Phase Contrast Microscopy- Definition, Principle, Parts, Uses

Phase-contrast microscopy is a technique that allows us to see transparent or colorless specimens that are otherwise difficult to observe with a conventional light microscope. These specimens include living cells, microorganisms, thin tissue slices, and other biological or non-biological materials that have little or no contrast in brightfield microscopy.

Phase-contrast microscopy was invented by Frits Zernike, a Dutch physicist who received the Nobel Prize in Physics in 1953 for his discovery. He realized that light waves passing through a transparent object undergo a phase shift, which means that they are delayed or advanced in time compared to the light waves that do not interact with the object. This phase shift is proportional to the thickness and refractive index of the object, and it can be used to create contrast in the image.

Phase contrast microscopy is based on the principle that light waves passing through a transparent specimen undergo phase shifts depending on the refractive index and thickness of the specimen. These phase shifts are invisible to the human eye, but they can be converted into changes in brightness or contrast by using a special optical device called a phase plate. The phase plate introduces a phase difference between the undeviated light rays (that pass through the specimen without being bent or scattered) and the diffracted light rays (that are bent or scattered by the specimen). This phase difference creates an interference pattern that enhances the contrast between different parts of the specimen. By adjusting the phase plate, different degrees of contrast can be achieved. Phase contrast microscopy allows us to see details of transparent specimens that would otherwise be invisible in brightfield microscopy.

Phase contrast microscopy works by translating small changes in the phase of light waves passing through a transparent specimen into changes in amplitude (brightness), which are then seen as differences in image contrast. Unstained specimens that do not absorb light are known as phase objects. These specimens cause a phase shift in the light waves that pass through them, but this phase shift is invisible to the human eye or photosensors. Phase contrast microscopy converts these phase shifts into amplitude shifts by using a specialized condenser and objective lens that interfere with the light waves.

The basic steps of phase contrast microscopy are as follows:

• Partially coherent illumination produced by the tungsten-halogen lamp is directed through a collector lens and focused on a specialized annulus (labeled condenser annulus) positioned in the substage condenser front focal plane.
• Wavefronts passing through the annulus illuminate the specimen and either pass through undeviated or are diffracted and retarded in phase by structures and phase gradients present in the specimen.
• Undeviated and diffracted light collected by the objective is segregated at the rear focal plane by a phase plate and focused at the intermediate image plane to form the final phase contrast image observed in the eyepieces.

The condenser annulus is a disk that projects the light in an annular way, creating a hollow cone of light that illuminates the specimen. The phase plate is a transparent disc that has either a thick circular area (negative phase plate) or a thin circular groove (positive phase plate) that corresponds to the condenser annulus. The phase plate introduces a phase shift and an amplitude attenuation to the undeviated light, while leaving the diffracted light unchanged. This creates an interference pattern between the two types of light, resulting in a contrast-enhanced image of the specimen.

A phase contrast microscope is a type of light microscope that has some additional parts to enhance the contrast of transparent specimens. The main parts of a phase contrast microscope are:

• Light source: This is usually a tungsten-halogen lamp that provides partially coherent illumination for the specimen.
• Collector lens: This is a lens that collects and focuses the light from the source onto the condenser annulus.
• Condenser annulus: This is a circular disc with a ring-shaped opening that is placed in the front focal plane of the substage condenser. It allows only a narrow ring of light to pass through and illuminate the specimen.
• Substage condenser: This is a lens system that concentrates and controls the amount of light reaching the specimen. It has an aperture diaphragm that can be adjusted to match the size of the condenser annulus.
• Specimen: This is the transparent object that is placed on the microscope stage and fixed with a cover slip. It can be a living cell, a microorganism, a tissue slice, or any other thin material that causes phase shifts in the light passing through it.
• Objective lens: This is a lens that collects and magnifies the light coming from the specimen. It has a phase plate in its rear focal plane that modifies the phase and amplitude of the light waves.
• Phase plate: This is a transparent disc with a ring-shaped area that corresponds to the condenser annulus. It can be either positive or negative, depending on whether it advances or retards the phase of the undeviated light relative to the diffracted light. It also reduces the brightness of the undeviated light to create contrast in the image.
• Intermediate lens: This is a lens that further magnifies and projects the image formed by the objective lens onto the eyepiece or camera.
• Eyepiece or camera: This is the device that allows the observer to view or capture the final phase contrast image. It can be either an ocular lens that magnifies and focuses the image on the retina, or a digital camera that converts the image into electrical signals.

The annular diaphragm is a circular disc with a ring-shaped opening that is placed below the condenser of the phase-contrast microscope. The purpose of the annular diaphragm is to produce a cone of light that illuminates the specimen with partially coherent light. The annular diaphragm is matched to the phase plate in the objective lens, so that the light passing through the ring-shaped opening corresponds to the light passing through the conjugate area of the phase plate. The annular diaphragm ensures that only the light rays that are diffracted by the specimen reach the phase plate, while the undeviated light rays pass through the clear area of the phase plate. This creates a phase difference between the diffracted and undeviated light rays, which results in contrast enhancement in the image. The annular diaphragm also controls the numerical aperture and resolution of the microscope, as well as the amount of light reaching the specimen. By adjusting the position and size of the annular diaphragm, different levels of contrast and brightness can be achieved. The annular diaphragm is one of the key components of phase-contrast microscopy that enables visualization of transparent specimens without staining or fixation.

The phase plate is a key component of the phase contrast microscope that converts the phase shifts of light waves into changes in amplitude or brightness. The phase plate is a transparent disc that is placed at the rear focal plane of the objective lens, where an image of the annular diaphragm is formed. The phase plate has a circular area that corresponds to the annular groove of the diaphragm, called the conjugate area, and a surrounding area that corresponds to the region outside the groove, called the non-conjugate area.

The conjugate area of the phase plate is either thicker or thinner than the non-conjugate area, depending on whether it is a negative or positive phase plate. A negative phase plate has a thicker conjugate area that retards the direct light rays by 1/4 wavelength, while a positive phase plate has a thinner conjugate area that advances the direct light rays by 1/4 wavelength. In both cases, the phase difference between the direct and diffracted light rays becomes 1/2 wavelength, which leads to destructive interference and contrast enhancement.

The phase plate also reduces the amplitude of the direct light rays by absorbing or scattering some of the light. This further increases the contrast between the specimen and the background. The material and thickness of the phase plate determine how much light is attenuated and how much phase shift is introduced. Common materials for phase plates are carbon, gold, silver, or platinum group. The optimal thickness of the phase plate depends on the wavelength and numerical aperture of the objective lens.

The phase plate can be adjusted to align with the annular diaphragm by using a centering screw on the objective lens. The alignment is critical for achieving optimal contrast and minimizing artifacts in phase contrast microscopy.

Phase contrast microscopy is widely used in biological research for observing living cells and their structures without staining or fixation. Some of the applications of phase contrast microscopy are:

• Visualizing unstained living cells. Phase contrast microscopy enables the visualization of transparent cells and their organelles, such as nuclei, mitochondria, vacuoles, and flagella. It can also reveal cellular movements, such as chromosomal and flagellar motions.
• Studying cellular events such as cell division. Phase contrast microscopy can help in monitoring the changes in cell shape, size, and morphology during cell division. It can also show the formation and separation of daughter cells.
• Examining intracellular components of living cells at relatively high resolution. Phase contrast microscopy can provide high-contrast images of intracellular components that are difficult to see with brightfield microscopy, such as cytoplasmic granules, vesicles, and filaments.
• Analyzing cell networks and populations. Phase contrast microscopy can also be used to observe extended cell networks and populations, such as tissues, cultures, and biofilms. It can show the interactions and communications between different cell types and their microenvironment.

Phase contrast microscopy is not only useful for biological applications, but also for some material and earth science applications, such as examining lithographic patterns, fibers, latex dispersions, glass fragments, and subcellular particles.

Phase contrast microscopy is a powerful technique that can enhance the contrast and visibility of transparent specimens, such as living cells, without the need for staining or fixation. Some of the advantages of using phase contrast microscopy are:

• It allows the observation of living cells in their natural state and environment, which can reveal more information about their structure, function and behavior than dead or fixed cells.
• It produces high-contrast, high-resolution images of cellular components, such as nuclei, organelles, membranes and cytoskeletons, that are otherwise difficult to see with brightfield microscopy.
• It is ideal for studying and interpreting thin specimens, such as tissue slices, microorganisms, fibers and latex dispersions.
• It can be used to monitor the dynamics of ongoing biological processes in live cells, such as cell division, motility, secretion and phagocytosis.
• It can be easily added to most brightfield microscopes by installing specialized phase objectives and condensers that conform to the tube length parameters.
• It does not require any special preparation or treatment of the specimens, which saves time and preserves the integrity of the samples.

Although phase contrast microscopy is a powerful technique for enhancing the visibility and contrast of transparent specimens, it also has some limitations and drawbacks. Some of them are:

• Halo effect: Phase images are often surrounded by bright or dark halos around the edges of details, especially those with a large phase shift. These halos are artifacts that can obscure the fine structure and reduce the resolution of the image.
• Reduced numerical aperture: The phase annuli in the condenser and the objective limit the working numerical aperture of the optical system to a certain degree, thus reducing the resolution and light-gathering ability of the microscope.
• Not suitable for thick specimens: Phase contrast microscopy does not work well with thick specimens or specimens with multiple layers, because phase shifts occur from areas slightly below or above the plane that is in focus. This can result in confusing and distorted images with poor contrast.
• Expensive and complex: Phase contrast microscopy requires specialized condensers and objective lenses that add considerable cost to a microscope. It also requires careful alignment of the light path and adjustment of the phase ring position to achieve optimal results.
• Limited color information: Phase contrast microscopy produces monochromatic images that do not reveal the natural color or pigmentation of the specimens. This can limit the identification and differentiation of some biological structures or organisms.

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