Kwan Jeong

Volumetric motility-contrast imaging of tissue response to cytoskeletal anti-cancer drugs

Microscopic imaging of cellular motility has recently advanced from two dimensions to three dimensions for applications in drug development. However, significant degradation in resolution occurs with increasing imaging depth, limiting access to motility information from deep inside the sample. Here, digital holographic optical coherence imaging is adapted to allow visualization of motility in tissue at depths inaccessible to conventional motility assay approaches. This method tracks the effect of cytoskeletal anti-cancer drugs on tissue inside its natural three-dimensional environment using time-course measurement of motility within tumor tissue.

Fourier-domain digital holographic optical coherence imaging of living tissue

Digital holographic optical coherence imaging is a full-frame coherence-gated imaging approach that uses a CCD camera to record and reconstruct digital holograms from living tissue. Recording digital holograms at the optical Fourier plane has advantages for diffuse targets compared with Fresnel off-axis digital holography. A digital hologram captured at the Fourier plane requires only a 2D fast Fourier transform for numerical reconstruction. We have applied this technique for the depth-resolved imaging of rat osteogenic tumor multicellular spheroids and acquired cross-section images of the anterior segment and the retinal region of a mouse eye. A penetration depth of 1.4 mm for the tumor spheroids was achieved.

Three-dimensional holographic imaging of living tissue using a highly sensitive photorefractive polymer device

Photorefractive materials are dynamic holographic storage media that are highly sensitive to coherent light fields and relatively insensitive to a uniform light background. This can be exploited to effectively separate ballistic light from multiply-scattered light when imaging through turbid media. We developed a highly sensitive photorefractive polymer composite and incorporated it into a holographic optical coherence imaging system. This approach combines the advantages of coherence-domain imaging with the benefits of holography to form a high-speed wide-field imaging technique. By using coherence-gated holography, image-bearing ballistic light can be captured in real-time without computed tomography. We analyzed the implications of Fourier-domain and image-domain holography on the field of view and image resolution for a transmission recording geometry, and demonstrate holographic depth-resolved imaging of tumor spheroids with 12 microm axial and 10 microm lateral resolution, achieving a data acquisition speed of 8 x 10(5) voxels/s.

Fourier-domain holographic optical coherence imaging of tumor spheroids and mouse eye

Fourier-domain holography (FDH) has several advantages over image-domain holography for optical coherence imaging of tissue. Writing the hologram in the Fourier plane significantly reduces background arising from reference light scattered from the photorefractive holographic film. The ability to use FDH is enhanced by the use of a diffuse target, such as scattering tissue, rather than specular targets, because the broader angular distribution from diffuse targets is transformed into a relatively uniform distribution in the Fourier plane. We demonstrate significantly improved performance for Fourier-domain optical coherence imaging on rat osteogenic sarcoma tumor spheroids and mouse eye. The sensitivity is documented at -95 dB.

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.

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.

Speckle fluctuation spectroscopy of intracellular motion in living tissue using coherence-domain digital holography

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.

Fourier-domain holography in photorefractive quantum-well films

Fourier-domain holography (FDH) is investigated as a candidate for holographic optical coherence imaging to produce real-time images of structure inside living tissue and turbid media. The effects of spatial filtering, the background intensity distributions, and the role of background noise in determining dynamic range are evaluated for both FDH and image-domain holography (IDH). The grating washout effect in FDH (edge enhancement) is removed by use of a vibrating diffuser that consequently improves the image quality. By comparing holographic images and background images of FDH and IDH we show that FDH provides a higher dynamic range and a higher image quality than IDH for this specific application of imaging diffuse volumetric objects.

Functional imaging by dynamic speckle in digital holographic optical coherence imaging

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 drugs.

Digital holography and tissue dynamics spectroscopy: on the road to high-content drug discovery

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.