Question

Can you please compare types of microscopic methods used in microbiology (bright-field, dark-field microscope, phase-contrast, differential-interference contrast microscope, fluorescence, confocal microscope, SEM and TEM). I appreciate it a lot. Thank you in advance!!

Answer 1

Ans:

• Brightfield Microscopes:

Compound microscope with two or more lenses that produce a dark image on a bright background.

A brightfield microscope creates an image by directing light from the illuminator at the specimen; this light is differentially transmitted, absorbed, reflected, or refracted by different structures.

At very high magnifications, resolution may be compromised when light passes through the small amount of air between the specimen and the lens.

To solve this problem, a drop of oil can be used to fill the space between the specimen and an oil immersion lens, a special lens designed to be used with immersion oils. Since the oil has a refractive index very similar to that of glass, it increases the maximum angle at which light leaving the specimen can strike the lens.

• Darkfield Microscopy: The resulting image typically shows bright objects on a dark background.]

The only light that reaches the objective is light that has been refracted or reflected by structures in the specimen.

Darkfield microscopy can often create high-contrast, high-resolution images of specimens without the use of stains, which is particularly useful for viewing live specimens that might be killed or otherwise compromised by the stains.

• Phase-Contrast Microscopes:

Phase-contrast microscopes use refraction and interference caused by structures in a specimen to create high-contrast, high-resolution images without staining.

It is the oldest and simplest type of microscope that creates an image by altering the wavelengths of light rays passing through the specimen.

Because it increases contrast without requiring stains, phase-contrast microscopy is often used to observe live specimens. Certain structures, such as organelles in eukaryotic cells and endospores in prokaryotic cells, are especially well visualized with phase-contrast microscopy.

• Differential interference contrast (DIC) microscopes

Similar to phase-contrast microscopes in that they use interference patterns to enhance contrast between different features of a specimen.

In a DIC microscope, two beams of light are created in which the direction of wave movement (polarization) differs.

DIC microscopes results in high-contrast images of living organisms with a three-dimensional appearance.

These microscopes are especially useful in distinguishing structures within live, unstained specimens.

• Fluorescence microscope:

A fluorescence microscope uses fluorescent chromophores called fluorochromes, which are capable of absorbing energy from a light source and then emitting this energy as visible light.

The microscope transmits an excitation light, generally a form of EMR with a short wavelength, such as ultraviolet or blue light, toward the specimen; the chromophores absorb the excitation light and emit visible light with longer wavelengths.

This microscope produces an image of the specimen in bright colors against a dark background.

This microscopy can be used to identify pathogens, to find particular species within an environment, or to find the locations of particular molecules and structures within a cell.

One of the most important applications of fluorescence microscopy is a technique called immunofluorescence, which is used to identify certain disease-causing microbes by observing whether antibodies bind to them.

• Confocal Microscopes:

A confocal microscope uses a laser to scan multiple z-planes successively.

his produces numerous two-dimensional, high-resolution images at various depths, which can be constructed into a three-dimensional image by a computer.

Image clarity is further enhanced by a narrow aperture that eliminates any light that is not from the z-plane

Confocal microscopes are thus very useful for examining thick specimens such as biofilms, which can be examined alive and unfixed.

• Transmission Electron Microscope (TEM):

The TEM is somewhat analogous to the brightfield light microscope in terms of the way it functions. However, it uses an electron beam from above the specimen that is focused using a magnetic lens

TEM requires that the beam and specimen be in a vacuum and that the specimen be very thin and dehydrated.

TEM usually use acceleration voltage in the range of 60-300kV.

The transmission electron microscope is used to view thin specimens (tissue sections, molecules, etc) through which electrons can pass generating a projection image.

• Scanning Electron Microscope (SEM):

SEMs form images of surfaces of specimens, usually from electrons that are knocked off of specimens by a beam of electrons. This can create highly detailed images with a three-dimensional appearance that are displayed on a monitor.

SEM provides information on the sample’s surface and its composition in 3D.

SEM resolution is limited to ~0.5 nm.

SEMs usually use acceleration voltages up to 30 kV