High-Resolution, Wide-Field, Forward-Viewing Spectrally Encoded Endoscope.

BACKGROUND AND OBJECTIVE: Spectrally encoded endoscopy (SEE) is an optical imaging technology that uses spatial wavelength multiplexing to conduct endoscopy in miniature, small diameter probes. Contrary to the previous side-viewing SEE devices, forward-viewing SEE probes are advantageous as they provide a look ahead that facilitates navigation and surveillance. The objective of this work was to develop a miniature forward-viewing SEE probe with a wide field of view and a high spatial resolution. MATERIALS AND METHODS: We designed and developed a forward-viewing SEE device with an overall total diameter of 1.27 mm, which consists of a monolithic illumination probe with a length of 3.87 mm and a diameter of 500 µm, 8 multimode detection fibers that were polished at a 17° angle, a rotational scanning mechanism, and a sheath. The SEE device was evaluated using a USAF resolution target and was used for preclinical imaging of a swine joint ex vivo. RESULTS: This design resulted in a high resolution probe (best spatial resolution of 20.3 µm), a wide total angular field of view of 100°, and an effective number of imaging elements of ~344,000 pixels. The SEE probe performance was compared to a commercial color chip-on-the-tip endoscope; while monochrome, results showed better spatial resolution and a wider field of view for the SEE device. CONCLUSION: These results demonstrate the potential of this forward-viewing SEE probe for visualization and navigation in medical imaging applications. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Single-beam spectrally encoded color imaging.

We have developed, to the best of our knowledge, a new method of conducting spectrally encoded color imaging using a single light beam. In our method, a single broadband light beam was incident on a diffraction grating, where the overlapped third order of the red, fourth order of the green, and fifth order of the blue spectral bands were focused on a line illuminating tissue. This configuration enabled each point on the line to be illuminated by three distinctive wavelengths, corresponding to red, green, and blue. A custom grating was designed and fabricated to achieve high diffraction efficiencies for the wavelengths and diffraction orders used for color spectrally encoded imaging. A bench system was built to test the new spectrally encoded color imaging method. For a beam diameter of 174 µm, the bench system achieved 89,000 effective pixels over a 70° circular field. Spectrally encoded color images of excised swine tissue revealed blood vessels with a similar color appearance to those obtained via a conventional color camera. The results suggest that this single-beam spectrally encoded color method is feasible and can potentially simplify color spectrally encoded endoscopy probe designs.

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.

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.

Use dependence of presynaptic tenacity.

Recent studies indicate that synaptic vesicles (SVs) are continuously interchanged among nearby synapses at very significant rates. These dynamics and the lack of obvious barriers confining synaptic vesicles to specific synapses would seem to challenge the ability of synapses to maintain a constant amount of synaptic vesicles over prolonged time scales. Moreover, the extensive mobilization of synaptic vesicles associated with presynaptic activity might be expected to intensify this challenge. Here we examined the ability of individual presynaptic boutons of rat hippocampal neurons to maintain their synaptic vesicle content, and the degree to which this ability is affected by continuous activity. We found that the synaptic vesicle content of individual boutons belonging to the same axons gradually changed over several hours, and that these changes occurred independently of activity. Intermittent stimulation for 1 h accelerated rates of vesicle pool size change. Interestingly, however, following stimulation cessation, vesicle pool size change rates gradually converged with basal change rates. Over similar time scales, active zones (AZs) exhibited substantial remodeling; yet, unlike synaptic vesicles, AZ remodeling was not affected by the stimulation paradigms used here. These findings indicate that enhanced activity levels can increase synaptic vesicle redistribution among nearby synapses, but also highlight the presence of forces that act to restore particular set points in terms of SV contents, and support a role for active zones in preserving such set points. These findings also indicate, however, that neither AZ size nor SV content set points are particularly stable, questioning the long-term tenacity of presynaptic specializations.

Tumor Treating Fields (TTFields) Reversibly Permeabilize the Blood-Brain Barrier In Vitro and In Vivo.

Despite the availability of numerous therapeutic substances that could potentially target CNS disorders, an inability of these agents to cross the restrictive blood-brain barrier (BBB) limits their clinical utility. Novel strategies to overcome the BBB are therefore needed to improve drug delivery. We report, for the first time, how Tumor Treating Fields (TTFields), approved for glioblastoma (GBM), affect the BBB's integrity and permeability. Here, we treated murine microvascular cerebellar endothelial cells (cerebEND) with 100-300 kHz TTFields for up to 72 h and analyzed the expression of barrier proteins by immunofluorescence staining and Western blot. In vivo, compounds normally unable to cross the BBB were traced in healthy rat brain following TTFields administration at 100 kHz. The effects were analyzed via MRI and immunohistochemical staining of tight-junction proteins. Furthermore, GBM tumor-bearing rats were treated with paclitaxel (PTX), a chemotherapeutic normally restricted by the BBB combined with TTFields at 100 kHz. The tumor volume was reduced with TTFields plus PTX, relative to either treatment alone. In vitro, we demonstrate that TTFields transiently disrupted BBB function at 100 kHz through a Rho kinase-mediated tight junction claudin-5 phosphorylation pathway. Altogether, if translated into clinical use, TTFields could represent a novel CNS drug delivery strategy.

In Vivo Safety of Tumor Treating Fields (TTFields) Applied to the Torso.

BACKGROUND: Tumor Treating Fields (TTFields) therapy is a non-invasive, loco-regional, anti-mitotic treatment modality that targets rapidly dividing cancerous cells, utilizing low intensity, alternating electric fields at cancer-cell-type specific frequencies. TTFields therapy is approved for the treatment of newly diagnosed and recurrent glioblastoma (GBM) in the US, Europe, Israel, Japan, and China. The favorable safety profile of TTFields in patients with GBM is partially attributed to the low rate of mitotic events in normal, quiescent brain cells. However, specific safety evaluations are warranted at locations with known high rates of cellular proliferation, such as the torso, which is a primary site of several of the most aggressive malignant tumors. METHODS: The safety of delivering TTFields to the torso of healthy rats at 150 or 200 kHz, which were previously identified as optimal frequencies for treating multiple torso cancers, was investigated. Throughout 2 weeks of TTFields application, animals underwent daily clinical examinations, and at treatment cessation blood samples and internal organs were examined. Computer simulations were performed to verify that the targeted internal organs of the torso were receiving TTFields at therapeutic intensities (≥ 1 V/cm root mean square, RMS). RESULTS: No treatment-related mortality was observed. Furthermore, no significant differences were observed between the TTFields-treated and control animals for all examined safety parameters: activity level, food and water intake, stools, motor neurological status, respiration, weight, complete blood count, blood biochemistry, and pathological findings of internal organs. TTFields intensities of 1 to 2.5 V/cm RMS were confirmed for internal organs within the target region. CONCLUSIONS: This research demonstrates the safety of therapeutic level TTFields at frequencies of 150 and 200 kHz when applied as monotherapy to the torso of healthy rats.

RGB-color forward-viewing spectrally encoded endoscope using three orders of diffraction.

Spectrally encoded endoscopy (SEE) is an ultra-miniature endoscopy technology that encodes each spatial location on the sample with a different wavelength. One challenge in SEE is achieving color imaging with a small probe. We present a novel SEE probe that is capable of conducting real-time RGB imaging using three diffraction orders (6th order diffraction of the blue spectrum, 5th of green, and 4th of red). The probe was comprised of rotating 0.5 mm-diameter illumination optics inside a static, 1.2 mm-diameter flexible sheath with a rigid distal length of 5 mm containing detection fibers. A color chart, resolution target, and swine tissue were imaged. The device achieved 44k/59k/23k effective pixels per R/G/B channels over a 58° angular field and differentiated a wide gamut of colors.

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.

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.

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.

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.

Neuroligin-1 loss is associated with reduced tenacity of excitatory synapses.

Neuroligins (Nlgns) are postsynaptic, integral membrane cell adhesion molecules that play important roles in the formation, validation, and maturation of synapses in the mammalian central nervous system. Given their prominent roles in the life cycle of synapses, it might be expected that the loss of neuroligin family members would affect the stability of synaptic organization, and ultimately, affect the tenacity and persistence of individual synaptic junctions. Here we examined whether and to what extent the loss of Nlgn-1 affects the dynamics of several key synaptic molecules and the constancy of their contents at individual synapses over time. Fluorescently tagged versions of the postsynaptic scaffold molecule PSD-95, the AMPA-type glutamate receptor subunit GluA2 and the presynaptic vesicle molecule SV2A were expressed in primary cortical cultures from Nlgn-1 KO mice and wild-type (WT) littermates, and live imaging was used to follow the constancy of their contents at individual synapses over periods of 8-12 hours. We found that the loss of Nlgn-1 was associated with larger fluctuations in the synaptic contents of these molecules and a poorer preservation of their contents at individual synapses. Furthermore, rates of synaptic turnover were somewhat greater in neurons from Nlgn-1 knockout mice. Finally, the increased GluA2 redistribution rates observed in neurons from Nlgn-1 knockout mice were negated by suppressing spontaneous network activity. These findings suggest that the loss of Nlgn-1 is associated with some use-dependent destabilization of excitatory synapse organization, and indicate that in the absence of Nlgn-1, the tenacity of excitatory synapses might be somewhat impaired.