The difference between positive and negative phase contrast in a microscope
Depending on the configuration and nature of the phase ring positioned at the objective back focal plane, samples can be observed in positive or negative phase contrast. This interactive tutorial studies the relationship between surround (S), diffraction (D), and resulting bright particles (P waves), as well as positive and negative phase contrast microscopy. In addition, the geometry of the phase plate and representative sample images are also presented.
When people use it in their work now, most of the researchers are in the negative difference, and now the positive difference does not play much role in the current scientific research work.
The tutorial initializes the phase image with a randomly selected sample that appears in the Phase Contrast Image window, and the corresponding wave relationship is shown in the left neighbor of the image window. In order to operate the tutorial, use the mouse cursor to move the translation between the positive and negative phase contrast or the bright lighting phase contrast mode slider. When the slider is translated, the images that appear in the phase contrast image window change how the specimen appears in the current imaging mode set by the slider. Also, below the waveform graph is a phase plate that changes shape to match the imaging mode selected by the slider. To view a new sample, use the Selected Sample drop-down menu to select another sample.
A plot of the phase plate configuration, wave relationships, and vectors associated with the generation of positive and negative phase contrast images is presented in Figure 1. Examples of specimens imaged by these techniques are also shown. In a positive phase-contrast optical configuration (upper row of the image in Figure 1), the surround (S)-wavefront passes through the phase plate, resulting in a net phase shift of 180° phase advance, by 1/4 wavelength (1 half wavelength ). Advanced surround wavefronts are now able to participate in destructive interference with diffracted (D) waves at the intermediate image plane. In most cases, simply advancing the relative phase of the surrounding wavefront alone is not sufficient to result in the generation of high-contrast images in Nikon microscopes. This is because the amplitude of the surround waves is significantly larger than that of the diffracted waves, and suppresses the resulting image produced by interference from a fraction of the total number of waves. In order to reduce the surrounding wavefront to a value closer to the amplitude of the diffracted waves (and perform interference in the image plane), the opacity in the phase ring of the objective is obtained by applying a semi-transparent metal (neutral increasing density) coating Floor. The surrounding light waves, which pass almost completely through the phase ring by design, under phase contrast microscopy, are significantly reduced in amplitude by the opacity of the phase plate to a value in the range of 10 to 30% of the original intensity.
Because the resulting particle wave is produced by the interference* of the surrounding and diffracted wavefronts, the amplitude of the particle (P) wave produced by the interference between the wavefronts arriving at the image plane is now much smaller than the surrounding one when in Sexual density coating applied. The net effect is to convert the relative phase difference introduced by the passage of light emerging from the image plane through the specimen to a difference in amplitude (intensity). Because the human eye will interpret the difference in intensity as a contrast, the specimen is now visible in the microscope eyepiece and can also be captured on the membrane with conventional camera systems, or digitally, using CCD or CMOS devices. All positive phase contrast systems selectively advance the phase of the linear surround (S)-wavefront relative to the spherical diffracted (D) wavefront. Specimens with a higher refractive index than the surrounding medium appear darker on a neutral gray background, while those with a lower refractive index than the swimming medium appear brighter than the gray background.
In order to modify the spatial separation of the diffracted wavefronts surrounding the phase and amplitude in a phase contrast optical system, a number of phase plate configurations have been introduced. Because the phase plate is located at or very close to the objective rear focal plane (diffraction plane) all light that passes through the microscope must travel through this component. The part of the phase plate in its condenser annular focus is called the conjugate region, while the remaining region is called the complementary region. The conjugate region contains the material responsible for changing the phase of the surrounding (undiffracted) light by either plus or minus 90 degrees with respect to the diffracted wavefront. In general, the phase-conjugate ring area is wider (about 25%) than the area defined by the condensing ring image to reduce the amount of surrounding light that propagates to the complementary area.
Most phase plates available from modern microscope manufacturers are one of those prepared by vacuum deposition of thin dielectric and metallic films on a glass plate or mounted directly on the lens surface of the microscope objective. The role of the dielectric film is to phase the light, while the metal film attenuates the intensity of the undiffracted light. Some manufacturers use multiple anti-reflective coatings combined with the film to reduce the amount of glare and reflection of stray light back into the optical system. If the phase plate is not formed on the surface of a lens, it is usually cemented between successive lenses that reside on the focal plane near the rear of the objective. The thickness and refractive index of the dielectric, metal, and antireflection coatings, as well as those of the optical cement, are carefully chosen to produce the desired phase shift between the complementary and conjugated regions of the phase plate. In optical terms, a phase plate that changes the phase relative to the surrounding light to diffract light by 90 degrees (either positive or negative) is called a quarter-wave plate because of the optical path difference effect on it.
An overview of the positive-phase inverse is shown in Figure 1. The positive-phase contrast plate (left side of Figure 1) propels the surround wave, by 1/4 wavelength, due to the erosion ring in the glass plate, which can be reduced by the upper pass in the high-index plate The wave physical path taken. Because of interaction with the sample, when the diffracted sample rays (D) are retarded, the optical path difference between the encircling and diffracted waves that emerge from the phase plate is half wavelength by 1/4 wavelength. The net result is a 180-degree optical path difference between the surrounding and diffracted waves, which results in destructive interference for high-refractive-index samples between the image planes. The amplitude curve for the positive phase opposite destructive interference wave is shown in the upper graph of Figure 1. The resulting particle (P) wave has a lower amplitude than the surround (S)-wave, thus making the object appear compared to a relatively darker background. Bottom, image of Zygnema green algae shown on the right (labeled DL). The vector represented by the progress of the 1/4 wavelength, which is shown as a 90-degree counterclockwise rotating surround wave in positive phase contrast, appears between the figure and the image in Figure 1.
Alternatively, the microscope optics can also be fabricated to produce a negative phase opposite, as shown in the lower part of Figure 1, in which case the surround (S)-waves are delayed (rather than as advanced) by a quarter wavelength relative on the one diffracted (D) wave. As a result, specimens with high refractive indices appear brighter against a darker grey background (see the lower image labeled BM in Figure 1). In negative phase opposite, the objective phase plate contains a raised ring that retards the phase (rather than advancing the phase as the positive phase opposite), passing a quarter wavelength relative to the phase of the diffracted wave as the zeroth order surround wave . Because the diffracted waves have been delayed by a quarter wavelength as they pass through the specimen, the optical path difference between the surrounding and diffracted waves is eliminated and the high-refractive-index sample interferes constructively at the image plane. Note that the resulting particle (P) wave is higher in amplitude than the surround (S) wave in negative phase contrast. Also shown is a negative phase reverse, where the circumnavigation wave vector passes through a 90 degree clockwise rotation of the vector diagram.
