopy
Confocal microscopy is a
powerful tool for visualizing and analysing complex biological and material
systems and it provide high-resolution, 3D images.
A conventional light
microscope, uses a mixed wavelength light source and illuminates a large area
of the specimen and thus will have a relatively great depth of field. Here,
images of bacteria from all levels (above, in, and below the plane of focus) in
the field will be visible, and thus image can be fuzzy and crowded.
This problem could be
solved by using Confocal Microscope and Fluorescently stained specimens
are usually examined.
Confocal
Microscopy
(https://images.app.goo.gl/NkU2EbyRL4cCidHaA)
- Laser Source: Provides the excitation light.
- Scanning Mirrors (Dichroic Mirror): Control the position of the laser beam on the sample.
- Objective Lens: Focuses the laser beam onto the sample and collects the emitted fluorescence.
- Pinhole Aperture (Emission pinhole): Blocks out-of-focus light.
- Detector: Detects the emitted fluorescence to form the image which will be done by a computer. Special computer software is used to create high-resolution, 3D images of cell structures and biofilms.
The confocal microscope
improves images in two ways. Firstly,
illumination of one spot at a time reduces interference from rest of the
specimen and secondly, the aperture above the objective lens blocks out the out
of focus light rays. So the image will
have excellent contrast and resolution.
Types of Confocal Microscopes
- Laser Scanning Confocal Microscope (LSCM): The most common type, using a single pinhole to block out-of-focus light.
- Spinning Disk Confocal Microscope (SDCM): Uses a rotating disk with multiple pinholes to increase imaging speed.
- Two-Photon Microscopy: Uses a longer wavelength laser to excite fluorophores, reducing photobleaching and increasing imaging depth.
- Stimulated Emission Depletion (STED) Microscopy: A super-resolution technique that can achieve resolutions beyond the diffraction limit.
Fluorescence Microscope
All
the microscopes produce an image from light that passed through a specimen. An
object that actually emits light can also be observed, and this is the basis of
fluorescence microscopy. Fluorescence is the phenomenon where a substance,
after absorbing light energy, emits light of a different color. Flourescent
molecules absorb radiant energy, become excited and later release much of their
trapped energy as light. The light emitted by this excited molecule will have a
longer wavelength (or lower energy) than the radiation originally absorbed.
In
fluorescence microscopy, the specimen is illuminated with light of a specific
wavelength (ultraviolet, violet, or blue light). Usually, the specimen will be stained with
dye molecules, called the fluorophores or fluorochromes. The light will be absorbed by the
fluorophores and they then emit light of a longer wavelength, which is used to
form an image.
- A mercury vapor arc lamp is used as source and the beam passes through a special infrared filter which limits heat transfer.
- The light passes through an exciter filter that transmits only the desired wavelength.
- A darkfield condenser is used to provide a black background
- The microscope forms an image of the fluorochrome-labelled microorganisms. A barrier filter is positioned after the objective lens to remove any remaining ultraviolet light, which could damage the viewer’s eyes, or to remove blue and violet light, which would reduce the image contrast
Electron
microscopy - TEM and SEM
Electron
Microscopy is a technique that uses a beam of electrons to create an image of a
specimen. It has a much higher resolution than light microscopy, allowing for
the visualization of smaller structures. There are two main types of electron
microscopy, Transmission Electron Microscopy (TEM) and Scanning Electron
Microscopy (SEM).
The
resolution of a light microscope increases with a decrease in the wavelength of
the light it uses for illumination. Electron beams behave like radiation and
can be focused and is used in an Electron Microscope. Wavelength of electron beams
is around 0.005 nm, approximately 100,000 times shorter than that of visible
light and thus the resolution is enormously increased. The transmission electron microscope has a resolution
roughly 1,000 times more than light microscope.
Transmission
electron microscope (TEM
A
heated tungsten filament in the electron gun generates a beam of electrons that
is then focused on the specimen by the condenser. Since electrons cannot pass
through a glass lens, doughnut-shaped electromagnets called magnetic lenses are
used to focus the beam. The column containing the lenses and specimen must be under
high vacuum to obtain a clear image because otherwise electrons will be
deflected by collisions with air molecules. The specimen scatters electrons
passing through it, and the beam is focused by magnetic lenses to form an
enlarged, visible image of the specimen on a fluorescent screen. A denser
region in the specimen scatters more electrons and therefore appears darker in
the image and electron-transparent regions will appear brighter.
Scanning
electron microscope (SEM)
This
is used to examine the surfaces of microorganisms. While TEM produces an
image from radiation that has passed through a specimen, the SEM produce an
image from electrons emitted by an object’s surface.
The
SEM scans a narrow, tapered electron beam back and forth over the specimen.
When the beam strikes a particular area, surface atoms discharge a tiny shower
of electrons called secondary electrons, and these are trapped by a special
detector. Secondary electrons entering the detector strike a scintillator causing
it to emit light flashes. These light
flashes are converted to an electrical current and amplified by a
photomultiplier. The signal is sent to a cathode-ray tube and produces an image.
|
Feature |
TEM |
SEM |
|
Image Formation |
Transmission of electrons through the
specimen |
Scanning of electrons across the surface of
the specimen |
|
Image Type |
2D projection of the internal structure |
3D image of the surface topography |
|
Specimen Preparation |
Requires thin sections of the specimen |
Can be used on bulk samples |
|
Resolution |
Higher resolution |
Lower resolution than TEM |
Specimen
Preparation for TEM and SEM
Specimen
preparation is a crucial step in electron microscopy, as it directly impacts
the quality of the images obtained. The specific techniques used vary depending
on the type of electron microscope (TEM or SEM) and the nature of the sample.
Specimen
Preparation for TEM
Since
electrons are quite easily absorbed and scattered by solid matter, only extremely
thin slices of a microbial specimen can be viewed in TEM. The specimen must be
around 20 to 100 nm thick (This is about 1⁄50 to 1⁄10 the diameter of a typical
bacterium) and this thin specimen must be able to maintain its structure when
bombarded with electrons under high vacuum.
Common method for preparing specimen
1. Fixation:
o Chemical
fixation: Using chemicals like glutaraldehyde and osmium tetroxide to preserve
the sample's structure.
o Cryofixation:
Rapid freezing the sample to preserve its native state.
2. Dehydration:
Removing water from the sample using a graded series of ethanol or acetone.
3. Embedding:
Infiltrating the sample with a resin (e.g., epoxy resin) and polymerizing it to
provide support.
4. Sectioning:
Using an ultramicrotome (with a glass or diamond knife) to cut thin sections
(50-100 nm) of the embedded sample.
5. Staining:
Enhancing contrast by staining the sections with heavy metal salts (e.g.,
uranyl acetate, lead citrate). The lead
and uranium ions bind to cell structures and make them more electron opaque,
thus increasing contrast in the material.
6. The
stained thin sections are then mounted on tiny copper grids and viewed.
Negative
staining - The specimen is spread out in a thin film
with either phosphotungstic acid or uranyl acetate. Heavy metals render the
background dark, whereas the specimen appears bright. Negative staining is an
excellent way to study the structure of viruses, bacterial gas vacuoles, etc.
Shadowing
- A microorganism
also can be viewed after shadowing with metal. It is coated with a thin
film of platinum or other heavy metal by evaporation at an angle of about 45°
from horizontal. Sothe metal strikes the
microorganism on only one side. The area coated with metal scatters electrons
and appears light in photographs, whereas the uncoated side and the shadow region
created by the object is dark. This technique is useful in studying virus
morphology, bacterial flagella, and plasmids.
Freeze-etching
procedure – To study shape of organelles within microorganisms,
specimens are prepared by the freeze-etching procedure. Cells are rapidly frozen in liquid nitrogen
and then warmed to -100°C in a vacuum chamber. A knife that has been precooled with
liquid nitrogen (-196°C) is used to fracture the frozen cells. Since the frozen cells are very brittle, they
break along lines of weakness, usually down the middle of internal membranes.
The specimen is left in the high vacuum for a minute so that some of the ice will
sublimate away and uncover more structural detail. Finally, the exposed surfaces
are shadowed and coated with layers of platinum and carbon to form a replica of
the surface. Then specimen to be removed chemically and this replica is studied
in the TEM to obtain a three-dimensional view of intracellular structure. Freeze-etching
minimizes the danger of forming artifacts because the cells are frozen quickly rather
than being subjected to chemical fixation, dehydration, and plastic embedding.
Specimen
Preparation for SEM
Specimen
preparation is easy, and in some cases air-dried material can be examined
directly. Sometimes, microorganisms must first be fixed, dehydrated, and dried
to preserve surface structure and prevent collapse of the cells when they are
exposed to the SEM high vacuum. Before viewing, dried samples are mounted and
coated with a thin layer of metal to prevent the buildup of an electrical
charge on the surface and to give a better image.
