Measuring the Acoustic Reflex through the Tympanic Membrane.

INTRODUCTION: The acoustic reflex is the active response of the middle ear to loud sounds, altering the mechanical transfer function of the acoustic energy into the inner ear. Our goal was to observe the effect of the acoustic reflex on the tympanic membrane by identifying a significant nonlinear increase in membrane oscillations. METHODS: By using interferometric spectrally encoded endoscopy, we record the membrane oscillations over time in response to a loud, 200-ms-long acoustic stimulus. RESULTS: A gradual reflex activation is measured between approximately 40 and 100 ms, manifested as a linear 42% increase in the umbo oscillation amplitude. CONCLUSION: The measured oscillations correlate well with those expected from a mechanical model of a damped harmonic oscillator, and the results of this work demonstrate the potential of interferometric spectrally encoded endoscopy to observe unique dynamical processes in the tympanic membrane and in the middle ear.

In vivo optical mapping of the tympanic membrane impulse response.

The wide frequency range of the human hearing could be narrowed by various pathologies in the middle ear and in the tympanic membrane that lead to conductive hearing loss. Diagnosing such hearing problems is challenging, however, often relying on subjective hearing tests supported by functional tympanometry. Here we present a method for in vivo 2D mapping of the impulse response of the tympanic membrane, and demonstrate its potential on a healthy human volunteer. The imaging technique is based on interferometric spectrally encoded endoscopy, with a handheld probe designed to scan the human tympanic membrane within less than a second. The system obtains high-resolution 2D maps of key functional parameters including peak response, rise and decay times, oscillation bandwidth and resonance frequency. We also show that the system can identify abnormal regions in the membrane by detecting differences in the local mechanical parameters of the tissue. We believe that by offering a full 2D mapping of broad-bandwidth dynamics of the tympanic membrane, the presented imaging modality would be useful for effective diagnosis of conductive hearing loss in patients.

Measuring the red blood cell shape in capillary flow using spectrally encoded flow cytometry.

Red blood cells in small capillaries exhibit a wide variety of deformations that reflect their true physiological conditions at these important locations. By applying a technique for the high-speed microscopy of flowing cells, termed spectrally encoded flow cytometry (SEFC), we image the light reflected from the red blood cells in human capillaries, and propose an analytical slipper-like model for the cell morphology that can reproduce the experimental in vivo images. The results of this work would be useful for studying the unique flow conditions in these vessels, and for extracting useful clinical parameters that reflect the true physiology of the blood cells in situ.

Optimization study of plasmonic cell fusion.

Artificial cell fusion often serves as a valuable tool for studying different applications in biology and medicine, including natural development, immune response, cancer metastasis and production of therapeutic molecules. Plasmonic cell fusion, a technique that uses specific cell labeling by gold nanoparticles and resonant femtosecond pulse irradiation for fusing neighboring cells, has been demonstrated useful for such applications, allowing high cell specificity and an overall low toxicity. Despite these advantages, the numerous experimental factors contributing to plasmonic fusion have often led to subpar fusion efficiencies, requiring repeated experiments and extensive calibration protocols for achieving optimal results. In this work we present a study that aims to improve the overall performance of plasmonic cell fusion in terms of fusion efficiency and cell viability. By varying the pulse fluence, nanoparticle concentration, incubation times, and culture handling protocols, we demonstrate up to 100% fusion of malignant epithelial cells across the entire irradiated area of the culture. We also show that some of the smaller cells may stay viable for up to several days. The results would allow plasmonic fusion to play a key role in numerous studies and applications that require specific, high-efficiency cell-cell fusion.

Plasmonic fusion between fibroblasts and skeletal muscle cells for skeletal muscle regeneration.

Normal regeneration of skeletal muscle takes place by the differentiation of muscle-specific stem cells into myoblasts that fuse with existing myofibers for muscle repair. This natural repair mechanism could be ineffective in some cases, for example in patients with genetic muscular dystrophies or massive musculoskeletal injuries that lead to volumetric muscle loss. In this study we utilize the effect of plasmonic cell fusion, i.e. the fusion between cells conjugated by gold nanospheres and irradiated by resonant femtosecond laser pulses, for generating human heterokaryon cells of myoblastic and fibroblastic origin, which further develop into viable striated myotubes. The heterokaryon cells were found to express the myogenic transcription factors MyoD and Myogenin, as well as the Desmin protein that is essential in the formation of sarcomeres, and could be utilized in various therapeutic approaches that involve transplantation of cells or engineered tissue into the damaged muscle.

Pinhole shifting for reducing speckle contrast in reflectance confocal microscopy.

The high speckle contrast in reflectance confocal microscopy is perhaps the most limiting factor on this imaging modality, particularly in high scattering samples such as biological tissues. In this Letter, we propose and numerically analyze a method for speckle reduction that uses simple lateral shifting of the confocal pinhole in several directions, which results in reduced speckle contrast and only a moderate penalty in both lateral and axial resolutions. By simulating free-space electromagnetic wave propagation through a high-numerical-aperture (NA) confocal imaging system, and assuming only single-scattering events, we characterize the 3D point-spread function (PSF) that results from full-aperture pinhole shifting. Simple summation of four different pinhole-shifted images resulted in a 36% reduction in speckle contrast, with reductions of only 17% and 60% in the lateral and axial resolutions, respectively. This method could be particularly useful in noninvasive microscopy for clinical diagnosis, where fluorescence labeling is impractical and high image quality is imperative for achieving accurate diagnosis.

Formation of Large Intracellular Actin Networks Following Plasmonic Cell Fusion.

Following fusion between two or more individual cells, the resulting cellular entity must undergo extensive restructuring of its plasma membrane and cytoskeleton in order to maintain its mechanical and physiological function. In artificial cell fusion that is executed by external triggering, such restructuring could be problematic due to the absence of preconditioning biological signals. In this work we study the reorganization of the actin filaments in adenocarcinoma cells that were fused using plasmonic triggering, i.e. the irradiation by resonant femtosecond laser pulses of cells specifically targeted by gold nanoparticles. Time-lapse confocal microscopy of the fusing cells has revealed the formation of large-scale actin networks that preserve the local orientations of the original actin cytoskeletons. The results confirm the local nature of the plasmonic interactions that were confined to the cells' plasma membranes and would help studying the development and dynamics of actin networks by offering a relatively stable, living cellular environment that supports large-scale actin growth.

Imaging the dynamics and microstructure of fibrin clot polymerization in cardiac surgical patients using spectrally encoded confocal microscopy.

During cardiac surgery with cardiopulmonary bypass (CPB), altered hemostatic balance may disrupt fibrin assembly, predisposing patients to perioperative hemorrhage. We investigated the utility of a novel device termed spectrally-encoded confocal microscopy (SECM) for assessing fibrin clot polymerization following heparin and protamine administration in CPB patients. SECM is a novel, high-speed optical approach to visualize and quantify fibrin clot formation in three dimensions with high spatial resolution (1.0 µm) over a volumetric field-of-view (165 × 4000 × 36 µm). The measurement sensitivity of SECM was first determined using plasma samples from normal subjects spiked with heparin and protamine. Next, SECM was performed in plasma samples from patients on CPB to quantify the extent to which fibrin clot dynamics and microstructure were altered by CPB exposure. In spiked samples, prolonged fibrin time (4.4 ± 1.8 to 49.3 ± 16.8 min, p < 0.001) and diminished fibrin network density (0.079 ± 0.010 to 0.001 ± 0.002 A.U, p < 0.001) with increasing heparin concentration were reported by SECM. Furthermore, fibrin network density was not restored to baseline levels in protamine-treated samples. In CPB patients, SECM reported lower fibrin network density in protaminized samples (0.055 ± 0.01 A.U. [Arbitrary units]) vs baseline values (0.066 ± 0.009 A.U.) (p = 0.03) despite comparable fibrin time (baseline = 6.0 ± 1.3, protamine = 6.4 ± 1.6 min, p = 0.5). In these patients, additional metrics including fibrin heterogeneity, length and straightness were quantified. Note, SECM revealed that following protamine administration with CPB exposure, fibrin clots were more heterogeneous (baseline = 0.11 ± 0.02 A.U, protamine = 0.08 ± 0.01 A.U, p = 0.008) with straighter fibers (baseline = 0.918 ± 0.003A.U, protamine = 0.928 ± 0.0006A.U. p < 0.001). By providing the capability to rapidly visualize and quantify fibrin clot microstructure, SECM could furnish a new approach for assessing clot stability and hemostasis in cardiac surgical patients.

Rapid imaging of tympanic membrane vibrations in humans.

Functional imaging of the human ear is an extremely challenging task because of its minute anatomic structures and nanometer-scale motion in response to sound. Here, we demonstrate noninvasive in vivo functional imaging of the human tympanic membrane under various acoustic excitations, and identify unique vibration patterns that vary between human subjects. By combining spectrally encoded imaging with phase-sensitive spectral-domain interferometry, our system attains high-resolution functional imaging of the two-dimensional membrane surface, within a fraction of a second, through a handheld imaging probe. The detailed physiological data acquired by the system would allow measuring a wide range of clinically relevant parameters for patient diagnosis, and provide a powerful new tool for studying middle and inner ear physiology.

Plasmonic targeting of cancer cells in a three-dimensional natural hydrogel.

Using specifically designed gold nanoparticles and local laser irradiation, individual cells and small cell clusters could be targeted on a microscopic scale with minimal toxicity to nearby tissue. To date, most scientific studies and technological demonstrations of this approach were conducted on two-dimensional cultures, while most feasibility tests and preclinical trials were conducted using animal models. For bridging the gap between two-dimensional cell cultures and animal experiments, we propose and demonstrate the use of a natural hydrogel for studying the effect of intense, ultrashort laser pulses on a gold nanoparticle targeted tissue. Using illumination parameters comparable to those used with two-dimensional cultures, we show the complete eradication of multilayered cell colonies comprising normal fibroblasts and malignant epithelial cells co-cultured on a hydrogel scaffold. By evaluating the extent of cell damage for various pulse durations at off-resonance irradiation, we find that the observed damage mechanism was dominated by rapid thermal transitions around the gold nanospheres, rather than by photoionization. The work provides a new tool for understanding the complex pulse-particle-tissue interactions and demonstrates the important role of nanoparticle mediated cavitation bubbles in a thick, multilayered tissue.

Hydrogel composition and laser micropatterning to regulate sciatic nerve regeneration.

Treatment of peripheral nerve injuries has evolved over the past several decades to include the use of sophisticated new materials endowed with trophic and topographical cues that are essential for in vivo nerve fibre regeneration. In this research, we explored the use of an advanced design strategy for peripheral nerve repair, using biological and semi-synthetic hydrogels that enable controlled environmental stimuli to regenerate neurons and glial cells in a rat sciatic nerve resection model. The provisional nerve growth conduits were composed of either natural fibrin or adducts of synthetic polyethylene glycol and fibrinogen or gelatin. A photo-patterning technique was further applied to these 3D hydrogel biomaterials, in the form of laser-ablated microchannels, to provide contact guidance for unidirectional growth following sciatic nerve injury. We tested the regeneration capacity of subcritical nerve gap injuries in rats treated with photo-patterned materials and compared these with injuries treated with unpatterned hydrogels, either stiff or compliant. Among the factors tested were shear modulus, biological composition, and micropatterning of the materials. The microchannel guidance patterns, combined with appropriately matched degradation and stiffness properties of the material, proved most essential for the uniform tissue propagation during the nerve regeneration process.

Measuring blood oxygen saturation along a capillary vessel in human.

Measuring oxygen saturation in capillary vessels could provide valuable information on oxygen transport and tissue viability. Most spectroscopic measurement techniques, however, lack the spatial resolution to account for the small vessel dimensions within a scattering tissue and the steep gradients of oxygen saturation levels. Here, we developed a noninvasive technique for image-guided confocal measurement of the optical absorption spectrum from a small region that is comparable in size to the cross section of a single capillary vessel. A wide range of oxygen saturation levels were measured in a single capillary in a human volunteer, with blood deoxygenation rates of 7.1% per hundred microns. The technique could help in studying oxygen exchange dynamics in tissues and could play a key role in future clinical diagnosis and therapeutic applications that require localized functional tissue inspection.

In vivo noninvasive microscopy of human leucocytes.

Leucocytes play a key role in our immune system, protecting the body against infections using a wide range of biological mechanisms. Effective imaging and identification of leucocytes within the blood stream in patients is challenging, however, because of their low volume fraction in the blood, the high tissue scattering and the rapid blood flow. Spectrally encoded flow cytometry (SEFC) has recently been demonstrated effective for label-free high-resolution in vivo imaging of blood cells using an optical probe that does not require mechanical scanning. Here, we use SEFC to noninvasively image leucocytes at different imaging depths within small vessels in human volunteers, and identify visual differences in cell brightness and nuclei shapes, that would help distinguish between the two most abundant leucocyte types. The observed differences match the in vitro characteristics of isolated granulocytes and mononuclear cells. The results prove the potential of the system for conducting differential leucocyte count and as an effective research tool for studying the function and distribution of leucocytes in humans.

Measuring sickle cell morphology during blood flow.

During a sickle cell crisis in sickle cell anemia patients, deoxygenated red blood cells may change their mechanical properties and block small blood vessels, causing pain, local tissue damage, and possibly organ failure. Measuring the structural and morphological changes in sickle cells is important for understanding the factors contributing to vessel blockage and for developing an effective treatment. In this work, we image blood cells from sickle cell anemia patients using spectrally encoded flow cytometry, and analyze the interference patterns between reflections from the cell membranes. Using a numerical simulation for calculating the interference pattern obtained from a model of a red blood cell, we propose an analytical expression for the three-dimensional shape of characteristic sickle cells and compare our results to a previously suggested model. Our imaging approach offers new means for analyzing the morphology of sickle cells, and could be useful for studying their unique physiological and biomechanical properties.

In vitro hematocrit measurement using spectrally encoded flow cytometry.

Measuring key physiological parameters of small blood samples extracted from patients could be useful for real-time clinical diagnosis at the point of care. An important parameter required from all blood tests is the blood hematocrit, a measure of the fractional volume occupied by the red cells within the blood. In this work, we present a method for in vitro evaluation of hematocrit based on the data acquired using spectrally encoded flow cytometry. Analysis of the reflectance confocal images of blood within a flow chamber resulted in an error as low as 1.7% in the measured hematocrit. The technique could be used as part of an in vitro diagnostic system that measures important blood parameters at the point of care.

Experimental Proof for the Role of Nonlinear Photoionization in Plasmonic Phototherapy.

Targeting individual cells within a heterogeneous tissue is a key challenge in cancer therapy, encouraging new approaches for cancer treatment that complement the shortcomings of conventional therapies. The highly localized interactions triggered by focused laser beams promise great potential for targeting single cells or small cell clusters; however, most laser-tissue interactions often involve macroscopic processes that may harm healthy nearby tissue and reduce specificity. Specific targeting of living cells using femtosecond pulses and nanoparticles has been demonstrated promising for various potential therapeutic applications including drug delivery via optoporation, drug release, and selective cell death. Here, using an intense resonant femtosecond pulse and cell-specific gold nanorods, we show that at certain irradiation parameters cell death is triggered by nonlinear plasmonic photoionization and not by thermally driven processes. The experimental results are supported by a physical model for the pulse-particle-medium interactions. A good correlation is found between the calculated total number and energy of the generated free electrons and the observed cell death, suggesting that femtosecond photoionization plays the dominant role in cell death.

Spectral imaging using forward-viewing spectrally encoded endoscopy.

Spectrally encoded endoscopy (SEE) enables miniature, small-diameter endoscopic probes for minimally invasive imaging; however, using the broadband spectrum to encode space makes color and spectral imaging nontrivial and challenging. By careful registration and analysis of image data acquired by a prototype of a forward-viewing dual channel spectrally encoded rigid probe, we demonstrate spectral and color imaging within a narrow cylindrical lumen. Spectral imaging of calibration cylindrical test targets and an ex-vivo blood vessel demonstrates high-resolution spatial-spectral imaging with short (10 µs/line) exposure times.

Reflectance confocal microscopy of red blood cells: simulation and experiment.

Measuring the morphology of red blood cells is important for clinical diagnosis, providing valuable indications on a patient's health. In this work, we have simulated the appearance of normal red blood cells under a reflectance confocal microscope and discovered unique relations between the morphological parameters and the resulting characteristic interference patterns of the cell. The simulation results showed good agreement with in vitro reflectance confocal images of red blood cells, acquired using spectrally encoded flow cytometry that imaged the cells in a linear flow without artificial staining. By matching the simulated patterns to confocal images of the cells, this method could be used for measuring cell morphology in three dimensions and for studying their physiology.

Miniature forward-viewing spectrally encoded endoscopic probe.

Spectrally encoded endoscopy is a promising technique for minimally invasive imaging, allowing high-quality imaging through small diameter probes that do not require rapid mechanical scanning. A novel optical configuration that employs broadband visible light and dual-channel imaging is used to demonstrate a miniature forward-viewing probe having a high number of resolvable points, low speckle contrast, negligible backreflections, and high signal-to-noise ratio. The system would be most suitable for imaging through narrow ducts and vessels for clinical diagnosis at hard-to-reach locations in the body.