Intracellular dynamics are dominated by active transport driven by energetic processes far from equilibrium. Cytoskeletal restructuring, membrane motions and molecular motors use GTP and ATP to drive directed transport that is quasi-one-dimensional with speeds from 10 nm/sec to 10 microns/sec and persistence times tp as large as several seconds. Light scattering under these conditions can be in the lifetime-broadened Doppler shift regime as opposed to a random diffusive regime. The isotropic distribution of 1D transport within cells and tissues produces broad-band signatures that do not produce specific Doppler spectral peaks, but produce Doppler spectral edges that can be related to the mean squared speeds inside cells. The wDtp = 1 product provides a natural dividing line between the Doppler and the diffusive regimes, with a broad cross-over range into which many tissue-based light scattering processes fall. In this talk, I will show how the intracellular Doppler character of dynamic light scattering is derived and modeled, and provide experimental support from biodynamic imaging. Biodynamic imaging uses low-coherence digital holography to capture dynamic spectra in three dimensions from living tissue samples. Biodynamic imaging, based on changes in intracellular dynamics caused by applied therapeutics or changing environments, is expanding into multiple applications, including the selection of chemotherapy for personalized cancer care, screening of potential new therapeutics, and the selection of embryos for artificial reproductive technology. I will give an overview of these applications, describing how changes in biophysical behavior provide actionable biomarkers for clinical applications.
The BioCD platform technology uses spinning-disk interferometry to detect molecular binding to target molecular probes in biological samples. Interferometric configurations have included differential phase contrast and in-line quadrature detection. For the detection of extremely low analyte concentrations, nano- or microparticles can enhance the signal through background-free diffraction detection. Diffraction signal measurements on BioCD biosensors are achieved by forming gratings on a disc surface. The grating pattern was printed with biotinylated bovine serum albumin (BSA) and streptavidin coated beads were deployed. The diameter of the beads was 1 micron and strong protein bonding occurs between BSA and streptavidin-coated beads at the printed location. The wavelength for the protein binding detection was 635 nm. The periodic pattern on the disc amplified scattered light into the first-order diffraction position. The diffracted signal contains Mie scattering and a randomly-distributed-bead noise contributions. Variation of the grating pattern periodicity modulates the diffraction efficiency. To test multiple spatial frequencies within a single scan, we designed a fan-shaped grating to perform frequency filter multiplexing on a diffraction-based BioCD.
Biodynamic imaging uses coherence-gated dynamic light scattering to create three dimensional maps of intracellular dynamics in living tissue biopsies. The technique is sensitive to changes in intracellular dynamics dependent on the mechanism of action (MoA) of therapeutics applied in vitro to the living samples. A preclinical trial in the assessment of chemotherapeutic response of dogs with B-cell lymphoma to the doxorubicin-based therapy CHOP has been completed using biodynamic imaging. The trial enrolled 19 canine patients presenting with non-Hodgkin’s B-cell lymphoma. Biopsies were acquired through surgery or through needle cores. The time-varying power spectrum of scattered light after drugs are applied ex vivo to the biopsies represent biodynamic biomarkers that are used in machine learning algorithms to predict the patient clinical outcome. Two distinct phenotypes emerged from the analysis that correlate with patient drug resistance or sensitivity. Cross validation of the algorithms perform with an accuracy of 90% in the prediction of dogs that will respond to treatment. Biodynamic imaging has the potential to help select chemotherapy for personalized cancer care.