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.

BackgroundMicroneedle is a method that creates transdermal microchannels across the stratum corneum barrier layer of skin. No previous study showed a therapeutic effect of microneedle itself on hair growth by wounding.ObjectiveThe aim of this study is to investigate the effect of repeated microwound formed by microneedle on hair growth and hair growth-related genes in a murine model.MethodsA disk microneedle roller was applied to each group of mice five times a week for three weeks. First, to identify the optimal length and cycle, microneedles of lengths of 0.15 mm, 0.25 mm, 0.5 mm, and 1 mm and cycles of 3, 6, 10, and 13 cycles were applied. Second, the effect of hair growth and hair-growth-related genes such as Wnt3a, β-catenin, vascular endothelial growth factor (VEGF), and Wnt10b was observed using optimized microneedle. Outcomes were observed using visual inspection, real-time polymerase chain reaction, and immunohistochemistry.ResultsWe found that the optimal length and cycle of microneedle treatment on hair growth was 0.25 mm/10 cycles and 0.5 mm/10 cycles. Repeated microneedle stimulation promoted hair growth, and it also induced the enhanced expression of Wnt3a, β-catenin, VEGF, and Wnt10b.ConclusionOur study provides evidence that microneedle stimulation can induce hair growth via activation of the Wnt/β-catenin pathway and VEGF. Combined with the drug delivery effect, we believe that microneedle stimulation could lead to new approaches for alopecia.

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.

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.

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.

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.

These results demonstrate that LED light irradiation induced hORSC proliferation and migration and inhibited apoptosis in vitro. The growth-promoting effects of LEDs on hORSCs appear to be associated with direct stimulation of the Wnt5a/β-catenin and ERK signaling pathway. Lasers Surg. Med. 49:940-947, 2017. © 2017 Wiley Periodicals, Inc.

Abstract. 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. Biological speckle has a dual character. On one hand, it is a parasitic effect that degrades the contrast of biomedical imaging, and many approaches seek speckle reduction.1 On the other hand, speckle provides strong statistical information about scattering media and can be used for interferometric imaging of cells.2 Dynamic speckle, in particular, has considerable information content. For instance, dynamic speckle can be used to monitor blood flow 3 and can assess the health of living tissue using intracellular motion as a fully endogenous imaging contrast agent.4 3-D imaging approaches are particularly important for assessing tissue viability, pharmacological toxicity, and cancer progression, 5 and can be provided by coherence-domain techniques 6 and digital holography. 7 Holography captures high-contrast depth-gated speckle statistics because of broad-field illumination. 8 We previously used coherence-gated digital holography to capture intracellular motion in 3-D tissue as an imaging contrast agent 9 based on the statistical fluctuations of dynamic speckle. However, there are many functional causes of subcellular motion, and an overall motility metric does not capture specific functions. In this work, we perform fluctuation spectroscopy on depth-gated dynamic speckle to generate frequency versus time spectrograms of tissue responding to various environmental and pharmacological perturbations. The spectral responses depend on the specific type of perturbation, and these spectral responses can serve as functional fingerprints for tissue-based screening.Holograms in short coherence digital holography were recorded and reconstructed from the experimental setup shown in Fig. 1. Lenses L1 and L2 expanded a reference beam, and lens L3 performed the Fourier transform of the object beam. Details of the experimental configuration are discussed in Ref.9. We used rat osteogenic sarcoma tumor spheroids as the target tissue samples.10 Multicellular tumor spheroids have approximately spherical geometry that facilitates comparison of structure to function. A typical pseudo-B-scan image is shown in Fig. 1, which is produced from 3-D holographic images of an 800-m-diam rat tumor spheroid.To generate frequency versus time spectrograms, we acquired 300 successive digital holograms at two acquisition rates ͑200 holograms for 20 s and 100 holograms for 200 s͒ every 4 min for three hou...