Phase contrast

Phase contrast microscopy is employed to examine samples that lack colour contrast resulting from differential absorption, such as unstained cells. This technique enhances contrast by leveraging the interference of diffracted light. It operates on the principle that a slight shift in the phase of light occurs when it passes through a specimen. The refractive index and density disparities between the specimen and its surrounding environment cause diffraction of the incident light, which, on average, is delayed by ¼ of a wavelength relative to undiffracted light. These shifts are subsequently converted into changes in amplitude, observable as variations in image contrast

Phase contrast microscopes consist of two crucial components: a phase annulus and a phase plate. The phase annulus, located in the condenser, focuses a hollow cone of light onto the specimen. Light rays passing through denser regions of the specimen undergo refraction and experience a phase delay compared to the undeviated rays. As light passes through the phase plate situated at the back focal plane of the objective lenses, it encounters a diffraction or phase retardation plate, contributing to image formation. Deviated or diffracted light bypasses the phase ring in the phase plate, while undeviated light rays intersect a phase ring and advance by 1/4 wavelength upon passing through it. Consequently, the deviated and undeviated waves become 1/2 wavelength out of phase with each other, resulting in their cancellation (destructive interference) when they combine to form an image. As a result, the background formed by undeviated light appears bright, while the unstained specimen appears dark and well-defined.