Phase contrast, DIC (differential interference contrast)

Phase Contrast Microscopy

Differences in light absorption are often negligible between living cells and their surrounding nutrient medium, as well as between the various intracellular components and plasma membranes, rendering these entities barely visible when observed by brightfield illumination. Phase contrast microscopy takes advantage of minute refractive index differences within cellular components and between unstained cells and their surrounding aqueous medium to produce contrast in these and similar transparent specimens.

 

Differential Interference Contrast Microscopy

An excellent mechanism for rendering contrast in transparent specimens, differential interference contrast (DIC) microscopy is a beam-shearing interference system in which the reference beam is sheared by a minuscule amount, generally somewhat less than the diameter of an Airy disk. The technique produces a monochromatic shadow-cast image that effectively displays the gradient of optical paths for both high and low spatial frequencies present in the specimen. Those regions of the specimen where the optical paths increase along a reference direction appear brighter (or darker), while regions where the path differences decrease appear in reverse contrast. As the gradient of optical path difference grows steeper, image contrast is dramatically increased.

The most obvious difference between DIC and phase contrast microscopy is the pseudo three-dimensional shadow-cast images formed by differential interference contrast optical systems. Specimen images are rendered in a manner reminiscent of the shadowing techniques utilized for many years in electron microscopy, which yield information on topographical properties of the specimen. However, in DIC, the apparent hills and valleys in images must not be mistaken as an indication of actual topographical profile in the specimen, and should always be interpreted with caution. Phase contrast does not produce images having significant three-dimensional character.

 

Characteristics of Phase Contrast and DIC Microscopy

Characteristic	Phase Contrast	DIC
Image Brightness (Brightfield = 100 Percent)	1.3 Percent	0.36 - 2.3 Percent
Epi-Fluorescence Light Loss (Brightfield = 0 Percent)	28 Percent	73 Percent
Lateral Resolution	Condenser Annulus Restricted	Superior
Axial Resolution (Depth Discrimination)	Poor	Superior
Illuminating Aperture	10 Percent of Objective NA	Variable
Phase Shift Detection Limit	< λ/100	< λ/100
Utility at High Phase Shifts	Not Useful	Useful
Azimuthal Effects	No	Yes
Halos and Shade-Off	Yes	No
Stained Specimens	Not Useful	Useful
Birefringent Specimens	Useful	Not Useful
Birefringent Specimen Containers	Yes	No
Brightfield Image Deterioration	Slight	None
Cost	Moderate	High