Differential Interference Contrast Microscopy (DIC) - Biomedical Optical Phase Microscopy and Nanoscopy - page 17

CHAPTER 2
Differential Interference
Contrast Microscopy (DIC)

Michael Shribak
Marine Biological Laboratory, Woods Hole, MA

Editor: Lisa L. Satterwhite

2.1 Introduction

Differential interference contrast (DIC) light microscopy is widely used to observe structure and
motion in unstained living cells and isolated organelles. DIC microscopy is a beam-shearing
interference system in which the reference beam is sheared by a small amount, generally less
than the diameter of an Airy disk. The technique produces a monochromatic shadow-cast image
that displays the gradient of optical paths. 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 grows
steeper, image contrast is significantly increased. Another important feature of the DIC
technique is that it produces effective optical sectioning. This is particularly obvious when high
numerical aperture (NA) objectives are used together with high NA condenser illumination.

The DIC technique was invented by Smith in 1947 [1,2] . He placed one Wollaston prism
between a pair of polarizers at the front focal plane of the condenser and another Wollaston
prism in the back focal plane of objective lens ( Figure 2.1 ). The first Wollaston prism splits
the input beam angularly into two orthogonally polarized beams. The condenser makes the
beam axes parallel with a small shear. Then the objective lens joins them in the back focal
plane where the second Wollaston prism introduces an angular deviation into the beam and
makes them parallel. The splitting angles

ε ε

2 are connected with the focal distances
of the condenser and objective f c and f ob and the shear amount d by the following relation:

1 and

f c ε 5 f ob ε 5 d

(2.1) 1 2

This optical configuration creates a polarizing-shearing interferometer, by which one
visualizes phase nonuniformity of the specimen. Smith originally called this device the