Identification of the Vibrational Optical Coherence Tomography Corneal Cellular Peak.

Purpose: Our team previously identified the presence of five corneal resonant frequency (RF) peaks in healthy volunteers using vibrational optical coherence tomography (VOCT). Prior studies have suggested that the ≤100 Hz RF peak represents the cellular element of tissue. The aim of this study was to confirm that this peak reflects the human corneal cellular component using VOCT and histological analysis. Methods: Two human research globes were obtained from the same donor, and VOCT measurements were collected from the full-thickness corneas. A microkeratome was then used to create serial-free corneal caps from each cornea, with VOCT performed on the residual stromal bed after each excision. All lamellar sections from both globes were sent for histological analysis to determine cellularity. Cell counts on the specimens were performed by two independent observers. Results: The average of the normalized ≤100 Hz peak values before lamellar sectioning was significantly higher than the average of this peak values after the first, second, and third cuts (P = 0.023), which was 33.9% less than before any cuts. The cell count values in the first slice were significantly higher than the average cell count values of the three deeper slices (P < 0.001), and the cell count dropped 84.4% after the first slice was removed. Conclusions: The findings of this study suggest that the ≤100 Hz corneal peak identified by VOCT corresponds to the cellular component of the cornea. Translational Relevance: This work furthers our understanding of the origin of the corneal ≤100 Hz peak identified using VOCT.

Melanin Stacking Differences in Pigmented and Non-Pigmented Melanomas: Quantitative Differentiation between Pigmented and Non-Pigmented Melanomas Based on Light-Scattering Properties.

Cutaneous melanoma is a cancer with metastatic potential characterized by varying amounts of pigment-producing melanocytes, and it is one of the most aggressive and fatal forms of skin malignancy, with several hundreds of thousands of cases each year. Early detection and therapy can lead to decreased morbidity and decreased cost of therapy. In the clinic, this often translates to annual skin screenings, especially for high-risk patients, and generous use of the ABCDE (asymmetry, border irregularity, color, diameter, evolving) criteria. We have used a new technique termed vibrational optical coherence tomography (VOCT) to non-invasively differentiate between pigmented and non-pigmented melanomas in a pilot study. The VOCT results reported in this study indicate that both pigmented and non-pigmented melanomas have similar characteristics, including new 80, 130, and 250 Hz peaks. Pigmented melanomas have larger 80 Hz peaks and smaller 250 Hz peaks than non-pigmented cancers. The 80 and 250 Hz peaks can be used to quantitative characterize differences between different melanomas. In addition, infrared light penetration depths indicated that melanin in pigmented melanomas has higher packing densities than in non-pigmented lesions. Using machine learning techniques, the sensitivity and specificity of differentiating skin cancers from normal skin are shown to range from about 78% to over 90% in this pilot study. It is proposed that using AI on both lesion histopathology and mechanovibrational peak heights may provide even higher specificity and sensitivity for differentiating the metastatic potential of different melanocytic lesions.

Dynamic Ocular Response to Mechanical Loading: The Role of Viscoelasticity in Energy Dissipation by the Cornea.

We have used vibrational optical coherence tomography (VOCT) to measure the resonant frequency, elastic modulus, and loss modulus of components of the anterior segment of pig eyes in vitro. Such basic biomechanical properties of the cornea have been shown to be abnormal not only in diseases of the anterior segment but also in posterior segment diseases as well. This information is needed to better understand corneal biomechanics in health and disease and to be able to diagnose the early stages of corneal pathologies. Results of dynamic viscoelastic studies on whole pig eyes and isolated corneas indicate that at low strain rates (30 Hz or less), the viscous loss modulus is as high as 0.6 times the elastic modulus for both whole eyes and corneas. This large viscous loss is similar to that of skin, which has been hypothesized to be dependent upon the physical association of proteoglycans with collagenous fibers. The energy dissipation properties of the cornea provide a mechanism to dissipate energy associated with blunt trauma, thereby preventing delamination and failure. The cornea possesses the ability to store impact energy and transmit excess energy to the posterior segment of the eye through its serial connection to the limbus and sclera. In this manner, the viscoelastic properties of the cornea, in concert with that of the posterior segment of the pig eye, function to prevent mechanical failure of the primary focusing element of the eye. Results of resonant frequency studies suggest that the 100-120 Hz and 150-160 Hz resonant frequency peaks reside in the anterior segment of the cornea since the removal of the anterior segment of the cornea decreases the peak heights at these resonant frequencies. These results suggest that there is more than one collagen fibril network found in the anterior portion of the cornea that provides structural integrity to prevent corneal delamination and that VOCT may be useful clinically to diagnose corneal diseases.

Measurement of the Elastic Modulus of Cornea, Sclera and Limbus: The Importance of the Corneal-Limbus-Scleral Biomechanical Unit.

BACKGROUND: Energy storage, transmission and dissipation are important considerations of normal mechanical homeostasis. In this paper we present a new technique termed vibrational optical coherence tomography (VOCT) to study the anterior anatomic structures of the pig eye to better understand how energy applied to the cornea is dissipated without delamination occurring. METHODS: VOCT uses infrared light and an applied sinusoidal audible sound wave to image and measure the resonant frequency and modulus of individual macromolecular components of tissue non-invasively. We have measured the resonant frequencies and calculated the moduli of tissues in the anterior portion of the pig eye using VOCT. RESULTS: While both pig and human eyes have similar resonant frequencies, they do differ in the peak amplitudes near the frequencies of 80, 120, 150 and 250 Hz. It is known that the stroma of pig cornea is much thicker than that of human corneas and these differences may explain the normalized peak height differences. The similarity of the resonant frequency peaks near 80, 120, 150 and 250 Hz of cornea, sclera and limbus suggest that the anatomically described layers in these tissues are connected into a single biomechanical unit that can store external mechanical energy and then transmit it for dissipation. Since the energy stored and dissipated is proportional to the modulus and the ability of the tissue to deform under stress, energy storage in these tissues is related to the stiffness. CONCLUSIONS: It is concluded that stored energy is transmitted to the posterior segment of the eye for dissipation through the attachment with the sclera. This mechanism of energy dissipation may protect the cornea from changes in shape, curvature, and refractive power. However, ultimately, energy dissipation through thinning of the sclera may cause globe elongation observed in subjects with myopia and glaucoma.

"Fingerprinting" Benign and Cancerous Skin Lesions Using Vibrational Optical Coherence Tomography: Differentiation among Cancerous Lesion Types Based on the Presence of New Cells, Blood Vessels, and Fibrosis.

In this study, we use vibrational optical coherence tomography (VOCT) to examine the morphology and stiffness of benign and cancerous lesions. Lesion images and 3D plots of weighted displacement versus frequency and depth were used to compare the cellular, dermal collagen, new blood vessels, and fibrotic composition of normal skin, actinic keratoses (AK), nodular and superficial basal cell carcinomas (BCCs), squamous cell carcinomas (SCCs), and melanomas. The results of this study suggest that benign and cancerous lesions differ based on the addition of new cells with increased resonant frequency and stiffness (80 Hz, 1.8 MPa), new blood vessel peaks (130 Hz, 4.10 MPa) that appear to be less stiff than normal blood vessels, and new fibrous tissue peaks (260 Hz, 15-17 MPa) that are present in carcinomas but not in normal skin and only partially present (80 Hz and 130 Hz only) in AKs. Results obtained by creating images based on the location of the 80 Hz, 130 Hz, and 260 Hz peaks of cancerous skin lesions suggest that the fibrous tissue appears to surround the new cells and new lesion blood vessels. The results of this study suggest that the morphology and location of the fibrous tissues in relation to the new cancer-associated cells and lesion blood vessels may provide information on the invasiveness and metastatic potential of skin cancers. The invasiveness and metastatic potential of melanomas may be a result of the cancer-associated cells laying down fibrous tissue that is used as a pathway for migration. The new cancer-associated blood vessels in the vicinity of the new cancer-associated cells may promote this migration and eventual metastasis. The ratios of peak heights 50/130 Hz and 80/130 Hz of normal cells, new lesion cells, new lesion blood vessels, and fibrotic tissue may be used as a "fingerprint" for detecting melanoma and to differentiate it from other skin cancers non-invasively using VOCT.

In Vivo Determination of the Human Corneal Elastic Modulus Using Vibrational Optical Coherence Tomography.

Purpose: To determine the in vivo elastic modulus of the human cornea using vibrational optical coherence tomography (VOCT). Methods: Vibrational analysis coupled with optical coherence tomography (OCT) was used to obtain the resonant frequency (RF) and elastic modulus of corneal structural components. VOCT corneal thickness values were measured using OCT images and correlated with corneal thickness determined with Pentacam (Oculus, Wetzlar, Germany). Moduli were obtained at two locations: central cornea (CC) and inferior cornea (IC). Measurements were obtained with and without anesthetic eye drops to assess their effect on the modulus measurements. Results: VOCT thickness values correlated positively (R2 = 0.97) and linearly (y = 1.039x-16.89) with those of Pentacam. Five RF peaks (1-5) were present, although their presence was variable across eyes. The RF for peaks 1 to 5 in the CC and IC ranged from 73.5 ± 4.9 to 239 ± 3 Hz and 72.1 ± 6.3 to 238 ± 4 Hz, respectively. CC and IC moduli for peaks 1 to 5 ranged from 1.023 ± 0.104 to 6.87 ± 0.33 MPa and 0.98 ± 0.15 to 6.52 ± 0.79 MPa, respectively. Topical anesthesia did not significantly alter the modulus (P > 0.05 for all), except for peak 2 in the CC (P < 0.05). Conclusions: This pilot study demonstrates the utility of VOCT as an in vivo, noninvasive technology to measure the elastic modulus in human corneas. The structural origin of these moduli is hypothesized based on previous reports, and further analyses are necessary for confirmation. Translational Relevance: This work presents VOCT as a novel approach to assess the in vivo elastic modulus of the cornea, an indicator of corneal structural integrity and health.

Identification of Cancerous Skin Lesions Using Vibrational Optical Coherence Tomography (VOCT): Use of VOCT in Conjunction with Machine Learning to Diagnose Skin Cancer Remotely Using Telemedicine.

In this pilot study, we used vibrational optical tomography (VOCT), along with machine learning, to evaluate the specificity and sensitivity of using light and audible sound to differentiate between normal skin and skin cancers. The results reported indicate that the use of machine learning, and the height and location of the VOCT mechanovibrational peaks, have potential for being used to noninvasively differentiate between normal skin and different cancerous lesions. VOCT data, along with machine learning, is shown to predict the differences between normal skin and different skin cancers with a sensitivity and specificity at rates between 78 and 90%. The sensitivity and specificity will be improved using a larger database and by using other AI techniques. Ultimately, VOCT data, visual inspection, and dermoscopy, in conjunction with machine learning, will be useful in telemedicine to noninvasively identify potentially malignant skin cancers in remote areas of the country where dermatologists are not readily available.

The "Virtual Biopsy" of Cancerous Lesions in 3D: Non-Invasive Differentiation between Melanoma and Other Lesions Using Vibrational Optical Coherence Tomography.

Early detection of skin cancer is of critical importance to provide five year survival rates that approach 99%. By 2050, one out of five Americans by age 70 will develop some form of skin cancer. This will result in a projected rate of 50 million skin biopsies per year given the current rate of escalation. In addition, the ability to differentiate between pigmented lesions and melanomas has proven a diagnostic challenge. While dermoscopy and visual analysis are useful in identifying many skin lesions, additional non-invasive techniques are needed to assist in the analysis of difficult to diagnose skin tumors. To augment dermoscopy data, we have developed 3D maps based on physical biomarker characteristics of benign and cancerous lesions using vibrational optical coherence tomography (VOCT). 3D images based on quantitative physical data involving changes in cellular and fibrous tissue stiffness along with changes in vascular quality are used to map and evaluate different types of cancers. 3D tumor maps constructed using quantitative VOCT data and OCT images have been used to characterize the differences between melanoma and other lesions. These characteristics can be used to plan the excision of difficult lesions where extensive surgery may be needed to remove the entire tumor in one step. In addition, it is now possible to use dermoscopy and VOCT to non-invasively differentiate between different cancerous lesion types using measurements of the resonant frequency of new cellular and vascular peaks. Quantitative VOCT information along with dermoscopic findings can be collected and analyzed remotely using artificial intelligence to improve cancerous tissue diagnosis.

Characterization of the Biomechanical Properties of Skin Using Vibrational Optical Coherence Tomography: Do Changes in the Biomechanical Properties of Skin Stroma Reflect Structural Changes in the Extracellular Matrix of Cancerous Lesions?

Early detection of skin cancer is of critical importance since the five-year survival rate for early detected skin malignancies is 99% but drops to 27% for cancer that has spread to distant lymph nodes and other organs. Over 2.5 million benign skin biopsies (55% of the total) are performed each year in the US at an alarming cost of USD ~2.5 B. Therefore there is an unmet need for novel non-invasive diagnostic approaches to better differentiate between cancerous and non-cancerous lesions, especially in cases when there is a legitimate doubt that a biopsy may be required. The purpose of this study is to determine whether the differences in the extracellular matrices among normal skin, actinic keratosis (AK), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) can be assessed non-invasively using vibrational optical coherence tomography (VOCT). VOCT is a new diagnostic technology that uses infrared light and audible sound applied transversely to tissue to measure the resonant frequencies and elastic moduli of cells, dermal collagen, blood vessels and fibrous tissue in skin and lesion stroma without physically touching the skin. Our results indicate that the cellular, vascular and fibrotic resonant frequency peaks are altered in AK, BCC and SCC compared to those peaks observed in normal skin and can serve as physical biomarkers defining the differences between benign and cancerous skin lesions. The resonant frequency is increased from a value of 50 Hz in normal skin to a value of about 80 Hz in pre- and cancerous lesions. A new vascular peak is seen at 130 Hz in cancerous lesions that may reflect the formation of new tumor blood vessels. The peak at 260 Hz is similar to that seen in the skin of a subject with Scleroderma and skin wounds that have healed. The peak at 260 Hz appears to be associated with the deposition of large amounts of stiff fibrous collagen in the stroma surrounding cancerous lesions. Based on the results of this pilot study, VOCT can be used to non-invasively identify physical biomarkers that can help differentiate between benign and cancerous skin lesions. The appearance of new stiff cellular, fragile new vessels, and stiff fibrous material based on resonant frequency peaks and changes in the extracellular matrix can be used as a fingerprint of pre- and cancerous skin lesions.

Molecular Basis for Mechanical Properties of ECMs: Proposed Role of Fibrillar Collagen and Proteoglycans in Tissue Biomechanics.

Collagen and proteoglycans work in unison in the ECM to bear loads, store elastic energy and then dissipate excess energy to avoid tissue fatigue and premature mechanical failure. While collagen fibers store elastic energy by stretching the flexible regions in the triple helix, they do so by lowering their free energy through a reduction in the entropy and a decrease in charge-charge repulsion. Entropic increases occur when the load is released that drive the reversibility of the process and transmission of excess energy. Energy is dissipated by sliding of collagen fibrils by each other with the aid of decorin molecules that reside on the d and e bands of the native D repeat pattern. Fluid flow from the hydration layer associated with the decorin and collagen fibrils hydraulically dissipates energy during sliding. The deformation is reversed by osmotic forces that cause fluid to reform a hydration shell around the collagen fibrils when the loads are removed. In this paper a model is presented describing the organization of collagen fibers in the skin and cell-collagen mechanical relationships that exist based on non-invasive measurements made using vibrational optical coherence tomography. It is proposed that under external stress, collagen fibers form a tensional network in the plane of the skin. Collagen fiber tension along with forces generated by fibroblasts exerted on collagen fibers lead to an elastic modulus that is almost uniform throughout the plane of the skin. Tensile forces acting on cells and tissues may provide a baseline for stimulation of normal mechanotransduction. We hypothesize that during aging, changes in cellular metabolism, cell-collagen interactions and light and UV light exposure cause down regulation of mechanotransduction and tissue metabolism leading to tissue atrophy.

Use of Vibrational Optical Coherence Tomography to Analyze the Mechanical Properties of Composite Materials.

Energy storage and dissipation by composite materials are important design parameters for sensors and other devices. While polymeric materials can reversibly store energy by decreased chain randomness (entropic loss) they fail to be able to dissipate energy effectively and ultimately fail due to fatigue and molecular chain breakage. In contrast, composite tissues, such as muscle and tendon complexes, store and dissipate energy through entropic changes in collagen (energy storage) and viscous losses (energy dissipation) by muscle fibers or through fluid flow of the interfibrillar matrix. In this paper we review the molecular basis for energy storage and dissipation by natural composite materials in an effort to aid in the development of improved substrates for sensors, implants and other commercial devices. In addition, we introduce vibrational optical coherence tomography, a new technique that can be used to follow energy storage and dissipation by composite materials without physically touching them.

Use of vibrational optical coherence tomography to measure viscoelastic properties of muscle and tendon: A new method to follow musculoskeletal injury and pathology In vivo.

The biomechanical properties of muscles and tendons in vivo are important parameters needed to understand musculoskeletal physiology and pathology. Values of the shear moduli reported for human musculoskeletal components using elastographic techniques range from several KPa to about 100 KPa and are much lower than the tensile moduli measured in vivo which are reported to be as high as several hundred MPa at high strains. In this paper we report the results of a pilot study to measure the mechanical properties of human muscles and tendons non-invasively and non-destructively in vivo using vibrational optical coherence tomography (VOCT). VOCT is a non-invasive technique that uses audible sound and reflected infrared light to measure the resonant frequency of each tissue component. Using VOCT we report that the moduli at the biceps muscle-tendon junction are about 24-30 MPa even though moduli in other anatomic locations of muscle and tendons can vary by as much as 10 MPa. It is concluded that the modulus and stress exerted by tendons and muscles at the muscle-tendon junction are similar and that deposition of fibrous tissue at the junction will lead to reduced values of the modulus leading to tissue pathology and muscle injury.

Mechano-vibrational spectroscopy of skin: Are changes in collagen and vascular tissue components early signs of basal cell carcinoma formation?

BACKGROUND: The modulus and resonant frequency of cancer cells and extracellular matrix are increased in both basal cell and squamous cell carcinomas, and in addition, the collagen stiffness is increased. The organization of the extracellular matrix surrounding cancer cells is clearly different than the extracellular matrix that is seen in normal skin. MATERIALS AND METHODS: We have used vibrational optical coherence tomography (VOCT) to measure the resonant frequency and stiffness of collagen, vascular, and reorganized fibrous extracellular matrix components. Measurements of vessels and fibrotic collagen content made on basal cell carcinomas (BCCs) are compared to similar measurements made on normal skin from different anatomical locations. RESULTS: Lesions with basal cell carcinomas exhibit characteristic resonant frequencies and moduli of reorganized extracellular matrix characteristic of fibrotic tissue. Cancerous lesions are characterized by dermal collagen (100 Hz), vascular (150 Hz), and fibrotic peaks (200-230 Hz). The fibrotic peak is not found in areas of normal skin. CONCLUSIONS: Based on this pilot study, it is hypothesized inflammation may promote precancerous lesion formation in the surrounding extracellular matrix. Measurement of the increases in vaculature and fibrotic content may be useful in early detection of BCCs.