Principle: In an inverted microscope, the source for transmitted light and the condenser are placed on the top of the stage, pointing down toward the stage. The objectives are located below the stage pointing up. The cells are observed through the bottom of the cell culture vessel.

It utilizes the difference between light rays propagating directly from the light source and light rays refracted by the specimen when light passes through it to add bright/ dark contrast to images of transparent specimens. The microscope is fitted with a phase-contrast objective and a condenser for observations.

Phase contrast is used to enhance the contrast of light microscopy images of transparent and colourless specimens. It enables visualisation of cells and cell components that would be difficult to see using an ordinary light microscope. Phase contrast does not require cells to be killed, fixed or stained.

The two components required to convert a traditional bright field microscope into a phase-contrast microscope are the annular diaphragm placed in the condenser back aperture, and the optically matched internal phase plate.

An amplitude specimen decreases the intensity (i.e. the amplitude) of the light. Phase specimens cause a phase shift of the light. Phase contrast microscopy is now capable of converting a difference in refractive index into a difference in brightness. …

Disadvantages and limitations of phase contrast: Annuli or rings limit the aperture to some extent, which decreases resolution. This method of observation is not ideal for thick organisms or particles. Thick specimens can appear distorted.

Phase contrast microscopy, first described in 1934 by Dutch physicist Frits Zernike, is a contrast-enhancing optical technique that can be utilized to produce high-contrast images of transparent specimens, such as living cells (usually in culture), microorganisms, thin tissue slices, lithographic patterns, fibers.

The following steps are recommended for the alignment of a phase contrast microscope.

1. Place a brightly stained specimen on the stage and rotate the 10x phase contrast objective into the optical pathway in brightfield illumination mode.
2. Remove the stained specimen and place a phase specimen on the microscope stage.

Phase-contrast microscopy is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations.

Uses of Phase Contrast Microscopy Phase contrast microscopy uses contrast to help illuminate details of living cells and other transparent micro-organisms. According to Rice University, there is little contrast between organelles within cells, making it difficult to see them using a bright light microscope.

The concept of resolution is inseparable from contrast, and is defined as the minimum separation between two points that results in a certain level of contrast between them. …

Since magnification increases the air gap and thereby its effect on scattered radiation, magnification is expected to affect the contrast of larger blood vessel images as well (> 0.5 mm), but quantitation of this effect is not known in the literature.

Brightfield Microscope is used in several fields, from basic biology to understanding cell structures in cell Biology, Microbiology, Bacteriology to visualizing parasitic organisms in Parasitology. Most of the specimens to viewed are stained using special staining to enable visualization.

Brightfield microscopy is the preferred method of pathologists for diagnosing solid tumors, utilizing common staining techniques such as hematoxylin and eosin staining and immunohistochemistry (IHC).

Only optical fluoresce microscopes can see inside a virus, and then only indirectly, using dye, which cannot actually penetrate a virus.

Fluorescent microscopy is often used to image specific features of small specimens such as microbes. It is also used to visually enhance 3-D features at small scales. When the reflected light and background fluorescence is filtered in this type of microscopy the targeted parts of a given sample can be imaged.

Intensified or cooled CCD cameras appear to be the most suitable device for quantitative imaging at low light levels in fluorescence microscopy.

It is believed that the primary culprit in the light-induced toxicity experienced by live cells is a result of free radicals produced by the excitation of fluorescent molecules. These free radicals are highly reactive and cause the destruction of cellular components, which can result in non-physiological behavior.

An electron microscope uses an ‘electron beam’ to produce the image of the object and magnification is obtained by ‘electromagnetic fields’; unlike light or optical microscopes, in which ‘light waves’ are used to produce the image and magnification is obtained by a system of ‘optical lenses’.

The principle behind fluorescence microscopy is simple. As light leaves the arc lamp it is directed through an exciter filter, which selects the excitation wavelength.

Electron microscopes have two key advantages when compared to light microscopes:

• They have a much higher range of magnification (can detect smaller structures)
• They have a much higher resolution (can provide clearer and more detailed images)

Electron Microscope Disadvantages The main disadvantages are cost, size, maintenance, researcher training and image artifacts resulting from specimen preparation. This type of microscope is a large, cumbersome, expensive piece of equipment, extremely sensitive to vibration and external magnetic fields.

Microscopes help the scientists to study the microorganisms, the cells, the crystalline structures, and the molecular structures, They are one of the most important diagnostic tools when the doctors examine the tissue samples.

Advantages of electron microscopy Magnification and higher resolution – as electrons rather than light waves are used, it can be used to analyze structures which cannot otherwise be seen. The resolution of electron microscopy images is in the range of up to 0.2 nm, which is 1000x more detailed than light microscopy.

ka. TEM) The advantage of using electron microscopes over light microscopes is that they can magnify objects up to a million times. Also electron microscopes can produce images of much smaller objects than light microscopes can.

Electron microscopes are the most powerful type of microscope, capable of distinguishing even individual atoms. However, these microscopes cannot be used to image living cells because the electrons destroy the samples.

The two main types of electron microscopes are the transmission electron microscope (TEM) and the scanning electron microscope (SEM).

There are two forms of phase contrast: positive and negative phase contrast. They mainly differ by the phase plates used for illumination. In positive phase contrast, the phase of light passing through the phase ring is advanced compared to the deviated light, whereas it is retarded in phase in negative phase contrast.

Positive Sequence: A balanced three-phase system with the same phase sequence as the original sequence. Negative sequence: A balanced three-phase system with the opposite phase sequence as the original sequence. Zero Sequence: Three phasors that are equal in magnitude and phase.

Brightfield microscopy can’t be used to observe living specimens of bacteria, although when using fixed specimens, bacteria have an optimum viewing magnification of 1000x.

It typically has a magnification power of up to 1000x. A light microscope might be used when examining individual cells within living tissue. This type of microscope might be used to study external features on an object or to examine structures not easily mounted onto flat slides.

What is the difference between bright-field and dark-field microscopes? bright-field is most widely used, specimen darker than surrounding field, dark-field is opposite with bright specimen surrounded by dark field. Do bright-field or dark-field microscopes reach better resolution?

Compared to permanently mounted slides, wet mounts do have certain advantages: Quick preparation: specimen fixation, dehydration and staining are not necessary (but possible, if required). For this reason, wet mounts are the first kind of mounts that students learn to make.

Disadvantages of wet mounts include: Movement: The advantage of observing movement can also be a disadvantage. Due to the movement of the organisms it may be more difficult to take pictures or to make drawings. Focus: Some organisms may swim vertically in the water and therefore move in and out of focus.

-the wet mount is a safer way to view pathogenic microorganisms. -the specimen can be viewed as living cells. -The motility of a specimen can be viewed under the microscope. -The specimen can be viewed as living cells.

Saline wet mount is used for the detection of trophozoites and cysts of protozoa, and eggs and larvae of helminths. It is particularly useful for the detection of live motile trophozoites of E. histolytica, Giardia lamblia, and Balantidium coli.