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Climate change increases sugar content in grapes, resulting in unwanted increase in ethanol content of wine. Lachancea thermotolerans ferments glucose and fructose into both ethanol and lactate, decreasing final ethanol content and positively affecting wine acidity. Reported Lachancea thermotolerans strains show big variation in lactate production during fermentation. However, a mechanistic understanding of this lactate producing phenotype is currently lacking. Through a combination of metabolomics, transcriptomics, genomics and computational methods we show that the lactate production is induced by amino acid limitation in a high lactate producing strain. We found in fermentations in synthetic grape juice media that lactate production starts in the last stages of growth, marked by decreased growth rate and increased expression levels of stress related genes. This onset of lactate production is specific for the high lactate producing strain and independent of oxygen availability. The onset of lactate production was changed by increased amino acid content of the media, and it is shown by both computational methods and amino acid measurements that at the onset of lactate production amino acids become limiting for growth. This study shows that lactate production of Lachancea thermotolerans is directly linked to nitrogen availability in the media, an insight that can further aid in the improvement of wine quality.

BackgroundTumor 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.MethodsThe 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).ResultsNo 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.ConclusionsThis 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.

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.

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.

INTRODUCTION Tumor Treating Fields (TTFields) therapy is an approved anti-cancer treatment for glioblastoma and mesothelioma. TTFields are delivered to patients continuously by two sets of arrays placed on opposite sides of the body at the tumor region to generate two perpendicular electric fields. Previously, in vivo studies of TTFields were limited due to the lack of a dedicated system that could maintain continuous and adequate contact of the arrays with the animal’s skin as well as the stress imposed on the animals by individual housing and the motility limitations they experience during treatment. METHODS Different electrode layouts were explored to optimize the intensity of the electric fields delivered to the target locations (therapeutic threshold >1 V/cm). The ability of various adhesive materials and wire coiling prevention strategies to increase TTFields device usage was examined. Stress reduction with different housing methods was evaluated via clinical examination of the animals. RESULTS Optimal array layouts were identified based on simulation data for TTFields delivery to the torso or the head of the mouse. Compacting conductors into a single printed circuit cable connected to a novel electric swivel machine resulted in fewer wire entanglements, and the improved adhesives resulted in fewer array replacements, overall elevating device usage. Improved cage design permitted pairs of mice to maintain social interactions while individually housed. Less weight loss was seen for animals housed in the dyadic relative to the standard solitary cages, indicating reduced stress. CONCLUSIONS The inovivo system provides means for continuous delivery of therapeutic levels of TTFields to the head and torso of mice while minimizing animal stress and increasing device usage. The new head arrays enable application of TTFields to the head of mice for the first time, allowing expansion of glioblastoma treatment research.

Purpose/objective: Tumor Treating Fields (TTFields) therapy is a noninvasive antineoplastic treatment modality that is FDA approved for treatment of glioblastoma and malignant pleural mesothelioma. TTFields are delivered to the patient continuously by 2 pairs of transducer arrays attached to the skin. In vivo TTFields experiments were so far limited due to the lack of a dedicated animal delivery system. Two main challenges are associated with TTFields application to the animal: 1) there is an absolute requirement for adequate and continuous contact between the electrodes and the animal skin throughout treatment; and 2) the wires connected to the electrodes require individual housing and limit animal movement, thus imposing stress. This work aimed to develop an in vivo system for continuous TTFields delivery to mice bearing cancer tumors in the torso or flank while addressing these challenges. Materials/Methods: To tackle the challenges and develop a viable in vivo system, several solutions were tested: 1) various electrode layouts; 2) a variety of adhesive materials; and 3) devices for preventing wire entanglement. Results: The final design of the transducer array electrodes included 2 adhesive layers, an inner layer for improved adherence, and an outer layer for securing the electrodes to the skin. Conductors were compacted into a single printed circuit cable connected to a novel electric swivel machine, that prevented cable coiling by sensing and rotating according to animal movement. These improvements resulted in fewer electrode entanglements and replacements, and thus in higher compliance (continuity) and less need for animal handling. To further reduce the impact of stress factors on the mice, a new cage was developed, that allows for 2 mice to be housed separately while still maintaining an interaction with one another. Indeed, animals treated with the inovivo system for 1-week displayed lower weight loss than animals treated with the previous non-dedicated system, indicative of reduced stress. Simulation were performed to ensure electric fields were indeed generated at the desired locations, showing above threshold TTFields intensities around the tumor for the flank subcutaneous model. For the torso orthotopic model, TTFields were shown to generate effective electric fields in the lung, liver, and pancreas, suggesting tumors in these organs may be treated effectively using the inovivo system. Conclusion: The new inovivo system provides means for continuous, 2 directions TTFields delivery to tumors in the torso or flank while minimizing stress on the mice. The inovivo system thus provides a tool for conducting TTFields experiments in mice, facilitating further in vivo studies for gaining additional mechanistical insight. The development of mouse head arrays to allow further research of the effect TTFields on glioblastoma, an application of widespread interest, is currently underway. Citation Format: Shiri Davidi, Roni Blatt, Mijal Munster, Anna Shteingauz, Yaara Porat, Adel Zeidan, Tal Marciano, Zeev Bomzon, Moshe Giladi, Uri Weinberg, Yoram Palti. inovivo: a dedicated system for delivery of therapeutic level Tumor Treating Fields (TTFields) to mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1317.

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.

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.

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 even organ failure. Measuring these cellular structural and morphological changes is important for understanding the factors contributing to vessel blockage and developing an effective treatment. In this work, we use spectrally encoded flow cytometry for confocal, high-resolution imaging of flowing blood cells from sickle cell anemia patients. A wide variety of cell morphologies were observed by analyzing the interference patterns resulting from reflections from the front and back faces of the cells’ membrane. Using numerical simulation for calculating the two-dimensional reflection pattern from the cells, we propose an analytical expression for the three-dimensional shape of a characteristic sickle cell and compare it to a previous from the literature. In vitro spectrally encoded flow cytometry offers new means for analyzing the morphology of sickle cells in stress-free environment, and could provide an effective tool for studying the unique physiological properties of these cells.

White blood cells (WBC) analysis is an important part of the complete blood count, providing good indication of the patient’s immune system status. The most common types of WBCs are the neutrophils and lymphocytes that comprise approximately 60% and 30% of the total WBC count, respectively; differentiating between these cells at the point of care would assist in accurate diagnosis of the possible source of infection (viral or bacterial) and in effective prescription of antibiotics. In this work, we demonstrate the potential of spectrally encoded flow cytometry (SEFC) to non-invasively image WBC in human patients, allowing morphology characterization of the main types of WBCs. The optical setup includes a broadband light that was diffracted and focused onto a single transverse line within the cross section of a small blood vessel at the inner patient lip. Light backscattered from the tissue was measured by a high-speed spectrometer, forming a two-dimensional reflectance confocal image of the flowing cells. By imaging at different depths into vessels of different diameters, we determine optimal imaging conditions (i.e. imaging geometry, speed and depth) for counting the total amount of WBCs and for differentiating between their main types. The presented technology could serve for analyzing the immune system status at the point of care, and for studying the morphological and dynamical characteristics of these cells in vivo.

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.

Values of blood oxygenation levels are useful for assessing heart and lung conditions, and are frequently monitored during routine patient care. Independent measurement of the oxygen saturation in capillary blood, which is significantly different from that of arterial blood, is important for diagnosing tissue hypoxia and for increasing the accuracy of existing techniques that measure arterial oxygen saturation. Here, we developed a simple, non-invasive technique for measuring the reflected spectra from individual capillary vessels within a human lip, allowing local measurement of the blood oxygen saturation. The optical setup includes a spatially incoherent broadband light that was focused onto a specific vessel below the lip surface. Backscattered light was imaged by a camera for identifying a target vessel and pointing the illumination beam to its cross section. Scattered light from the vessel was then collected by a single-mode fiber and analyzed by a fast spectrometer. Spectra acquired from small capillary vessels within a volunteer lip showed the characteristic oxyhemoglobin absorption bands in real time and with a high signal-to-noise ratio. Measuring capillary oxygen saturation using this technique would potentially be more accurate compared to existing pulse oximetry techniques due to its insensitivity to the patient’s skin color, pulse rate, motion, and medical condition. It could be used as a standalone endoscopic technique for measuring tissue hypoxia or in conjunction with conventional pulse oximetry for a more accurate measurement of oxygen transport in the body.

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.

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.

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.

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.

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.

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.