Polarization and Spectral Interferometric Techniques for Quantitative Phase Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 277

Although widely used for sample characterization, conventional DIC microscopy is
inherently qualitative due to the nonlinear relationship between the image intensity and the
optical path-length (OPL) gradient. Such a difficulty is further complicated by intrinsic
intensity variations due to scattering or absorption within the sample. Recently, quantitative
DIC microscopy has received growing attention, with a number of implementations
proposed to isolate and quantify the OPL gradient from the conventional DIC intensity
images [15 17] . From these gradient measurements, quantitative OPL or phase maps of the
sample can be reconstructed and analyzed [16,18] . In most of these systems, partially
coherent light sources were used in order to reduce speckle noise, but the broadband nature
of these sources has not been fully exploited.

On the other hand, broadband light sources have found key applications in spectral-domain
low-coherence interferometry (SD-LCI), perhaps best exemplified by SD-optical coherence
tomography (SD-OCT). SD-LCI has been shown to have a sensitivity advantage over its
time-domain counterparts [19] . Furthermore, when combined with common-path
interferometry, it can produce superior sensitivity when measuring path length or path-
length gradient [20 22] .

In Section 14.3.1 , we demonstrate SD-DIC microscopy as a method that combines the
common-path nature of DIC microscopy with the high sensitivity of SD-LCI to produce
high-resolution, quantitative measurement of the OPL gradient across a sample. We
introduce its principles, demonstrate imaging of both reflective objects (a USAF resolution
target) and transparent objects (live neonatal cardiomyocytes), and study the dynamics of
cardiomyocyte contraction at selected cellular locations.

14.3.1 Principles of Spectral Domain-DIC Microscopy

Figure 14.3A shows a first generation SD-DIC system. The broadband light from a single-
mode superluminescent diode (Superlum, Inc.; 40 nm) is split equally in

power into slow and fast axes of a 32.5 cm long polarization-maintaining (PM) fiber using a
polarization controller. The two copropagating orthogonal polarization components are then
passed through a Nomarski prism, which splits them into spatially separated o- and e-waves.
The axes of the PM fiber are aligned with the o- and e-polarizations of the Nomarski prism;
therefore the o- and e-waves experience a differential optical delay through the PM fiber.
An objective lens (Carl Zeiss, Inc.; 40

λ5 840 nm, Δλ5

3

, 0.75) focuses both beams onto the sample with a
slight lateral separation, which is smaller than the size of diffraction-limited spot. The
sample is mechanically scanned in xy using motorized actuators for two-dimensional
imaging. A CCD is used to monitor the sample and the beam location.

In this particular SD-DIC implementation, the sample could be configured into two
different modes. For surface profiling applications shown in Figure 14.3B , photons directly