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.

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.

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.

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.

Measuring blood velocity using correlative spectrally encoded flow cytometry.

Spectrally encoded flow cytometry (SEFC) is a promising technique for imaging blood in the microcirculation. Yet, the dependency of one of the axes of the image on time prevents effective quantification of essential clinical parameters. Here, we address this challenge by splitting the optical path in an SEFC system into two parallel imaging lines, followed by straightforward data analysis for recovering the flow speed from the multiplexed data. The method is demonstrated by measuring the flow velocity of latex beads and blood cells in vitro. The system allows real-time velocity measurements of up to 11.7 mm/s at high spatial resolution, and could be integrated into existing SEFC systems for effectively measuring blood parameters in small capillary vessels.

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.

Spectral imaging using single-axis spectrally dispersed illumination.

Spectral imaging is a powerful tool for a wide variety of applications; however, low imaging rates and signal-to-noise ratios (SNRs) often limit its use for many biomedical applications. Here, we present a technique for spectral imaging using a unique two-dimensional illumination pattern having spectral dispersion in one axis. The method, which is called spectrally dispersed illumination spectral imaging (SDISI), allows high-speed, high-resolution acquisition of spectral data from specimens that often cannot tolerate high illumination intensities or require fast imaging for avoiding motion artifacts. The technique is demonstrated by capturing spectral data cubes of the finger of a human volunteer using short exposure durations and a high (33.5 dB) SNR.

Optically induced cell fusion using bispecific nanoparticles.

Redirecting the immune system to eliminate tumor cells is a promising alternative to traditional cancer therapies, most often requiring direct interaction between an immune system effector cell and its target. Herein, a novel approach for selective attachment of malignant cells to antigen-presenting cells by using bispecific nanoparticles is presented. The engaged cell pairs are then irradiated by a sequence of resonant femtosecond pulses, which results in widespread cell fusion and the consequent formation of hybrid cells. The dual role of gold nanoparticles as conjugating agents and fusion promoters offers a simple yet effective means for specific fusion between different cells. This technology could be useful for a variety of in vitro and in vivo applications that call for selective fusion between cells within a large heterogenic cell population.

Phase-sensitive imaging of tissue acoustic vibrations using spectrally encoded interferometry.

Acoustic vibrations in tissue are often difficult to image, requiring high-speed scanning, high sensitivity and nanometer-scale axial resolution. Here we use spectrally encoded interferometry to measure the vibration pattern of two-dimensional surfaces, including the skin of a volunteer, at nanometric resolution, without the need for rapid lateral scanning and with no prior knowledge of the driving acoustic waveform. Our results demonstrate the feasibility of this technique for measuring tissue biomechanics using simple and compact imaging probes.

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.

Optical nanomanipulations of malignant cells: controlled cell damage and fusion.

Specifically targeting and manipulating living cells is a key challenge in biomedicine and in cancer research in particular. Several studies have shown that nanoparticles irradiated by intense lasers are capable of conveying damage to nearby cells for various therapeutic and biological applications. In this work ultrashort laser pulses and gold nanospheres are used for the generation of localized, nanometric disruptions on the membranes of specifically targeted cells. The high structural stability of the nanospheres and the resonance pulse irradiation allow effective means for controlling the induced nanometric effects. The technique is demonstrated by inducing desired death mechanisms in epidermoid carcinoma and Burkitt lymphoma cells, and initiating efficient cell fusion between various cell types. Main advantages of the presented approach include low toxicity, high specificity, and high flexibility in the regulation of cell damage and cell fusion, which would allow it to play an important role in various future clinical and scientific applications.

High-speed interferometric spectrally encoded flow cytometry.

Spectrally encoded flow cytometry (SEFC) is a promising technique for noninvasive in vivo microscopy of blood cells. Here, we introduce a novel SEFC system for label-free confocal imaging of blood cells flowing at velocities of up to 10 mm/s within 65 µm-diameter vessels. The new system employs interferometric Fourier-domain detection and a high-speed wavelength-swept source, allowing 100 kHz line rate, sufficient for sampling the rapidly flowing cells 80 µm below the tissue surface. The large data sets obtained by this technique would improve diagnosis accuracy, reduce imaging time, and open new possibilities for noninvasive monitoring of blood in patients.

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.

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.

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.

Dispersion management for controlling image plane in Fourier-domain spectrally encoded endoscopy.

Spectrally encoded endoscopy (SEE) uses single optical fiber and miniature diffractive optics to allow imaging through a miniature probe. Utilizing Fourier-domain interferometry, SEE was shown capable of video-rate three-dimensional imaging, albeit at limited depth of field due to the limited spectral resolution of the detection spectrometer. We show that by using dispersion management at the reference arm of the interferometer, the tilt and curvature of the field of view could be adjusted without modifying the endoscopic probe itself. By controlling the group velocity dispersion, this technique is demonstrated useful for imaging specimen regions which reside outside the system's depth of field. This approach could be used to improve usability, functionality and image quality of SEE without affecting probe size and flexibility.

Spectrally encoded spectral imaging.

Spectral imaging, i.e. the acquisition of the spectrum emitted from each sample location, is a powerful tool for a wide variety of applications in science and technology. For biomedical applications, spectral imaging is important for accurate analysis of a biological specimen and for assisting clinical diagnosis, however it could be challenging mainly due to the typically low damage thresholds and strict time constraints. Here, we present a fiber-based technique termed spectrally encoded spectral imaging (SESI), in which a fully emitted spectrum is captured from each resolvable point of a specimen using an additional lateral scanning of the spectrally encoded line. The technique is demonstrated by capturing spectral data cubes of a color print and of a green leaf, and its potential advantage in signal-to-noise ratio is theoretically discussed. Using a miniaturized grating-lens configuration, SESI could be conducted endoscopically, allowing minimally invasive color and spectral imaging in remote locations of the body.

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.

Multiple-channel spectrally encoded imaging.

Spectrally encoded endoscopy (SEE) uses miniature diffractive optics to encode space with wavelength, allowing video-rate three-dimensional imaging through sub-millimeter, flexible endoscopic probes. Here we present a new approach for SEE in which the illumination and the collection channels are separated in space, and spectral encoding is present only in the collection channel. Bench-top experiments using spatially incoherent white light illumination reveal significant improvement in image quality and considerable reduction of speckle noise compared to conventional techniques, and show that the new system is capable of high sensitivity fluorescence imaging of single cells. The presented new approach would allow improved functionality and usability of SEE.

Flow cytometry using spectrally encoded confocal microscopy.

Flow cytometry techniques often rely on detecting fluorescence from single cells flowing through the cross section of a laser beam, providing invaluable information on vast numbers of cells. Such techniques, however, are often limited in their ability to resolve clusters of cells or parallel cell flow through large vessels. We present a confocal imaging technique that images unstained cells flowing in parallel through a wide channel, using spectrally encoded reflectance confocal microscopy that does not require mechanical scanning. Images of red blood cells from our system are compared to conventional transmission microscopy, and imaging of flowing red blood cells in vitro is experimentally demonstrated.