For example, two seagulls floating on the sea may be at different heights — the wavelength and maximum amplitude of the waves are the same in both cases, but the phase is different.
The phase difference between the red and blue waves in Fig. Light waves can interfere with each other if they have the same frequency and well-ordere d phases , with the result dependant upon the phase difference between waves. If the light is moving through a denser substance it moves slower, depending on the refractive index RI of the medium the light is moving through.
The higher the RI , the slower the light. We could alternatively represent this delay as if the light has had to travel further at the same speed. This is the optical path length OPL , which is given by the distance traveled through a substance, multiplied by its RI. If two waves with the same wavelength start from the same point and take different paths before reaching the same point , there will likely be a difference in OPL.
Unless this difference is a precise multiple of the wavelength, there will be then a difference in phase between the two waves. This means that phase differences in light from the same sample can be introduced by forcing some of the light to travel further. This is the concept behind phase-contrast. Phase-contrast microscopy was first described by Dutch physicist Frits Zernike in the s, for which he later was awarded the Nobel Prize in Physics Phase contrast is a technique that exploits the ability of some microscope samples to alter the OPL of light passing through it, adding contrast through the interference of light of different phases.
Transparent unstained samples such as cells do not absorb light and are called phase objects. When light passes through a sample area with no phase object , there is no significant change in the RI or OPL, so no significant diffraction occurs Fig. This light that is not diffracted is often referred to as direct or zero-order light as it continues unmodified through the sample. On the other hand, when light passes through an area of the sample with a phase object such as cellular structures , small changes in the RI will diffract and scatter some light and cause changes to the OPL, depending on the thickness and RI of each structure.
The thicker the structure, the greater the diffraction of the light. The diffracted light is a small proportion of the total light that has passed through the sample.
This diffracted light that passed through a phase object arrives at the detector out of phase with the direct light that did not pass through a phase object.
This small phase shift is not enough to cause significant interference between direct and diffracted light, which along with the poor absorption of transparent structures means there is little amplitude difference between areas where such structures are present and where they are not. Phase-contrast microscopy is a method that manipulates this property of phase objects to introduce additional interference between the direct and diffracted.
This technique transforms differences in phase into differences in brightness , increasing contrast in images of non-absorbing samples. There are two main issues when implementing phase-contrast microscopy: How to phase shift the scattered light or the direct light but not both , and how to get light with well-ordered phase to illuminate the sample.
It was known that constraining light through a small pinhole generated an expanding light wave with well-organized phase but at the expense of a great loss of intensity. This circular wave was easily converted to a flat wave with a lens. Phase-contrast compromises between light intensity and uniform phase by using a circular ring annulus of illumination. This annulus acted similarly to a ring of pinholes , with any particular direction around the ring having the same phase, even though the phase would vary irregularly around the ring.
To phase shift either the scattered or direct light, a phase-shifting optic like a glass disk is placed in the light path where it would predominantly affect the direct light. As the light hits the sample the phase and direction of the diffracted solid lines to the right of the sample and direct dashed lines light changes. The objective lens takes the scattered light and focuses it to ordered waves, while the direct light is focused to the optical center, where the phase-shifting material is placed.
This brings the scattered light and direct light back into phase , allowing for the generation of contrast through interference upon arrival at the detector. A phase-contrast microscope with annular illumination is depicted in Fig. Implemented in a modern infinity-corrected microscope, the phase-shifting ring is located at the objective rear focal plane. 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.
The phase plate is introduced into the light path at the rear focal plane, often permanently etched to one of the internal lens elements of the objective such as the tube lens from Fig. This phase plate decreases the direct light to more closely match the intensity of the diffracted light, and thus further reduce the contribution of background light to the image. Light passing through the sample is refracted or diffracted due to features with different refractive indices, creating new optical paths.
Almost all of these new optical paths will not pass through the attenuating areas of the phase plate but instead will pass through the non-attenuating center of the phase ring. Phase contrast is a light microscopy technique used to enhance the contrast of 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. As phase contrast microscopy does not require cells to be killed, fixed or stained, the technique enables living cells, usually in culture, to be visualised in their natural state. This means biological processes can be seen and recorded at high contrast and specimen detail can be observed.
Fluorescence staining can be used in combination with phase contrast to further improve the visualisation of samples. Phase contrast is ideal for thinner samples, therefore an inverted microscope system can be used. This provides the additional advantage of having more working space. Phase contrast can also be installed on upright microscopes. If thicker samples need to be visualised in high-resolution, differential interference contrast DIC is a more suitable technique to use.
Phase contrast is used to visualise transparent specimens, when high-resolution is not required, including:. It is relatively simple and inexpensive to adapt an inverted or upright light microscope for phase contrast. The following components need to be installed:. Care must be taken with the condenser annulus and the phase rings, as they need to be matched in diameter and optically conjugated.
Phase contrast microscopy translates small changes in the phase 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. Our eyes are unable to detect these slight phase differences as they can only detect variations in the frequency and intensity of light. Phase contrast enables high contrast images to be produced by further increasing the difference of the light phase.
It is this characteristic that enables background light to be separated from specimen diffracted light. When the light is focused on the image plane, the diffracted and background light cause destructive or constructive interference which decreases or increases the brightness of the areas that contain the sample, in comparison to the background light.
Light from a tungsten-halogen lamp goes through the condenser annulus in the substage condenser before it reaches the specimen. This allows the specimen to be illuminated by parallel light that has been defocused. Some of the light that passes through the specimen will not be diffracted bright yellow in the picture. These light waves form a bright image on the rear aperture of the objective.
The light waves that are diffracted by the specimen pass the diffracted plane and focus on the image plane only. This allows the background light and the diffracted light to be separated. When the light is focused on the image plane, the diffracted and background light will cause destructive or constructive interference, which changes the brightness of the areas that contain the sample in comparison to the background light.
All of the components required for phase contrast need to be aligned and centred. Some phase sliders are pre-centred, we therefore recommend that you check beforehand. At the image plane, the phase of the diffracted light would be out of phase with the direct light, but the amplitude of their interference would be almost the same as that of the direct light.
This would result in very little specimen contrast. To speed up the direct undeviated zeroth order light, a phase plate is installed with a ring shaped phase shifter attached to it at the rear focal plane of the objective. The narrow area of the phase plate is optically thinner than the rest of the plate. As a result, undeviated light passing through the phase ring travels a shorter distance in traversing the glass of the objective than does the diffracted light.
The diffracted and direct light can now interfere destructively so that the details of the specimen appear dark against a lighter background just as they do for an absorbing or amplitude specimen. This is a description of what takes place in positive or dark phase contrast. In this case, the zeroth order light arrives at the image plane in step or in phase with the diffracted light, and constructive interference takes place. The image would appear bright on a darker background.
The image appears bright on a darker background. This type of phase contrast is described as negative or bright contrast. Because the undeviated light of the zeroth order is much brighter than the faint diffracted light, a thin absorptive transparent metallic layer is deposited on the ring to bring the direct and diffracted light into better balance of intensity in order to increase contrast.
Such a green filter also helps achromatic objectives produce their best images, since achromats are spherically corrected for green light. The accessories needed for phase contrast work are a substage phase contrast condenser equipped with annuli and a set of phase contrast objectives, each of which has a phase plate installed.
The condenser usually has a brightfield position with an aperture diaphragm and a rotating turret of annuli each phase objective of different magnification requires an annulus of increasing diameter as the magnification of the objective increases. Each phase objective has a darkened ring on its back lens.
Such objectives can also be used for ordinary brightfield transmitted light work with only a slight reduction in image quality. Practice aligning a phase contrast microscope and discover how improper alignment affects specimen appearance. The phase outfit, as supplied by the manufacturer, usually includes a green filter and a phase telescope. The latter is used to enable the microscopist to alight the condenser annulus to superimpose it onto the ring of the phase plate.
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