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.
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.
Dynamic speckle from 3-D coherence-gated optical sections provides a sensitive label-free measure of cellular activity up to 1 mm deep in living tissue. However, specificity to cellular functionality has not previously been demonstrated. In this work, we perform fluctuation spectroscopy on dynamic light scattering captured using coherence-domain digital holography to obtain the spectral response of tissue that is perturbed by temperature, osmolarity, and antimitotic cytoskeletal drugs. Different perturbations induce specific spectrogram response signatures that can show simultaneous enhancement and suppression in different spectral ranges.
Motility contrast imaging (MCI) detects dynamic speckle from living tissue using digital holography. It detects sub-cellular motion in living tissue as a fully endogenous imaging contrast agent. Three-dimensional imaging assays of anti-mitotic cancer drugs extract label-free functional signatures in tumors.
Digital holographic optical coherence imaging (DHOCI) is a full-frame coherence-gated imaging approach that uses a
CCD camera to record and reconstruct a digital hologram from inside tissue. Our recording of digital holograms at the
optical Fourier plane has advantages for diffuse targets compared with Fresnel off-axis digital holography. DHOCI is
capable of performing functional imaging by using dynamic image speckle as a contrast agent to locate regions of high
metabolic activity characterized by high cellular motility. We show strong dynamic speckle difference between three
metabolic states of a tumor, and demonstrate that functional imaging in DHOCI can capture motility information with
high contrast. We apply functional imaging to track the effect on cell motility by temperature changes or cytoskeletal
The post-genomic promise of a plethora of new therapeutic drugs has remained largely unfulfilled because of two principal bottlenecks: insufficient high-throughput drug toxicity assays, and acceptable in vitro surrogates to in vivo testing. In this paper, we report coherence-domain functional imaging of bulk tissue response to drug toxicity using cellular motility as both a contrast agent for imaging and as a biomarker for metabolic activity. Osteogenic sarcoma tumor spheroids treated with sodium azide exhibit a rapid onset of increased cellular motility, followed by cellular exhaustion. This behavior correlates with the known biological progression of azide poisoning. These specific findings for azide poisoning are relevant in general because of the common action of many drug candidates on the same oxidative phosphorylation pathways affected by azide. Furthermore, azide poisoning is generally representative of hypoxia, including ischemic hypoxia, which is the most common cause of tissue damage in disease and trauma. The OCI motility mapping technique could therefore introduce a new and general approach to the study of toxicity and pathology in vitro.
Holographic Optical Coherence Imaging (OCI) uses spatial heterodyne detection in direct analogy with the temporal heterodyne detection of time-domain OCT. The spatial demodulator can be a sensitive dynamic holographic film or can be a CCD array placed directly at the hologram plane. We show that a digital hologram captured at the Fourier plane requires only a simple 2D inverse FFT of the digital hologram to compute the real image and its conjugate. Our recording on the optical Fourier plane has an advantage for diffuse targets because the intensity distribution of diffuse targets is relatively uniform at the Fourier plane and hence uses the full dynamic range of CCD camera. We applied this technique to human liver tumor spheroids and produced depth-resolved images to depth of 1.4 mm.
Holographic optical coherence imaging of diffuse targets is an en face direct imaging modality that simultaneously illuminates and detects several hundred spatial modes. The interferences among these modes contain information on long-range structure. By using Fourier spatial filtering in Fourier-domain holography, we demonstrate the first phase-contrast en face imaging of extended tissue. This ability represents a fundamental difference between holographic optical coherence imaging (OCI) and conventional optical coherence tomography (that illuminates only a single spatial mode at a time). Channel cross-talk is separated into "interesting" speckle that carries information on long-range spatial coherences in tissue, and "uninteresting" speckle that arises from multiple scattering. Spatial coherence control of the illuminating beam can separate these two contributions. Data on multicellular tumor spheroids obtained from Fourier-domain OCI operating in a phase-contrast mode, using the knife-edge technique, are presented. We achieve -95 dB of sensitivity and nearly 50 dB of dynamic range in tissue reflection.
This paper reviews the physical basis of holographic optical coherence imaging (OCI) applied in image-domain holography (IDH) and Fourier-domain holography (FDH). Holographic OCI is a multi-spatial-channel direct imaging approach that is closely related to short-coherence speckle interferometry and speckle holography, drawing in addition from laser-ranging concepts and techniques of optical coherence tomography (OCT). It produces a series of en face images at successive depths that can be presented in a so-called video "fly-through". Interchannel cross-talk is described as multichannel spatial heterodyne that produces image-bearing speckle. The speckle holograms are proposed to relate to specific structure in the tissue and may be useful as a clinical diagnostic. For instance, sub-cellular motility (a metric of the vitality of a cell and a means to quantify the response to inter-cellular signaling) can be detected with wide field of view without the need for cellular-scale optical resolution. This can be applied across biologically significant areas of tissue with potential for intraoperative applications to asses the state of health beneath the surface of broad areas of excised tissue.