DIC microscopes can detect objects much smaller than the theoretical diffraction limit of light, such as unstained microtubules of 25 nm in diameter (10) and noble metal nanoparticles of <10 nm in diameter (11) (see Note 1). In addition, DIC microscopy makes full use of the numerical apertures (NA) of the objective and condenser simultaneously to create a shallow depth of focus, which allows optical sectioning with minimal interference from obscuring structures above and below the focal plane (12). Compared to other wide-field light microscopy techniques, such as bright-field, dark-field, and phase contrast microscopies, DIC microscopy provides the best image quality in terms of contrast, and lateral and axial resolutions.

A commercial DIC microscope normally comes with a green filter centered at around 540 nm, because the human eye is more sensitive to green light than other wavelengths in the visible range. However, 540-nm light is not the best wavelength to image many non-fluorescent nano-objects. As shown in Fig. 2a, the unstained microtubules of 25 nm in diameter are visible with good contrast at wavelengths shorter than 465 nm, but are barely visible at wavelengths longer than 565 nm. Another example, as shown in Fig. 2b, 30-nm silver nanospheres generate high DIC image contrasts when the sample is illuminated at the localized surface plasmon resonance (LSPR) wavelength of 420 nm.

The underlying mechanism of the wavelength-dependent DIC image contrast is based on the fact that samples made of different materials show varying apparent refractive indices at different wavelengths. Thus the refractive index difference between a nanoparticle and its local environment changes at different wavelengths, which gives rise to a unique DIC contrast spectrum. By taking advantage of this effect, different nanoparticles and cellular features in the same sample can be differentiated by simply comparing the DIC contrasts at certain signature wavelengths (13).

Furthermore, wavelength-dependent DIC microscopy is capable of resolving the rotational motion of anisotropic noble metal nanoparticles (14–16). Gold nanorods appear in different DIC image patterns when positioned in different orientations and illuminated at the longitudinal and transverse plasmon resonance wavelengths (Fig. 2c). This phenomenon is quite different from the optically isotropic nanoparticles which always appear as half bright and half dark dots in DIC images. The unique DIC image patterns of gold nanorods result from the asymmetrical phase retardations imposed on the two orthogonally polarized beams split by the first Nomarski prism. The wavelength-dependent DIC image patterns can conveniently be used to distinguish gold nanorods from other cellular nano-features. The 2D orientation of gold nanorods relative to the two orthogonally polarized beams can be estimated directly from the bright and dark DIC intensities (16) or by calculating the DIC polarization anisotropy (17), which is defined as , where I _B and I _D are the normalized bright and dark intensities at the longitudinal plasmon resonance wavelength, respectively. The 3D orientation of gold nanorods can also be determined in each DIC image because the bright and dark intensities are the projections of the dipole of the nanorod onto the two polarization directions (16).