David Levitz

Quantitative characterization of developing collagen gels using optical coherence tomography

Nondestructive optical imaging methods such as optical coherence tomography (OCT) have been proposed for characterizing engineered tissues such as collagen gels. In our study, OCT was used to image collagen gels with different seeding densities of smooth muscle cells (SMCs), including acellular gels, over a five-day period during which the gels contracted and became turbid with increased optical scattering. The gels were characterized quantitatively by their optical properties, specified by analysis of OCT data using a theoretical model. At 6 h, seeded cell density and scattering coefficient (mu(s)) were correlated, with mu(s) equal to 10.8 cm(-1)(10(6) cells mL). Seeded cell density and the scattering anisotropy (g) were uncorrelated. Over five days, the reflectivity in SMC gels gradually doubled with little change in optical attenuation, which indicated a decrease in g that increased backscatter, but only a small drop in mu(s). At five days, a subpopulation of sites on the gel showed substantially higher reflectivity (approximately a tenfold increase from the first 24 h). In summary, the increased turbidity of SMC gels that develops over time is due to a change in the structure of collagen, which affects g, and not simply due to a change in number density of collagen fibers due to contraction.

Non-destructive label-free monitoring of collagen gel remodeling using optical coherence tomography

Matrix remodeling plays a fundamental role in physiological and pathological processes, as well as in tissue engineering applications. In this paper, optical coherence tomography (OCT), a non-destructive optical imaging technology, was used to image collagen gel remodeling by smooth muscle cells (SMCs). The optical scattering properties of collagen-SMC gels were characterized quantitatively by fitting OCT data to a theoretical model. Matrix remodeling over 5 days produced a 10-fold increase in the reflectivity of the collagen gels, corresponding to a decrease in scattering anisotropy from 0.91 to 0.46. The increase in reflectivity was corroborated in confocal mosaic images. Blocking matrix degradation in collagen-SMC gels with doxycycline, a non-specific matrix metalloproteinases (MMPs) inhibitor, impeded the decrease in scattering anisotropy and resulted in few macroscopic signs of remodeling. Causing matrix degradation in acellular gels with a 3 h treatment of MMP-8 (collagenase 2) partially mimicked the decrease in anisotropy measured in collagen-SMC gels after 5 days. These results suggest that the decrease in scattering anisotropy in the collagen-SMC gels was due to MMP activity that degrades collagen fibrils into smaller fragments.

Measuring tissue optical properties in vivo using reflectance-mode confocal microscopy and OCT

The ability to separately measure the scattering coefficient (μs [cm-1]) and the anisotropy (g) is difficult, especially when measuring an in vivo site that can not be excised for bench-top measurements. The scattering properties (μs and g) can characterize the ultrastructure of a biological tissue (nuclear size, mitochondra, cytoskeletion, collagen fibers, density of membranes) without needing an added contrast agent. This report describes the use of reflectance-mode confocal scanning laser microscopy (rCSLM) to measure optical properties. rCSLM is the same as optical coherence tomography (OCT) when the OCT is conducted in focus-tracking mode. The experimental measurement involves translating the depth of focus, zf, of an objective lens, down into a tissue. As depth z increases, the reflected signal R decreases due to attenuation by the tissue scattering (and absorption, μa). The experimental data behaves as a simple exponential, R(z) = ρ exp(-μzf) where ρ is the local reflectivity (dimensionless) and μ [cm-1] is an attenuation coefficient. The relationship between (ρ,μ) and (μs,g) is: μ = (μs a(g) + μa) 2 G(g,NA) ρ = μs Lf b(g,NA) where a(g) is a factor that drops from 1 to 0 as g increases from 0 to 1 (determined by Monte Carlo simulations) allowing photons to reach the focus despite scattering, G is a geometry factor describing the average photon pathlength that depends on the numerical aperture (NA) of the lens and the anisotropy (g), Lf is the axial extent of the focus, and b(g,NA) is the fraction of scattered light that backscatters into the lens for detection.

Dermal reflectivity determined by optical coherence tomography is an indicator of epidermal hyperplasia and dermal edema within inflamed skin.

Psoriasis is a common inflammatory skin disease resulting from genetic and environmental alterations of cutaneous immune responses. While numerous therapeutic targets involved in the immunopathogenesis of psoriasis have been identified, the in vivo dynamics of inflammation in psoriasis remain unclear. We undertook in vivo time course focus-tracked optical coherence tomography (OCT) imaging to noninvasively document cutaneous alterations in mouse skin treated topically with Imiquimod (IMQ), an established model of a psoriasis-like disease. Quantitative appraisal of dermal architectural changes was achieved through a two parameter fit of OCT axial scans in the dermis of the form A(x, y, z) = ρ(x, y)exp [-μ(x, y)z]. Ensemble averaging over 2000 axial scans per mouse in each treatment arm revealed no significant changes in the average dermal attenuation rate, , however the average local dermal reflectivity , decreased significantly following 1, 3, and 6 days of IMQ treatment (p < 0.001) in comparison to vehicle-treated control mice. In contrast, epidermal and dermal thickness changes were only significant when comparing controls and 6-day IMQ treated mice. This suggests that dermal alterations, attributed to collagen fiber bundle enlargement, occur prior to epidermal thickness changes due to hyperplasia and dermal thickness changes due to edema. Dermal reflectivity positively correlated with epidermal hyperplasia (r(epi)(2) = 0.78) and dermal edema (r(derm)(2) = 0.86). Our results suggest that dermal reflectivity as measured by OCT can be utilized to quantify a psoriasis-like disease in mice, and thus has the potential to aid in the quantitative assessment of psoriasis in humans.

Optical design of low cost imaging systems for mobile medical applications

Colposcopes, the gold standard devices for imaging the cervix at high magnfication, are expensive and sparse in low resource settings. Using a lens attachment, any smartphone camera can be turned into an imaging device for tissues such as the cervix. We create a smartphone-based colposcope using a simple lens design for high magnification. This particular design is useful because it allows parameters such as F-number, depth of field, and magnification to be controlled easily. We were therefore able to determine a set of design steps which are general to mobile medical imaging devices and allow them to maintain requisite image quality while still being rugged and affordable.

Characterizing matrix remodeling in collagen gels using optical coherence tomography

Optical coherence tomography (OCT) has shown promise at non-destructively characterizing engineered tissues such as collagen gels. However, as the collagen gels develop, the OCT images lose contrast of structures as the gels develop, making visual assessment difficult. Our group proposed quantitatively characterizing these gels by fitting the optical properties from the OCT signals. In this paper, we imaged collagen gels seeded with smooth muscle cells (SMCs) over a 5-day period and used the data to measure their optical properties. Our results showed that over time, the reflectivity of the samples increased 10-fold, corresponding to a decrease in anisotropy factor g, without much change in the scattering coefficient μs. Overall, the optical properties appeared to be dominated by scattering from the collagen matrix, not the cells. However, SMCs remodeled the collagen matrix, and this collagen remodeling by the cells is what causes the observed changes in optical properties. Moreover, the data showed that the optical properties were sensitive to the activity of matrix metalloproteinases (MMPs), enzymes that break down local collagen fibrils into smaller fragments. Blocking MMPs in the SMC gels greatly impeded both the remodeling process and change in optical properties at day 5. Treating day 1 acellular gels with MMP-8 for 3 hr managed to partially reproduce the remodeling observed in SMC gels at day 5. Altogether, we conclude that matrix remodeling in general, and MMPs specifically, greatly affect the local optical properties of the sample, and OCT is a unique tool that can assess MMP activity in collagen gels both non-destructively and label free.

Optically characterizing collagen gels made with different cell types

The ability of optical imaging techniques such as optical coherence tomography (OCT) to non-destructively characterize tissue-engineered constructs has generated enormous interest recently. Collagen gels are 3D structures that represent a simple common model of many engineered tissues that contain 2 primary scatterers: collagen and cells. We are testing the ability of OCT data to characterize the remodeling of such collagen-based constructs by 3 different types of cells: vascular smooth muscle cells (SMCs), endothelial cells (ECs), and osteoblasts (OBs). Collagen gels were prepared with SMCs, ECs, and OBs with a seeding density of 1×106 cells/ml; additionally, acellular controls were also prepared. The disk-shaped constructs were allowed to remodel in the incubator for 5 days, with OCT imaging occurring on days 1 and 5. From the OCT data, the attenuation and reflectivity were evaluated by fitting the data to a theoretical model that relates the tissue optical properties (scattering coefficient and anisotropy factor) and imaging conditions to the OCT signal. The degree of gel compaction was determined from the volume of the culture medium that feeds the constructs. We found that gel compaction (relative to the acellular control) occurred in the SMC constructs, but not in the OB or EC constructs. The optical property data showed that at day 5 the SMC constructs had an overall higher reflectivity (lower g) relative to day 1, whereas there was no obvious change in reflectivity of the EC, OB constructs and acellular controls relative to day 1. Moreover, there was a difference in the attenuation of the OB constructs on day 5 relative to day 1, but not in the other constructs. The apparent decrease in anisotropy observed in the SMC constructs, but not in the OB and EC constructs and acellular controls, suggests that OCT is sensitive to the remodeling of the collagen matrix that accompanies gel compaction, and can offer highly localized information on the construct microstructure. The apparent increase in the scattering coefficient of the OB constructs is believed to be caused by a higher rate of proliferation by these cell types relative to the others. Overall, these results suggest that the optical properties of collagen gels contain information on both cell number and collagen gel microstructure.

Optically characterizing vascular tissue constructs made with soluble versus homogenized collagen

The ability of optical imaging techniques such as optical coherence tomography (OCT) to non-destructively characterize tissue-engineered constructs has generated enormous interest recently. We are testing the hypothesis that OCT data can be used to characterize the cellularity of collagen-based vascular constructs made from 2 types of collagen scaffold matrix: soluble collagen and homogenized collagen. Smooth muscle cells were seeded in these 2 scaffold matrices at a seeding density of 1×106 cells/ml. The disk-shaped constructs were allowed to remodel and compact in the incubator for 96 hours. OCT imaging of the constructs occurred at 24 hour intervals. From the OCT data, the attenuation and reflectivity were evaluated by fitting the data to a theoretical model that relates the tissue optical properties (scattering coefficient and anisotropy factor) and imaging conditions to the OCT signal. The fitted optical properties were compared to the construct volume. Representative H&E histological sections of the constructs were used to assess cell proliferation. Our data showed that the optical properties of the solubilized constructs changed over time while those of the homogenized constructs did not, in agreement with the histology and compaction observations.

Smartphone-coupled rhinolaryngoscopy at the point of care

Rhinolaryngoscopy remains difficult to perform in resource-limited settings due to the high cost of purchasing and maintaining equipment as well as the need for specialists to interpret exam findings. While the lack of expertise can be obviated by adopting telemedicine-based approaches, the capture, storage, and sharing of images/video is not a common native functionality of medical devices. Most rhinolaryngoscopy systems consist of an endoscope that interfaces with the patient’s naso/oropharynx, and a tower of modules that record video/images. However, these expensive and bulky modules can be replaced by a smartphone that can fulfill the same functions but at a lower cost. To demonstrate this, a commercially available rhinolaryngoscope was coupled to a smartphone using a 3D-printed adapter. Software developed for other clinical applications was repurposed for ENT use, including an application that controls image and video capture, a HIPAA-compliant image/video storage and transfer cloud database, and customized software features developed to improve practitioner competency. Audio recording capabilities to assess speech pathology were also integrated into the smartphone rhinolaryngoscope system. The illumination module coupled onto the endoscope remained unchanged. The spatial resolution of the rhinolaryngoscope system was defined by the fiber diameter of endoscope fiber bundle, rather than the smartphone camera. The mobile rhinolaryngoscope system was used with appropriate patients by a general practitioner in an office setting. The general practitioner then consulted with an ENT specialist via the HIPAA compliant cloud database and workflow modules on difficult cases. These results suggest the smartphone-based rhinolaryngoscope holds promise for use in low-resource settings.

Scattering and absorption measurements of cervical tissues measures using low cost multi-spectral imaging

Cervical cancer is a leading cause of death for women in low resource settings. In order to better detect cervical dysplasia, a low cost multi-spectral colposcope was developed utilizing low costs LEDs and an area scan camera. The device is capable of both traditional colposcopic imaging and multi-spectral image capture. Following initial bench testing, the device was deployed to a gynecology clinic where it was used to image patients in a colposcopy setting. Both traditional colposcopic images and spectral data from patients were uploaded to a cloud server for remote analysis. Multi-spectral imaging (~30 second capture) took place before any clinical procedure; the standard of care was followed thereafter. If acetic acid was used in the standard of care, a post-acetowhitening colposcopic image was also captured. In analyzing the data, normal and abnormal regions were identified in the colposcopic images by an expert clinician. Spectral data were fit to a theoretical model based on diffusion theory, yielding information on scattering and absorption parameters. Data were grouped according to clinician labeling of the tissue, as well as any additional clinical test results available (Pap, HPV, biopsy). Altogether, N=20 patients were imaged in this study, with 9 of them abnormal. In comparing normal and abnormal regions of interest from patients, substantial differences were measured in blood content, while differences in oxygen saturation parameters were more subtle. These results suggest that optical measurements made using low cost spectral imaging systems can distinguish between normal and pathological tissues.