Clinical trials of coherence-gated Doppler infrared spectroscopy of intracellular dynamics in living tumor tissue seek to identify the efficacy of prescribed chemotherapy for cancer patients. Changes in intracellular dynamics have specific Doppler signatures that depend on the applied cancer drugs and the sensitivity of the patient to treatment. A challenging feature of these assays is a strong intra-tumor heterogeneity that poses a significant challenge to machine-learning classifiers. We train a Twin Deep Network (TDN) to identify these signatures in the presence of strong heterogeneous background to accurately predict patient response to therapy. The TDN is being applied to two ongoing clinical trials: a clinical trial of HER2neg breast-cancer patients, and esophageal cancer patients, all undergoing neoadjuvant therapy or chemoradiation therapy, respectively. This work provides insight into the value of Deep Learning for advanced data analytics as the volume and variety of data from optics-based assays grows
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.
Cellular adhesions play a critical role in cell behavior, and modified expression of cellular adhesion compounds has been linked to various cancers. We tested the role of cellular adhesions in drug response by studying three cellular culture models: three-dimensional tumor spheroids with well-developed cellular adhesions and extracellular matrix (ECM), dense three-dimensional cell pellets with moderate numbers of adhesions, and dilute three-dimensional cell suspensions in agarose having few adhesions. Our technique for measuring the drug response for the spheroids and cell pellets was biodynamic imaging (BDI), and for the suspensions was quasi-elastic light scattering (QELS). We tested several cytoskeletal chemotherapeutic drugs (nocodazole, cytochalasin-D, paclitaxel, and colchicine) on three cancer cell lines chosen from human colorectal adenocarcinoma (HT-29), human pancreatic carcinoma (MIA PaCa-2), and rat osteosarcoma (UMR-106) to exhibit differences in adhesion strength. Comparing tumor spheroid behavior to that of cell suspensions showed shifts in the spectral motion of the cancer tissues that match predictions based on different degrees of cell-cell contacts. The HT-29 cell line, which has the strongest adhesions in the spheroid model, exhibits anomalous behavior in some cases. These results highlight the importance of using three-dimensional tissue models in drug screening with cellular adhesions being a contributory factor in phenotypic differences between the drug responses of tissue and cells.
Freshly-harvested porcine oocytes are invested with cumulus granulosa cells in cumulus-oocyte complexes (COCs). The
cumulus cell layer is usually too thick to image the living oocyte under a conventional microscope. Therefore, it is
difficult to assess the oocyte viability. The low success rate of implantation is the main problem for in vitro fertilization.
In this paper, we demonstrate our dynamic imaging technique called motility contrast imaging (MCI) that provides a
non-invasive way to monitor the COCs before and after maturation. MCI shows a change of intracellular activity during
oocyte maturation, and a measures dynamic contrast between the cumulus granulosa shell and the oocytes. MCI also
shows difference in the spectral response between oocytes that were graded into quality classes. MCI is based on shortcoherence
digital holography. It uses intracellular motility as the endogenous imaging contrast of living tissue. MCI
presents a new approach for cumulus-oocyte complex assessment.
The detection of cellular mitosis inside three-dimensional living tissue at depths up to 1 mm has been beyond
the detection limits of conventional microscopies. In this paper, we demonstrate the use of motility contrast
imaging and fluctuation spectroscopy to detect motional signatures that we attribute to mitotic events within
groups of 100 cells in multicellular tumor spheroids. Motility contrast imaging is a coherence-domain
speckle-imaging technique that uses low-coherence off-axis holography as a coherence gate to localize
dynamic light scattering from selected depths inside tissue. Fluctuation spectroscopy is performed on a pervoxel
basis to generate micro-spectrograms that display frequency content vs. time. Mitosis, especially in Telophase and Cytokinesis, is a relatively fast and high-amplitude phenomenon that should display energetic features within the micro-spectrograms. By choosing an appropriate frequency range and threshold, we detect energetic events with a density and rate that are comparable to the expected mitotic fraction in the UMR cell line. By studying these mitotic events in tumors of two different sizes, we show that micro-spectrograms contain characteristically different information content than macro-spectrograms (averaged over many voxels) in which the mitotic signatures (which are overall a low-probability event) are averaged out. The detection of mitotic fraction in thick living tissue has important consequences for the use of tissue-based assays for drug discovery.
In the cell cycle, mitosis is the most dramatic phase, especially in Telophase and Cytokinesis. For single cells and cell
monolayer, there are precise microscopic studies of mitosis, while for 3-D tissue such as tumor spheroids the light signal
is obscured by the high background of diffusely scattered light. Therefore, the mitosis phase cannot be detected deep
inside 3-D tissue using conventional microscopic techniques. In this work, we detect mitosis in living tissue using Tissue
Dynamic Imaging (TDI). We trace depth-gated dynamic speckles from a tumor spheroid (up to 1mm in diameter) using
coherence-gated digital holography imaging. Frequency-versus-time spectrograms depend on specific types of
perturbation such as cell shape change, membrane undulation and cell organelles movements. By using these spectral
responses as functional finger prints, we can identify mitosis events from different voxels at a specified depth inside
tumor spheroids. By performing B-scans of the tumor spheroid, we generate 3-D mitosis maps (or movies) for the entire
tumor spheroids. We show that for healthy tumor spheroids, the mitosis events only happen within the proliferating shell.
We also compare results when anti-cancer drugs are applied to arrest, release and synchronize mitosis. This shows the
application of TDI for drug screening. The technique can identify and monitor complex motilities inside 3-D tissue with
a strong potential for drug diagnosis and developmental biology studies.
KEYWORDS: Speckle, Tissue optics, Tumors, Spectroscopy, Tissues, Digital holography, Drug discovery, Coherence imaging, Network on a chip, Imaging spectroscopy
Digital holography, Fourier optics and speckle are combined to enable a new direction in drug discovery.
Optical coherence imaging (OCI) is a coherence-gated imaging approach that captures dynamic speckle from inside
living tissue. The speckle temporal fluctuations arise from internal motions in the biological tissue, and the changes in
these motions caused by applying drugs can be captured and quantified using tissue dynamics spectroscopy (TDS). A
phenotypic profile of many reference drugs provides a training set that would help classify new compounds that may be
candidates as new anti-cancer drugs.
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.
We have developed motility contrast imaging (MCI) as a coherence-domain volumetric imaging approach that uses
subcellular dynamics as an endogenous imaging contrast agent of living tissue. Fluctuation spectroscopy analysis of
dynamic light scattering (DLS) from 3-D tissue has identified functional frequency bands related to organelle transport,
membrane undulations and cell shape change. In this paper, we track the behavior of dynamic light scattering as we
bridge the gap between the two extremes of 2-D cell culture on the one hand, and 3-D tissue spheroids on the other. In a
light backscattering geometry, we capture speckle from 2-D cell culture consisting of isolated cells or planar rafts of cells
on cell-culture surfaces. DLS from that cell culture shows differences and lower sensitivity to intra-cellular dynamics
compared with the 3-D tissue. The motility contrast is weak in this limit. As the cellular density increases to cover the
surface, the motility contrast increases. As environmental perturbations or pharmaceuticals are applied, the fluctuation
spectral response becomes more dramatic as the dimensionality of the cellular aggregations increases. We show that
changing optical thickness of the cellular-to-tissue targets usually causes characteristic frequency shifts in the
spectrograms, while changing cellular dimensionality causes characteristic frequencies to be enhanced or suppressed.
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