Chapter 3 – Making Light Work in Biology  77

The difference in OPLs of the sample and reference beam results in an interference pattern

at the image plane (normally a camera detector), and it is this wave interference that creates

contrast.

Since the sample and reference beams emerge at different angles from the first Wollaston/​

Nomarski prism, they generate two bright-​field images of orthogonal polarization that are lat­

erally displaced from each other by typically a few hundred nanometers, with corresponding

regions of the two images resulting from different OPLs, or phases. Thus, the resultant inter­

ference pattern depends on the variations of phase between lateral displacements of the

sample, in other words with the spatial gradient of refractive index of across a biological

sample. It is therefore an excellent technique for identifying the boundaries of cells and also

of cell organelles.

A related technique to DIC using polarized illumination is Hoffmann modulation contrast

(HMC) microscopy. HMC systems consist of a condenser and objective lens, which have a

slit aperture and two coupled polarizers instead of the first Wollaston/​Nomarski prism and

polarizer of DIC, and a modulator filter in place of the second Wollaston/​Nomarski prism,

which has a spatial dependence on the attenuation of transmitted light. This modulator filter

has usually three distinct regions of different attenuation, with typical transmittance values of

T =​ 100% (light), 15% (gray), and 1% (dark). The condenser slit is imaged onto the gray zone

of the modulator. In regions of the sample where there is a rapid spatial change of sample

optical path, refraction occurs, which deviates the transmitted light path. The refracted light

will be attenuated either more or less in passing through the modulator filter, resulting in an

image whose intensity values are dependent on the spatial gradient of the refractive index

of the sample, similar to DIC. HMC has an advantage over DIC in that it can be used with

birefringent specimens, which would otherwise result in confusing images in DIC, but has a

disadvantage in that DIC can utilize the whole aperture of the condenser resulting in higher

spatial resolution information from the transmitted light.

Quantitative phase imaging (QPI) (Popescu, 2011) utilizes the same core physics

principles as phase microscopy but renders a quantitative image in which each pixel intensity

is a measure of the absolute phase difference between the scattered light from a sample rela­

tive to a reference laser beam and has the same advantages of being label-​free and thus less

prone to potential physiological artifacts due to the presence of a contrast-​enhancing label

such as a fluorescent dye. It can thus in effect create a map of the variation of the refraction

index across a sample, which is a proxy for local biomolecular concentration—​for example, as

a metric for the spatial variation of biomolecular concentration across a tissue or in a single

cell. 2D and 3D imaging modalities exist, with the latter also referred to as holotomograpy.

The main drawback of QPI is the lack of specificity since it is non-​trivial to deconvolve the

respective contributions of different cellular biomolecules to the measured refractive index.

To mitigate this issue, QPI can be also combined with other forms of microscopy such as

fluorescence microscopy in which specific fluorescent dye labeling can be used with multi­

color microscopy to map out the spatial distribution of several different components (see

section 3.5.3), while QPI can be used to generate a correlated image of the total biomolecular

concentration in the same region of the cell or tissue sample. Similarly, QPI can be correlated

with several other light microscopy techniques, for example including optical tweezers (see

Chapter 6).

3.4.3  DIGITAL HOLOGRAPHIC MICROSCOPY

Digital holographic microscopy is emerging as a valuable tool for obtaining 3D spatial infor­

mation for the localization of swimming cells, for example, growing cultures of bacteria,

as well as rendering time-​resolved data for changes to cellular structures involved in cell

motility during their normal modes of action, for example, flagella of bacteria that rotate to

enable cells to swim by using a propeller type action, and similarly cilia structures of certain

eukaryotic cells. The basic physics of hologram formation involves an interference pattern

between a laser beam, which passes through (or some variant of the technique is reflected

from) the sample, and a reference beam split from the same coherent source that does not