Coherent light scattering from cellular dynamics in living tissues.

This review examines the biological physics of intracellular transport probed by the coherent optics of dynamic light scattering from optically thick living tissues. Cells and their constituents are in constant motion, composed of a broad range of speeds spanning many orders of magnitude that reflect the wide array of functions and mechanisms that maintain cellular health. From the organelle scale of tens of nanometers and upward in size, the motion inside living tissue is actively driven rather than thermal, propelled by the hydrolysis of bioenergetic molecules and the forces of molecular motors. Active transport can mimic the random walks of thermal Brownian motion, but mean-squared displacements are far from thermal equilibrium and can display anomalous diffusion through Lévy or fractional Brownian walks. Despite the average isotropic three-dimensional environment of cells and tissues, active cellular or intracellular transport of single light-scattering objects is often pseudo-one-dimensional, for instance as organelle displacement persists along cytoskeletal tracks or as membranes displace along the normal to cell surfaces, albeit isotropically oriented in three dimensions. Coherent light scattering is a natural tool to characterize such tissue dynamics because persistent directed transport induces Doppler shifts in the scattered light. The many frequency-shifted partial waves from the complex and dynamic media interfere to produce dynamic speckle that reveals tissue-scale processes through speckle contrast imaging and fluctuation spectroscopy. Low-coherence interferometry, dynamic optical coherence tomography, diffusing-wave spectroscopy, diffuse-correlation spectroscopy, differential dynamic microscopy and digital holography offer coherent detection methods that shed light on intracellular processes. In health-care applications, altered states of cellular health and disease display altered cellular motions that imprint on the statistical fluctuations of the scattered light. For instance, the efficacy of medical therapeutics can be monitored by measuring the changes they induce in the Doppler spectra of livingex vivocancer biopsies.

Biodynamic prediction of neoadjuvant chemotherapy response: Results from a prospective multicenter study of predictive accuracy among muscle-invasive bladder cancer patients.

BACKGROUND: Biodynamic signatures (temporal patterns of microscopic motion within a 3-dimensional tumor explant) offer phenomic biomarkers that are highly predictive for therapeutic response. OBJECTIVE: By utilizing motility contrast tomography, which provides a simple, fast assessment of motion patterns in living tissue, we evaluated the predictive accuracy of a biodynamic drug response classifier in muscle-invasive bladder cancer (MIBC) patients undergoing neoadjuvant chemotherapy (NAC). DESIGN, SETTING, AND PARTICIPANTS: One hundred five consecutive bladder cancer patients suspected of having MIBC were screened in a multi-institutional prospective observational study (NCT03739177) from July 2018 to June 2020, of whom, 30 completed NAC and radical cystectomy. INTERVENTION(S): Biodynamic signatures from treatment-naïve fresh bladder tumor specimens obtained after transurethral resection were measured in living tumor fragments challenged by standard-of-care cytotoxins. Patients received gemcitabine and cisplatin or dose-dense methotrexate, vinblastine, doxorubicin, and cisplatin per institutional guidelines and were followed through radical cystectomy. OUTCOMES MEASUREMENTS AND STATISTICAL ANALYSIS: A 4-level classifier was developed to predict pathologic complete response (pCR) vs. incomplete response utilizing a one-left-out cross-validation protocol to minimize over-fitting. Area under the curve evaluated predictive utility. RESULTS: Thirty percent (9 of 30) achieved pCR. Utilizing the 4-level classifier, biodynamically "favored" (scoring ≥ 3) and "strongly favored" (scoring 4) regimens accurately predicted pCR at rates of 66.7% (4 of 6 patients) and 100% (4 of 4 patients), respectively. Biodynamically "favored" scores predicted pCR with 88% sensitivity and 95% negative predictive value, P < 0.0001. Only 5.0% (1 of 20 patients) achieved pCR from regimens scoring 1 or 2, indicating poor to no response from NAC. Area under the receiver operating curve was 96% (95% Confidence Interval: 79%-99%, P < 0.0001). Future direction involves validating this model prospectively. PRINCIPAL CONCLUSIONS: Biodynamic scoring accurately predicts response in MIBC patients receiving NAC and holds promise to substantially improve the scope of appropriate management intervention.

Biodynamic digital holographic speckle microscopy for oocyte and embryo metabolic evaluation.

Assisted reproductive technologies seek to improve the success rate of pregnancies. Morphology scoring is a common approach to evaluate oocyte and embryo viability prior to embryo transfer in utero, but the efficacy of the method is low. We apply biodynamic imaging, based on dynamic light scattering and low-coherence digital holography, to assess the metabolic activity of oocytes and embryos. A biodynamic microscope, developed to image small and translucent biological specimens, is inserted into the bay of a commercial inverted microscope that can switch between conventional microscopy channels and biodynamic microscopy. We find intracellular Doppler spectral features that act as noninvasive proxies for embryo metabolic activity that may relate to embryo viability.

Doppler imaging detects bacterial infection of living tissue.

Living 3D in vitro tissue cultures, grown from immortalized cell lines, act as living sentinels as pathogenic bacteria invade the tissue. The infection is reported through changes in the intracellular dynamics of the sentinel cells caused by the disruption of normal cellular function by the infecting bacteria. Here, the Doppler imaging of infected sentinels shows the dynamic characteristics of infections. Invasive Salmonella enterica serovar Enteritidis and Listeria monocytogenes penetrate through multicellular tumor spheroids, while non-invasive strains of Escherichia coli and Listeria innocua remain isolated outside the cells, generating different Doppler signatures. Phase distributions caused by intracellular transport display Lévy statistics, introducing a Lévy-alpha spectroscopy of bacterial invasion. Antibiotic treatment of infected spheroids, monitored through time-dependent Doppler shifts, can distinguish drug-resistant relative to non-resistant strains. This use of intracellular Doppler spectroscopy of living tissue sentinels opens a new class of microbial assay with potential importance for studying the emergence of antibiotic resistance.

Cancer Holography for Personalized Medicine.

Digital holography can measure the 3D physiology and motion of cancer cells, allowing identification of effective chemotherapies for patients.

Biodynamic signatures from ex vivo bone marrow aspirates are associated with chemotherapy-induced neutropenia in cancer-bearing dogs.

BACKGROUND: Neutropenia is the most common dose-limiting side effect of cytotoxic chemotherapy in cancer-bearing dogs. Biodynamic imaging (BDI) is a functional imaging technology that measures dynamic light scattering from living, three-dimensional tissues to characterize intracellular motion within those tissues. Previous studies have associated BDI biomarkers with tumour sensitivity to chemotherapy agents in dogs with naturally occurring cancer. We hypothesized that BDI, performed ex vivo on bone marrow aspirate samples, would identify dynamic biomarkers associated with the occurrence of specific degrees of neutropenia in tumour-bearing dogs receiving doxorubicin chemotherapy. MATERIALS AND METHODS: Bone marrow aspirates were collected from 10 dogs with naturally occurring cancers prior to initiation of doxorubicin treatment. BDI was performed on bone marrow samples treated ex vivo with doxorubicin at 0.1, 1, 10 and 100 µM along with 0.1% DMSO as a control. Dogs then were treated with doxorubicin (30 mg/m2 , intravenously). Peripheral blood neutrophil counts were obtained on the day of treatment and again 7 days later. Receiver operating characteristic curves identified provisional breakpoints for BDI biomarkers that correlated with specific changes in neutrophil counts between the two time points. RESULTS: Provisional breakpoints for several BDI biomarkers were identified, specifying dogs with the largest proportionate change in neutrophils and with neutropenia that was grade 2 or higher following doxorubicin treatment. CONCLUSIONS: Biodynamic imaging of bone marrow aspirates may identify those dogs at greater risk for neutropenia following doxorubicin chemotherapy. This approach may be useful for pre-emptively modifying chemotherapy dosing in dogs to avoid unacceptable side effects.

Probing complex geophysical geometries with chattering dust.

The modern energy economy and environmental infrastructure rely on the flow of fluids through fractures in rock. Yet this flow cannot be imaged directly because rocks are opaque to most probes. Here we apply chattering dust, or chemically reactive grains of sucrose containing pockets of pressurized carbon dioxide, to study rock fractures. As a dust grain dissolves, the pockets burst and emit acoustic signals that are detected by distributed sets of external ultrasonic sensors that track the dust movement through fracture systems. The dust particles travel through locally varying fracture apertures with varying speeds and provide information about internal fracture geometry, flow paths and bottlenecks. Chattering dust particles have an advantage over chemical sensors because they do not need to be collected, and over passive tracers because the chattering dust delineates the transport path. The current laboratory work has potential to scale up to near-borehole applications in the field.

Intracellular Doppler Spectroscopy detects altered drug response in SKOV3 tumor spheroids with silenced or inhibited P-glycoprotein.

Intracellular Doppler spectroscopy is a form of low-coherence digital holography based upon Doppler detection of scattered light that measures drug response/resistance in tumor spheroids, xenografts, and clinical biopsies. Multidrug resistance (MDR) is one of the main causes of ineffective cancer treatment. One MDR mechanism is mediated by the MDR1 gene that encodes the drug efflux pump P-glycoprotein (Pgp). Overexpression of Pgp in some cancers is associated with poor chemotherapeutic response. This paper uses intracellular Doppler spectroscopy to detect Pgp-mediated changes to drug response in 3D tissues grown from an ovarian cancer cell line (SKOV3). The SKOV3 cell line was incrementally exposed to cisplatin to create a cell line with increased Pgp expression (SKOV3cis). Subsequently, MDR1 in a subset of these cells was silenced in SKOV3cis using shRNA to create a doxycycline inducible, Pgp-silenced cell line (SKOV3cis-sh). A specific Pgp inhibitor, zosuquidar, was used to study the effects of Pgp inhibition on the Doppler spectra. Increased drug sensitivity was observed with Pgp silencing or inhibition as determined by drug IC50s of paclitaxel-response of silenced Pgp and doxorubicin-response of inhibited Pgp, respectively. These results indicate that intracellular Doppler spectroscopy can detect changes in drug response due to silencing or inhibition of a single protein associated with drug resistance with important consequences for personalized medicine.

Doppler fluctuation spectroscopy of intracellular dynamics in living tissue.

Intracellular dynamics in living tissue are dominated by active transport driven by bioenergetic processes far from thermal equilibrium. Intracellular constituents typically execute persistent walks. In the limit of long mean free paths, the persistent walks are ballistic, exhibiting a "Doppler edge" in light scattering fluctuation spectra. At shorter transport lengths, the fluctuations are described by lifetime-broadened Doppler spectra. Dynamic light scattering from transport in the ballistic, diffusive, or the crossover regimes is derived analytically, including the derivation of autocorrelation functions through a driven damped harmonic oscillator analog for light scattering from persistent walks. The theory is validated through Monte Carlo simulations. Experimental evidence for the Doppler edge in three-dimensional (3D) living tissue is obtained using biodynamic imaging based on low-coherence interferometry and digital holography.

Biodynamic imaging for phenotypic profiling of three-dimensional tissue culture.

Three-dimensional (3-D) tissue culture represents a more biologically relevant environment for testing new drugs compared to conventional two-dimensional cancer cell culture models. Biodynamic imaging is a high-content 3-D optical imaging technology based on low-coherence interferometry and digital holography that uses dynamic speckle as high-content image contrast to probe deep inside 3-D tissue. Speckle contrast is shown to be a scaling function of the acquisition time relative to the persistence time of intracellular transport and hence provides a measure of cellular activity. Cellular responses of 3-D multicellular spheroids to paclitaxel are compared among three different growth techniques: rotating bioreactor (BR), hanging-drop (HD), and nonadherent (U-bottom, UB) plate spheroids, compared with ex vivo living tissues. HD spheroids have the most homogeneous tissue, whereas BR spheroids display large sample-to-sample variability as well as spatial heterogeneity. The responses of BR-grown tumor spheroids to paclitaxel are more similar to those of ex vivo biopsies than the responses of spheroids grown using HD or plate methods. The rate of mitosis inhibition by application of taxol is measured through tissue dynamics spectroscopic imaging, demonstrating the ability to monitor antimitotic chemotherapy. These results illustrate the potential use of low-coherence digital holography for 3-D pharmaceutical screening applications.

Approaching a universal scaling relationship between fracture stiffness and fluid flow.

A goal of subsurface geophysical monitoring is the detection and characterization of fracture alterations that affect the hydraulic integrity of a site. Achievement of this goal requires a link between the mechanical and hydraulic properties of a fracture. Here we present a scaling relationship between fluid flow and fracture-specific stiffness that approaches universality. Fracture-specific stiffness is a mechanical property dependent on fracture geometry that can be monitored remotely using seismic techniques. A Monte Carlo numerical approach demonstrates that a scaling relationship exists between flow and stiffness for fractures with strongly correlated aperture distributions, and continues to hold for fractures deformed by applied stress and by chemical erosion as well. This new scaling relationship provides a foundation for simulating changes in fracture behaviour as a function of stress or depth in the Earth and will aid risk assessment of the hydraulic integrity of subsurface sites.

Digital holography of intracellular dynamics to probe tissue physiology.

Digital holography provides improved capabilities for imaging through dense tissue. Using a short-coherence source, the digital hologram recorded from backscattered light performs laser ranging that maintains fidelity of information acquired from depths much greater than possible by traditional imaging techniques. Biodynamic imaging (BDI) is a developing technology for live-tissue imaging of up to a millimeter in depth that uses the hologram intensity fluctuations as label-free image contrast and can study tissue behavior in native microenvironments. In this paper BDI is used to investigate the change in adhesion-dependent tissue response in 3D cultures. The results show that increasing density of cellular adhesions slows motion inside tissue and alters the response to cytoskeletal drugs. A clear signature of membrane fluctuations was observed in mid-frequencies (0.1-1 Hz) and was enhanced by the application of cytochalasin-D that degrades the actin cortex inside the cell membrane. This enhancement feature is only observed in tissues that have formed adhesions, because cell pellets initially do not show this signature, but develop this signature only after incubation enables adhesions to form.

Biodynamic imaging of live porcine oocytes, zygotes and blastocysts for viability assessment in assisted reproductive technologies.

The success of assisted reproductive technologies relies on accurate assessment of reproductive viability at successive stages of development for oocytes and embryos. The current scoring system used to select good-quality oocytes relies on morphologically observable traits and hence is indirect and subjective. Biodynamic imaging may provide an objective approach to oocyte and embryo assessment by measuring physiologically-relevant dynamics. Biodynamic imaging is a coherence-gated approach to 3D tissue imaging that uses digital holography to perform low-coherence speckle interferometry to capture dynamic light scattering from intracellular motions. The changes in intracellular activity during cumulus oocyte complex maturation, before and after in vitro fertilization, and the subsequent development of the zygote and blastocyst provide a new approach to the assessment of preimplant candidates.

Phenotypic profiling of Raf inhibitors and mitochondrial toxicity in 3D tissue using biodynamic imaging.

The existence of phenotypic differences in the drug responses of 3D tissue relative to 2D cell culture is a concern in high-content drug screening. Biodynamic imaging is an emerging technology that probes 3D tissue using short-coherence dynamic light scattering to measure the intracellular motions inside tissues in their natural microenvironments. The information content of biodynamic imaging is displayed through tissue dynamics spectroscopy (TDS) but has not previously been correlated against morphological image analysis of 2D cell culture. In this article, a set of mitochondria-affecting compounds (FCCP, valinomycin, nicardipine, ionomycin) and Raf kinase inhibitors (PLX4032, PLX4720, GDC, and sorafenib) are applied to multicellular tumor spheroids from two colon adenocarcinoma cell lines (HT-29 and DLD-1). These were screened by TDS and then compared against conventional image-based high-content analysis (HCA). The responses to the Raf inhibitors PLX4032 and PLX4720 are grouped separately by cell line, reflecting the Braf/Kras difference in these cell lines. There is a correlation between TDS and HCA phenotypic clustering for most cases, which demonstrates the ability of dynamic measurements to capture phenotypic responses to drugs. However, there are significant 2D versus 3D phenotypic differences exhibited by several of the drugs/cell lines.

Active holography in InGaAs/InP quantum-well microcavities.

High-efficiency dynamic holography at 1.55 µm is demonstrated in a broad-area InGaAs/InP multiple-quantum-well vertical microcavity. The design places single quantum wells at the cavity antinodes, reducing mode-pulling and enabling a higher Q-factor. The device is pumped by interference fringes through an amorphous mirror that is transparent to a high-energy hologram writing pulse at a wavelength of 1.06 µm. Optically pumped free carrier gratings are probed by a tunable 1.5 µm laser in a four-wave mixing configuration. Diffraction efficiency into both m=±1 diffraction orders of 35% (70% total) has been obtained with a phase grating contribution approaching the maximum π phase shift by combining absorption bleaching with asymmetric Fabry-Perot reflectivity. The diffracted signal exhibits rise/fall times of 5 ns, demonstrating the high speed capabilities of this device.

Tissue dynamics spectroscopy for phenotypic profiling of drug effects in three-dimensional culture.

Coherence-gated dynamic light scattering captures cellular dynamics through ultra-low-frequency (0.005-5 Hz) speckle fluctuations and Doppler shifts that encode a broad range of cellular and subcellular motions. The dynamic physiological response of tissues to applied drugs is the basis for a new type of phenotypic profiling for drug screening on multicellular tumor spheroids. Volumetrically resolved tissue-response fluctuation spectrograms act as fingerprints that are segmented through feature masks into high-dimensional feature vectors. Drug-response clustering is achieved through multidimensional scaling with simulated annealing to construct phenotypic drug profiles that cluster drugs with similar responses. Hypoxic vs. normoxic tissue responses present two distinct phenotypes with differentiated responses to environmental perturbations and to pharmacological doses.

Molecular layer detection on a diffractive optical balance.

Diffraction-based molecular detection is achieved by etching optical gratings into thermal oxide on silicon. The gratings perform as a stable common-path diffractive optical balance (DOB) designed to operate near a missing diffraction order. The biosensor is operated in an off-null condition with a phase bias to produce a high-contrast responsivity that is linear in accumulated molecules but with a low background. The DOB linear responsivity is a factor of 20 larger than the reflectometric responsivity of planar thermal oxide.

Pinned films and capillary hysteresis in microfluidic channels.

Pinned water films in a microfluidic channel act as elastic membranes under tension that increase capillary pressures while preserving the mechanical work dissipated around capillary pressure-saturation, P(c)-S(w), hysteresis cycles. High-resolution two-photon laser micromachining of SU-8 photoresist was used to fabricate wedge-shaped microfluidic channels that included sharp edge features to pin wetting films during drainage. The films were measured using confocal fluorescence microscopy. The tension in the film acts as an elastic tether that shifts the P(c)-S(w) hysteresis cycle higher in pressure relative to the hysteresis cycle in the same sample when films are not pinned. The film tension is strongly nonlinear as the restoring force decreases with increasing displacement. The contribution of elastic forces to hysteresis has important consequences for pressure and saturation control in microfluidics.

Tissue dynamics spectroscopy for three-dimensional tissue-based drug screening.

Tissue dynamics spectroscopy combines dynamic light scattering with short-coherence digital holography to capture intracellular motion inside multicellular tumor spheroid tissue models. The cellular mechanical activity becomes an endogenous imaging contrast agent for motility contrast imaging. Fluctuation spectroscopy is performed on dynamic speckle from the proliferating shell and hypoxic core to generate drug-response spectrograms that are frequency versus time representations of the changes in spectral content induced by an applied compound or an environmental perturbation. A combination of 28 reference compounds and conditions applied to rat osteogenic UMR-106 spheroids generated spectrograms that were crosscorrelated in a similarity matrix used for unsupervised hierarchical clustering of similar compound responses. This work establishes the feasibility of tissue dynamics spectroscopy for three-dimensional tissue-based phenotypic profiling of drug response as a fully endogenous probe of the response of tissue to reference compounds.

Holographic tissue dynamics spectroscopy.

Tissue dynamics spectroscopy uses digital holography as a coherence gate to extract depth-resolved quasi-elastic dynamic light scattering from inside multicellular tumor spheroids. The temporal speckle contrast provides endogenous dynamical images of proliferating and hypoxic or necrotic tissues. Fluctuation spectroscopy similar to diffusing wave spectroscopy is performed on the dynamic speckle to generate tissue-response spectrograms that track time-resolved changes in intracellular motility in response to environmental perturbations. The spectrograms consist of several frequency bands that range from 0.005 to 5 Hz. The fluctuation spectral density and temporal autocorrelations show the signature of constrained anomalous diffusion, but with large fluctuation amplitudes caused by active processes far from equilibrium. Differences in the tissue-response spectrograms between the proliferating outer shell and the hypoxic inner core differentiate normal from starved conditions. The differential spectrograms provide an initial library of tissue-response signatures to environmental conditions of temperature, osmolarity, pH, and serum growth factors.