The microscope condenser images the annular diaphragm at infinity, while the objective produces an image at the rear focal plane where a conjugate phase plate is positioned, as discussed below. Large, extended specimens are also easily visualized with phase contrast optics due to diffraction and scattering phenomena that occur at the edges of these objects. These small optical path differences produce a linear reduction in intensity with increasing phase shift the image grows progressively darker up to a point depending upon phase plate configuration , after which, the specimen image becomes brighter through reversal of contrast. For arguments in phase contrast microscopy, the role of the specimen in altering the optical path length in effect, the relative phase shift of waves passing through is of paramount importance. The major manufacturers all provide phase contrast accessories for their research and teaching-level microscopes, both in upright and inverted tissue culture configurations.
If a microscope can delineate change of phase as a change in brightness or color, the eye, photographic plate, or photocell will be able to detect the microscopic areas causing the phase changes. 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. Thus, the diffracted specimen waves passing through the phase plate remain 90-degrees a quarter-wavelength out of phase relative to the zeroth-order undeviated or surround light. In phase contrast microscopy under the conditions of the light waves which are not interacting with the specimen are focused as a bright ring in the back focal plane of the objective. The remaining light is unaffected by the specimen and forms the background light red. The sample is effectively illuminated by two light sources, one with 0° polarisation and the other with 90° polarisation.
It is impossible to distinguish between high and low refractive index components in a phase contrast image without information pertaining to the relative thickness of the components. He first received evidence of the phase contrast phenomenon in a study of diffraction gratings, when he was able to selectively detect transparent materials with different refractive indices. In addition, the intensity profile Figure 8 f is reversed from that observed with positive phase contrast. The destructive interference is illustrated in the figure. It is made up of a circular disc having a circular annular groove. The net result is that regions with very high optical path differences begin to appear bright. Figure 1 - Phase Contrast Microscope Configuration In effect, the phase contrast technique employs an optical mechanism to translate minute variations in phase into corresponding changes in amplitude, which can be visualized as differences in image contrast.
These differential absorption properties of stained specimen modify the intensity or amplitude of the light waves transmitted by different regions of the cells and this ultimately creates contrast in the image. In a phase contrast microscope, the image contrast is improved in two steps. The different polarisations prevent interference between these two images at this point. The light rays passing through the denser region of the specimen get regarded and they run with a delayed phase than the undeviated rays. Many staining reagents are toxic for living cells, however. This leads to a decreased amplitude of the resulting wave.
As explained above, the image is generated from two identical bright field images being overlaid slightly offset from each other typically around 0. In some phase contrast objectives, the thin phase plate contains a ring etched into the glass that has reduced thickness in order to differentially advance the phase of the surround S wave by a quarter-wavelength. At the back focal plane of the objective develops an image. Stanley Schwartz - Bioscience Department, Nikon Instruments, Inc. These scales are commonly found in the majority of bony fishes referred to as the Teleostei. With a conventional biological microscope, it is difficult to observe colorless, transparent cells while they are alive.
In phase contrast microscopy, the intensity of an image does not bear a simple linear relationship to the optical path difference produced by the specimen for the entire thickness and refractive index range. Ø Enable the study of membrane permeability of cells and different organelles. Ø It enables visualization of unstained cells. If two waves interfere, the amplitude of the resulting light wave will be equal to the vector sum of the amplitudes of the two interfering waves. In most cases, merely advancing the relative phase of the surround wavefront alone is insufficient to result in the generation of high-contrast images in the microscope.
Similar to a normal microscope, it possesses a light source, condenser system, objective lens system and ocular lens system. To create the colour intensities, the specimen is first stained with suitable dyes which will impart specific colour. Murphy - Department of Cell Biology and Anatomy and Microscope Facility, Johns Hopkins University School of Medicine, 725 N. Both the retarded and unretarded light has to pass through the phase plate kept on the back focal plane of the objective to form the final image. As a general rule, when objective numerical aperture and magnification is increased, the phase plate width and diameter both decrease.
Because the direct, zeroth order light and diffracted light are spatially separated in the diffraction plane, the phase of either wave component surround, S or diffracted, D can be selectively manipulated without interfering with the other. Something else that might be of interest to readers is X-Ray Phase-Contrast Imaging. The negative phase plate also contains both phase retarding and partially absorbing materials. Features - Transparent cells can be observed without staining them because the phase contrast can be converted into brightness differences. When light rays pass through an area of high refractive index, it deviates from its normal path and such a light ray experiences phase change or phase retardation. Phase contrast is also insensitive to polarization and birefringence effects, which is a major advantage when examining living cells growing in plastic tissue culture vessels. This microscopy lecture explains how phase contrast microscopy works.
A phase plate is mounted in or near the objective rear focal plane see Figures 4 and 5 in order to selectively alter the phase and amplitude of the surround or undeviated light passing through the specimen. The phase plate configurations, wave relationships, and vector diagrams associated with the generation of positive and negative phase contrast images are presented in Figure 6. The cell is surrounded by a nutrient medium having a refractive index of 1. Halos occur in phase contrast microscopy because the circular phase-retarding and neutral density ring located in the objective phase plate also transmits a small degree of diffracted light from the specimen it is not restricted to passing surround waves alone. In addition, phase plate geometry and representative specimen images are also presented. 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, latex dispersions, glass fragments, and subcellular particles including nuclei and other organelles.