Phase Contrast Microscopy (PMC) Vs Differential Interference Contrast Microscopy (DIC)

What Is Phase Contrast Microscopy?

Phase-contrast microscopy was first described in 1934 by Dutch physicist Frits Zernike. It a technique used for gaining contrast in a translucent specimen without staining the specimen. The optics of the microscope converts the differences in the refractive index of the specimen into brightness differences. This causes the translucent object to appear brighter than the dark background. 

Phase contrast is used to enhance the contrast of light microscopy images of transparent and colorless specimens.  It enables visualization of cells and cell components that would be difficult to see using an ordinary light microscope.

In a phase-contrast microscope, image contrast is increased in two ways: By generating constructive interference between  scattered and background light rays in regions of the field of view that contain the specimen and by reducing the amount of background light that reaches the image plane.

What You Need To Know About Phase Contrast Microscope (PCM)

• Phase contrast microscope was developed by Zernike in 1934.
• Phase contrast microscope utilizes two light beams without beam splitters.
• Phase contrast microscope works by the detection of sharp changes in the refractive index.
• Produces image by the incomplete separation of direct and diffracted light.
• Optical sectioning is not possible in PMC.
• Images in phase contrast microscope are not sensitive to the orientation of specimen.
• Optical system of phase contrast microscope does not require beam recombiners.
• Nomarski’s prism is not used in the optical path.
• Full numerical aperture of the condenser and objective lenses cannot be utilized because of the masking effect of phase plate used in the light path.
• No three dimensional effects on the images.
• Has a low axial resolution.
• It uses annular diaphragm and phase plate to create contrast.
• Images from the phase contrast microscopy only provide qualitative data.
• Produce a ‘halo’ around edges of the image.
• Does not use a polarizer.

Advantages of Phase Contrast

• Phase contrast does not require cells to be killed, fixed or stained.
• High-contrast, high-resolution images
• Ability to combine with other means of observation such as fluorescence.

Limitation Of Phase Contrast Microscopy

• Phase images are usually surrounded by halos around the outlines of details.
• Phase images appear gray if white light is used and green if a green filter is used.
• Phase contrast does not work well with thick specimens because of shifts in phase occur from areas slightly below or slightly above the plane that is in focus.  Therefore this method is not ideal for thick organisms or particles.
• The phase rings/annuli do limit the working numerical aperture of the optical system to a certain degree, thus reducing resolution.
• Thick specimen can appear distorted.

What Is Differential Interference Contrast Microscope (DIC)?

Differential interference contrast microscopy, also referred to as Nomarski Interference contrast was first described by Francis Smith in 1955. It is a light microscopic technique based on an interference principle involving two coherent beams of light (from the same small light source) and image contrast achieved with gradients in optical path.

DIC introduces contrast to images of specimens which have little or no contrast when viewed using bright-field microscopy. The images produced using DIC have a 3D-like effect, making the technique ideal for electrophysiology experiments.

DIC is used for imaging live and unstained biological samples such as a smear from a tissue culture or individual water borne single-celled organisms. Its resolution and clarity in conditions such as this are unrivaled among standard optical microscopy techniques.

What You Need To Know About Interference Contrast Microscope 

• Differential interference contrast microscope was developed by Nomarski in 1952.
• Differential interference contrast microscope utilizes two light beams with beam splitters.
• Differential interference contrast microscope works by the detection of continous change of refractive index.
• Produce image by the complete separation of direct and diffracted light rays.
• Optical sectioning is possible due to the high axial resolution.
• Images in the differential interference contrast microscope are highly sensitive to the orientation of the specimen.
• Optical system of differential interference contrast microscope does require beam recombiners.
• Nomarski’s prisms are used in the optical path.
• DCI microscopes can utilize the full numerical aperture of objective and condenser lenses.
• Images are with a pseudo three dimensional effect.
• Has a high axial resolution.
• Does not use annular diaphragm and phase plate.
• Images from differential contrast microscopy provide both qualitative and quantitative data.
• No ‘halo’ is produced around the edges on the image.
• Uses a polarizer.

Advantages Of Differential Interference Contrast Microscopy

• The specimen will appear bright in contrast to the dark background.
• No halo effect occurs with differential interference contrast and it can be used to produce very clear images of thick specimen.
• DIC can be used together with digital imaging systems to add further definition to the image.

Disadvantages Of Differential Interference Contrast Microscopy

• Three-dimensional image of a specimen may not be accurate
• The enhanced light and shadow might add distortion to the appearance of the image.

Difference Between Phase Contrast Microscopy And Differential Interference Contrast Microscopy In Tabular Form