We demonstrate the use of a modified Rayleigh–Lamb frequency equation in conjunction with noncontact optical coherence elastography to quantify the viscoelastic properties of the cornea. Phase velocities of air-pulse-induced elastic waves were extracted by spectral analysis and used for calculating the Young’s moduli of the samples using the Rayleigh–Lamb frequency equation (RLFE). Validation experiments were performed on 2% agar phantoms (n=3) and then applied to porcine corneas (n=3) in situ. The Young’s moduli of the porcine corneas were estimated to be ∼60  kPa with a shear viscosity ∼0.33  Pa·s. The results demonstrate that the RLFE is a promising method for noninvasive quantification of the corneal biomechanical properties and may potentially be useful for clinical ophthalmological applications.

We irradiated neuroblastoma SH-SY5Y cell line with low-level light-emitting diode (LED) illumination at a visible wavelength of 520 nm (green) and intensity of 100  mW/cm². We captured and analyzed the cell morphology before LED treatment, immediately after, and 12 and 24 h after treatment. Our study demonstrated that LED illumination increases the amount of sprouting dendrites in comparison to the control untreated cells. This treatment also resulted in more elongated cells after treatment in comparison to the control cells and higher levels of expression of a differentiation related gene. This result is a good indication that the proposed method could serve in phototherapy treatment for increasing sprouting and enhancing neural network formation.

Human atherosclerotic samples collected by carotid endarterectomy were investigated using electronic paramagnetic resonance imaging (EPRI) for visualization of reactive oxygen species, and nonlinear optical microscopy (NLOM) to study structural features. Regions of strong EPRI signal, indicating a higher concentration of reactive oxygen species and increased inflammation, were found to colocalize with regions dense in cholesterol crystals as revealed by NLOM.

Red blood cell (RBC) rouleaux formation was experimentally studied using a light scattering technique. The suspensions of RBCs were obtained from the blood of healthy donors. Hematocrit of the samples was adjusted ranging from 1% to 4%. Measurements of the intensity of the coherent component of light scattered by the suspensions were performed and the scattering coefficient of the suspensions was determined. The number of RBCs per rouleaux was obtained using anomalous diffraction theory. The technique was used to show the effect of time, hematocrit, and sample thickness on the process. The number of cells per rouleaux first increases linearly, reaches a critical value at ∼3 cells per rouleaux, and then a further increase in the rouleaux size is observed. The kinetic constant of the rouleaux growth in the linear region is found to be independent of hematocrit. The aggregation rate increases as the sample thickness increases. The time at which the critical region appears strongly decreases as the hematocrit of the suspension increases.

We present a decoupled fluorescence Monte Carlo (dfMC) model for the direct computation of the fluorescence in turbid media. By decoupling the excitation-to-emission conversion and transport process of the fluorescence from the path probability density function and associating the corresponding parameters involving the fluorescence process with the weight function, the dfMC model employs the path histories of the excitation photons and the corresponding new weight function to directly calculate the fluorescence. We verify the model’s accuracy using phantom experiments and compare it with that of the perturbation fluorescence Monte Carlo model. The results indicate that the model is accurate for the direct fluorescence calculation and, thus, has great potential for application in fluorescence-based in vivo tomography.

Malignant skin tumors of different types were studied in vivo using optical coherence tomography (OCT), backscattering (BS), and Raman spectroscopy (RS). A multimodal method is proposed for early cancer detection based on complex analysis of OCT images by their relative alteration of scattered-radiation spectral intensities between malignant and healthy tissues. An increase in average accuracy of diagnosis was observed for a variety of cancer types (9% sensitivity, 8% specificity) by a multimodal RS-BS-OCT system in comparison with any of the three methods used separately. The proposed approach equalizes the processing rates for all methods and allows for simultaneous imaging and classification of tumors.

The development and ultimate operation of a nanocomposite high-aspect-ratio photoinjection (HARP) device is presented in this work. The device makes use of a nanocomposite material as the optically active layer and the device achieves a large optical penetration depth with a high aspect ratio which provides a strong actuation force far away from the point of photoinjection. The nanocomposite material can be continuously illuminated and the position of the microdroplets can, therefore, be controlled to diffraction limited resolution. The nanocomposite HARP device shows great potential for future on-chip applications.

Stenosis and occlusion of the superficial femoral artery (SFA) are most common in arterial occlusive disease. There are numerous interventional, surgical, and combined approaches to reconstitute maximum blood supply to the lower limb; however, despite intense clinical research, the long-term success rates are still poor. We present the first results with a catheter prototype for laser-based minimal invasive endarterectomy, called laser scoop desobliteration (LSD). The tip of a glass fiber containing a catheter was modified with a spatula head design and connected to an ultraviolet laser. It was tested in cadavers fixed with the Thiel embalming technique preserving tissue consistency, flexibility, and plasticity. After longitudinal arteriotomy of the SFA, a circular dissection between media and adventitia was performed. Then the LSD catheter was inserted and propagated with a progress of 1  mm/s. Afterward, the atheroma core, which showed a plain surface without substantial attaching tissue debris, was removed. Histological examination of the vessel wall showed that the dissection was performed at the media/adventitia interface. In summary, the constructed LSD catheter allowed a rapid and easy way to perform an endarterectomy, thereby offering an innovative approach in the treatment of chronic occluded SFA.

The outcome of light-based therapeutic approaches depends on light propagation in biological tissues, which is governed by their optical properties. The objective of this study was to quantify optical properties of brain tissue in vivo and postmortem and assess changes due to tissue handling postmortem. The study was carried out on eight female New Zealand white rabbits. The local fluence rate was measured in the VIS/NIR range in the brain in vivo, just postmortem, and after six weeks’ storage of the head at −20°C or in 10% formaldehyde solution. Only minimal changes in the effective attenuation coefficient μ_eff were observed for two methods of sacrifice, exsanguination or injection of KCl. Under all tissue conditions, μ_eff decreased with increasing wavelengths. After long-term storage for six weeks at −20°C, μ_eff decreased, on average, by 15 to 25% at all wavelengths, while it increased by 5 to 15% at all wavelengths after storage in formaldehyde. We demonstrated that μ_eff was not very sensitive to the method of animal sacrifice, that tissue freezing significantly altered tissue optical properties, and that formalin fixation might affect the tissue’s optical properties.

To adapt the complex tissue structure, laser propagation in a two-layered skin model is simulated to compare voxel-based Monte Carlo (VMC) and tetrahedron-based MC (TMC) methods with a geometry-based MC (GMC) method. In GMC, the interface is mathematically defined without any discretization. GMC is the most accurate but is not applicable to complicated domains. The implementation of VMC is simple because of its structured voxels. However, unavoidable errors are expected because of the zigzag polygonal interface. Compared with GMC and VMC, TMC provides a balance between accuracy and flexibility by the tetrahedron cells. In the present TMC, the body-fitted tetrahedra are generated in different tissues. No interface tetrahedral cells exist, thereby avoiding the photon reflection error in the interface cells in VMC. By introducing a distance threshold, the error caused by confused optical parameters between neighboring cells when photons are incident along the cell boundary can be avoided. The results show that the energy deposition error by TMC in the interfacial region is one-tenth to one-fourth of that by VMC, yielding more accurate computations of photon reflection, refraction, and energy deposition. The results of multilayered and n-shaped vessels indicate that a laser with a 1064-nm wavelength should be introduced to clean deep-buried vessels.

The fabrication of submicron-height sine-like relief of a trifocal diffractive zone plate using a nanoimprinting technique is studied. The zone plate is intended for use in combined trifocal diffractive–refractive lenses and provides the possibility to form trifocal intraocular lenses with predetermined light intensity distribution between foci. The optical properties of the designed zone plate having the optical powers 3 D, 0, −3 D in the three main diffraction orders are theoretically and experimentally investigated. The results of the theoretical investigations are in good agreement with experimental measurements. The effects of the pupil size (lens diameter) as well as the wavelength-dependent behavior of the zone plate are also discussed.

The ability to image targeted tracer binding to epidermal growth factor receptor (EGFR) was studied in vivo in orthotopically grown glioma tumors of different sizes. The binding potential was quantified using a dual-tracer approach, which employs a fluorescently labeled peptide targeted to EGFR and a reference tracer with similar pharmacokinetic properties but no specific binding, to estimate the relative bound fraction from kinetic compartment modeling. The recovered values of binding potential did not vary significantly as a function of tumor size (1 to 33  mm³), suggesting that binding potential may be consistent in the U251 tumors regardless of size or stage after implantation. However, the fluorescence yield of the targeted fluorescent tracers in the tumor was affected significantly by tumor size, suggesting that dual-tracer imaging helps account for variations in absolute uptake, which plague single-tracer imaging techniques. Ex vivo analysis showed relatively high spatial heterogeneity in each tumor that cannot be resolved by tomographic techniques. Nonetheless, the dual-tracer tomographic technique is a powerful tool for longitudinal bulk estimation of receptor binding.

A diffuse imaging method is presented that enables wide-field estimation of the depth of fluorescent molecular markers in turbid media by quantifying the deformation of the detected fluorescence spectra due to the wavelength-dependent light attenuation by overlying tissue. This is achieved by measuring the ratio of the fluorescence at two wavelengths in combination with normalization techniques based on diffuse reflectance measurements to evaluate tissue attenuation variations for different depths. It is demonstrated that fluorescence topography can be achieved up to a 5 mm depth using a near-infrared dye with millimeter depth accuracy in turbid media having optical properties representative of normal brain tissue. Wide-field depth estimates are made using optical technology integrated onto a commercial surgical microscope, making this approach feasible for real-world applications.

Early identification of diabetic foot complications and their precursors is essential in preventing their devastating consequences, such as foot infection and amputation. Frequent, automatic risk assessment by an intelligent telemedicine system might be feasible and cost effective. Infrared thermography is a promising modality for such a system. The temperature differences between corresponding areas on contralateral feet are the clinically significant parameters. This asymmetric analysis is hindered by (1) foot segmentation errors, especially when the foot temperature and the ambient temperature are comparable, and by (2) different shapes and sizes between contralateral feet due to deformities or minor amputations. To circumvent the first problem, we used a color image and a thermal image acquired synchronously. Foot regions, detected in the color image, were rigidly registered to the thermal image. This resulted in 97.8%±1.1% sensitivity and 98.4%±0.5% specificity over 76 high-risk diabetic patients with manual annotation as a reference. Nonrigid landmark-based registration with B-splines solved the second problem. Corresponding points in the two feet could be found regardless of the shapes and sizes of the feet. With that, the temperature difference of the left and right feet could be obtained.

Tumor collagen characteristics influence tumor malignancy, invasion, and metastasis. This study investigates the effects of trastuzumab (Tz) on the collagen of Tz-responsive (BT474) and Tz-resistant (HR6) breast cancer xenografts. Collagen content was assessed by in vivo second harmonic generation (SHG) imaging and histological trichrome staining of tumor sections. Collagen SHG imaging of control BT474 and HR6 tumors demonstrated increased collagen density after 14 days of treatment (p<0.05). Trichrome staining revealed decreased collagen in Tz-treated BT474 and HR6 tumors at 2, 5, and 14 days of treatment, suggesting that Tz affects the tumor microenvironment independent of epithelial cell response. Additionally, collagen alignment analysis revealed significantly less aligned collagen in the Tz-treated BT474 tumors at day 14 compared with control BT474 tumors. There was no correlation between SHG endpoints (collagen density and alignment) and trichrome staining (p<0.05), consistent with the physically distinctive nature of these measurements. There was also no correlation between tumor size and collagen endpoints (p<0.05). These results identify changes within the collagen compartment of the tumor microenvironment following Tz treatment, which are independent from the tumor cell response to Tz, and demonstrate that intravital collagen SHG imaging is capable of measuring dynamic changes in tumor microenvironment following treatment that complements trichrome staining.

Intravital multiphoton imaging of arteries is technically challenging because the artery expands with every heartbeat, causing severe motion artifacts. To study leukocyte activity in atherosclerosis, we developed the intravital live cell triggered imaging system (ILTIS). This system implements cardiac triggered acquisition as well as frame selection and image registration algorithms to produce stable movies of myeloid cell movement in atherosclerotic arteries in live mice. To minimize tissue damage, no mechanical stabilization is used and the artery is allowed to expand freely. ILTIS performs multicolor high frame-rate two-dimensional imaging and full-thickness three-dimensional imaging of beating arteries in live mice. The external carotid artery and its branches (superior thyroid and ascending pharyngeal arteries) were developed as a surgically accessible and reliable model of atherosclerosis. We use ILTIS to demonstrate Cx3cr1^GFP monocytes patrolling the lumen of atherosclerotic arteries. Additionally, we developed a new reporter mouse (Apoe^−/−Cx3cr1^GFP/+Cd11c^YFP) to image GFP+ and GFP+YFP+ macrophages “dancing on the spot” and YFP+ macrophages migrating within intimal plaque. ILTIS will be helpful to answer pertinent open questions in the field, including monocyte recruitment and transmigration, macrophage and dendritic cell activity, and motion of other immune cells.

Structured illumination microscopy provides twice the linear resolution of conventional fluorescence microscopy, but in thick samples, aberrations degrade the performance and limit the resolution. Here, we demonstrate structured illumination microscopy through 35 µm of tissue using adaptive optics (AO) to correct aberrations resulting in images with a resolution of 140 nm. We report a 60% minimum improvement in the signal-to-noise ratio of the structured illumination reconstruction through thick tissue by correction with AO.

Diffuse reflectance spectroscopy (DRS) can be used to noninvasively measure skin properties. To extract skin properties from DRS spectra, you need a model that relates the reflectance to the tissue properties. Most models are based on the assumption that skin is homogenous. In reality, skin is composed of multiple layers, and the homogeneity assumption can lead to errors. In this study, we analyze the errors caused by the homogeneity assumption. This is accomplished by creating realistic skin spectra using a computational model, then extracting properties from those spectra using a one-layer model. The extracted parameters are then compared to the parameters used to create the modeled spectra. We used a wavelength range of 400 to 750 nm and a source detector separation of 250  μm. Our results show that use of a one-layer skin model causes underestimation of hemoglobin concentration [Hb] and melanin concentration [mel]. Additionally, the magnitude of the error is dependent on epidermal thickness. The one-layer assumption also causes [Hb] and [mel] to be correlated. Oxygen saturation is overestimated when it is below 50% and underestimated when it is above 50%. We also found that the vessel radius factor used to account for pigment packaging is correlated with epidermal thickness.

Collagen is the key target of nonenzymatic glycation during physiopathological processes such as diabetes. The induced changes in the biochemical property of collagen by nonenzymatic glycation remain a major challenge to probe. This study investigated the use of confocal Raman microspectroscopy to label-free monitor the nonenzymatic glycation of collagen scaffolds from type 2 diabetic (T2D) mice at different timepoints (0, 4, 8, and 12 weeks). The glycated collagen scaffolds were obtained through the decellularized dermal matrix method to remove the epidermis layer, subcutaneous tissue, and cells in the dermis and to retain the collagen fibrils. Raman spectra showed no changes in Raman peak positions, which indicated that nonenzymatic glycation could produce no significant changes in the triple-helix structure of collagen in T2D mice. However, the relative intensity of the Raman bands at 921, 1033, 1244, 1274, 1346, 1635, and 1672  cm⁻¹ increased as diabetic time progressed. Correlation analysis suggested that the spectra of these bands had a high positive correlation with the expression of anti-advanced glycation end products obtained by immunofluorescence imaging of the same collagen scaffolds. Confocal Raman microspectroscopy proves a potential tool to label-free monitor the collagen changes caused by nonenzymatic glycation in T2D mice.

Diffuse reflectance spectroscopy using a fiber optic probe is a promising technique for evaluating the optical properties of biological tissue. We herein present a method for determining the reduced scattering coefficient, μ^′_s, the absorption coefficient, μ_a , and the tissue oxygen saturation, StO ₂ , of in vivo brain tissue using a single-reflectance fiber probe with two source-collector geometries. We performed in vivo recordings of diffuse reflectance spectra and of the electrophysiological signals for exposed rat brain during the cortical spreading depression evoked by the topical application of KCl. The time courses of μ_a at 500, 570, and 584 nm indicated the hemodynamic change in the cerebral cortex as well as StO ₂ . At 570 nm, the time course of μ^′_s was well correlated with that of μ_a , which also reflects the scattering by RBCs. On the other hand, increases in μ′_s at 500 and 584 nm and a decrease in μ′_s at 800 nm were observed before the profound increase in μ_a, and these occurrences were synchronized with the negative dc shift of the local field potential. The resultant change in the slope of μ^′_s(λ) is indicative of the morphological changes in the cellular and subcellular structures induced by the depolarization due to the temporal depression of the neuronal bioelectrical activity. The results of the present study indicate the potential application of the proposed method in evaluating the pathophysiological conditions of in vivo brain.

We report on the combined far-field measurement of the three involuntary eye movements, drift, microsaccades, and ocular microtremor (OMT), using a noncontact far-field optical method. We review the significance of the smallest and least measured, and thus least understood, of the three, OMT. Using modern digital imaging techniques, we perform detailed analysis, present experimental results, and examine the extracted parameters using a noncontact far-field sensor. For the first time, in vivo noncontact measurements of all three fixational in-plane movements of the human eye are reported, which simultaneously provide both the horizontal (left-right) and vertical (up-down) displacement results.

A tip nanobiosensor for monitoring DNA replication was presented. The effects of excitation power and polarization on tip-enhanced fluorescence (TEF) were assessed with the tip immersed in fluorescein isothiocyanate solution first. The photon count rose on average fivefold with radially polarized illumination at 50 mW. We then used polymerase-functionalized tips for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA. The amplicon-SYBR® Green I complex was detected and compared to real-time loop-mediated isothermal amplification. The signals of the reaction using 4 and 0.004  ng/μl templates were detected 10 and 30 min earlier, respectively. The results showed the potential of TEF in developing a nanobiosensor for real-time DNA amplification.

A laser-induced breakdown spectroscopy (LIBS) guided smart surgical tool using a femtosecond fiber laser is developed. This system provides real-time material identification by processing and analyzing the peak intensity and ratio of atomic emissions of LIBS signals. Algorithms to identify emissions of different tissues and metals are developed and implemented into the real-time control system. This system provides a powerful smart surgical tool for precise robotic microsurgery applications with real-time feedback and control.

High-grade dysplasia (HGD) in Barrett’s esophagus (BE) poses increased risk for developing esophageal adenocarcinoma. To date, early detection and treatment of HGD regions are still challenging due to the sampling error from tissue biopsy and relocation error during the treatment after histopathological analysis. In this study, CP-A (metaplasia) and CP-B (HGD) cell lines were used to investigate the “seek-and-treat” potential using 5-aminolevulinic acid-induced protoporphyrin IX (PpIX). The photodynamic therapy photosensitizer then provides both a phototoxic effect and additional image contrast for automatic detection and real-time laser treatment. Complementary to our studies on automatic classification, this work focused on characterizing subcellular irradiation and the potential phototoxicity on both metaplasia and HGD. The treatment results showed that the HGD cells are less viable than metaplastic cells due to more PpIX production at earlier times. Also, due to mitochondrial localization of PpIX, a better killing effect was achieved by involving mitochondria or whole cells compared with just nucleus irradiation in the detected region. With the additional toxicity given by PpIX and potential morphological/textural differences for pattern recognition, this cellular platform serves as a platform to further investigate real-time “seek-and-treat” strategies in three-dimensional models for improving early detection and treatment of BE.

Infections with pathogens could cause serious health problems, such as septicemia and subsequent death. Some of these deaths are caused by nosocomial, chronic, or burn-related wound infections. Photodynamic therapy (PDT) can be useful for the treatment of these infections. Our aim was to investigate the antibacterial effect of indocyanine green (ICG) and 808-nm laser on a rat abrasion wound model infected with the multidrug resistant Staphylococcus aureus strain. Abrasion wounds were infected with a multidrug resistant clinical isolate of S. aureus. ICG concentrations of 500, 1000, and 2000  μg/ml were applied with a 450  J/cm² energy dose. Temperature change was monitored by a thermocouple system. The remaining bacterial burden was determined by the serial dilution method after each application. Wounds were observed for 11 days posttreatment. The recovery process was assessed macroscopically. Tissue samples were also examined histologically by hematoxylin–eosin staining. Around a 90% reduction in bacterial burden was observed after PDT applications. In positive control groups (ICG-only and laser-only groups), there was no significant reduction. The applied energy dose did not cause any thermal damage to the target tissue or host environment. Results showed that ICG together with a 808-nm laser might be a promising antibacterial method to eliminate infections in animals and accelerate the wound-healing process.

The objective was to study the relationship between laser fluence and ablation efficiency of a femtosecond laser with a Gaussian-shaped pulse used to ablate dentin and enamel for prosthodontic tooth preparation. A diode-pumped thin-disk femtosecond laser with wavelength of 1025 nm and pulse width of 400 fs was used for the ablation of dentin and enamel. The laser spot was guided in a line on the dentin and enamel surfaces to form a groove-shaped ablation zone under a series of laser pulse energies. The width and volume of the ablated line were measured under a three-dimensional confocal microscope to calculate the ablation efficiency. Ablation efficiency for dentin reached a maximum value of 0.020  mm³/J when the laser fluence was set at 6.51  J/cm². For enamel, the maximum ablation efficiency was 0.009  mm³/J at a fluence of 7.59  J/cm². Ablation efficiency of the femtosecond laser on dentin and enamel is closely related to the laser fluence and may reach a maximum when the laser fluence is set to an appropriate value.