Phase Contrast Microscopy, Dark Field Microscopy and Electron Microscopy

Phase Contrast Microscopy

The unstained cells of living organisms absorb very little light. If light absorption is poor, the differences in intensity distribution will be very small. As a result of this phenomenon, the cells cannot be seen under a brightfield microscope. Using phase-contrast microscopy, light passing through a transparent specimen is phase-shifted, resulting in changes in brightness in an image. In 1934, Frits Zernike, a Dutch physicist, described it for the first time.

Principle

Light is affected by phase shifts when passing through cells, but these changes cannot be seen by the human eye. Phase-contrast microscopes can detect these phase shifts by noticing changes in image contrast caused by changes in amplitude.

Working

• Through a collector lens, a tungsten-halogen lamp produces partially coherent illumination that is directed onto a specially designed annulus (labeled condenser annulus) built into the front focal plane of the substage condenser.
• A wavefront passing through an annulus illuminates a specimen and either passes through it un-deviated, or is diffracted and delayed in phase by structures within the sample.
• It is focused at the intermediate image plane, where phase-contrast images are observed, with the use of a phase plate to separate reflected and diffracted light collected by the objective.

Parts

As an example, phase-contrast microscopy uses a light microscope that is specially designed with a phase plate and annular diaphragm in addition to the basic components.

The annular diaphragm

• Above the condenser, it is located.
• A circular disk with a circular annular groove makes up the device.
• This groove allows light to pass through.
• An annular groove on the annular diaphragm allows light to fall into the annular groove of the specimen or object to be studied.
• Consequently, an image appears at the back focal plane of the objective.
• On this annular phase plate is located each phase plate.

The phase plates

• The plate either has a thick circular area or a thin circular groove, depending on whether it is a negative phase plate.
• Phase plates are made up of thick and thin areas called conjugate areas.
• It is a disc-shaped transparent surface.
• An annular diaphragm and phase plate are used in this microscope to obtain phase contrast.
• The difference between direct and diffracted rays is obtained by separating them.
• A light ray that travels straight through an annular groove goes outside the groove, but a light ray whose path has been diffracted goes outside the groove.
• A microscope shows a different degree of contrast depending on the refractive indices of different aspects of a cell.

Dark Field Microscopy

• A vast portion of our knowledge of the living world comes from the field of microbiology, an area in which Antoni van Leeuwenhoek made important contributions.
• A vast portion of our knowledge of the living world comes from the field of microbiology, an area in which Antoni van Leeuwenhoek made important contributions.
• The microscope has evolved over the years from simple Leeuwenhoek instruments with magnifications of 300X to electron microscopes capable of magnifying more than 250,000X.
• A microscope is either a light microscope or an electron microscope.
• Illuminating specimens is done through the use of visible light or ultraviolet rays. A wide range of instruments is available, such as brightfield, darkfield, phase-contrast, and fluorescent.
• Compared to the ordinary light microscope, this microscope has a modified condenser system, which does not directly illuminate the specimen.
• As light is deflected or scattered from a specimen obliquely directed toward a dark background, the specimen appears bright.
• In contrast to brightfield microscopy, darkfield microscopy allows observation of living specimens.

Principle

• As light strikes the specimen in a dark field microscope, it scatters from being blocked off from the light source.
• If the background is dark, light objects with a similar refractive value will appear bright on the dark background.
• Objects can scatter light in every azimuth or direction when they are struck. The dark field microscope is designed so that the sample is only illuminated by scattered light, eliminating dispersed light or zeroth order illumination.
• This is possible by introducing a condenser and/or stop below the stage so that light rays will be reflected off the object at different angles rather than being reflected directly above or below it.
• An individual can view a specimen in a dark field by diffracting, reflecting, and refracting light off of an object, resulting in “a cone of light”.

• Microscopy on dark-grounds involves the use of a compound light microscope called a dark ground microscope.
• The most important component of the dark-ground microscope is the cone-shaped condenser with a central circular stop, which provides light to illuminate objects in the dark.
• Transmitted light in the ordinary light microscope is replaced by reflected light in this microscope.
• It makes it so that light does not come directly into contact with the objective lens.
• The reflection or scattering of light rays from the object into the objective lens causes the microorganisms to appear bright, stained, against a dark background.

Electron Microscopy

A high-resolution image of a biological or non-biological specimen can be obtained with electron microscopy (EM). A number of biomedical research techniques are based on this method including discovering detailed structures of tissues, cells, organelles and macromolecular complexes. The high resolution of EM imaging is caused by the use of electrons, which have short wavelengths. When ancillary techniques (e.g., thin sectioning, immunolabeling, negative staining) are used in conjunction with electron microscopy, specific questions can be answered. A cell's structure provides important clues as to how it functions and how it behaves under disease conditions.

A transmission electron microscope (TEM) and a scanning electron microscope (SEM) can be differentiated. Using a transmission electron microscope, thin specimens can be viewed (tissue sections, molecules, etc.), which allow electrons to pass through so an image is formed. There are several similarities between the TEM and a conventional light microscope (compound). Additionally to imaging the interiors of cells (in thin sections), metal shadows allow investigators to observe the structure of protein molecules, and negative stains allow introspection of molecule assembly in viruses and filaments of cytoskeletal networks, as well as how proteins are arranged in cell membranes (through freeze-fracture).

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