Mechanisms of SARS-CoV-2 Inactivation Using UVC Laser Radiation.

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has had a tremendous impact on humanity. Prevention of transmission by disinfection of surfaces and aerosols through a chemical-free method is highly desirable. Ultraviolet C (UVC) light is uniquely positioned to achieve inactivation of pathogens. We report the inactivation of SARS-CoV-2 virus by UVC radiation and explore its mechanisms. A dose of 50 mJ/cm2 using a UVC laser at 266 nm achieved an inactivation efficiency of 99.89%, while infectious virions were undetectable at 75 mJ/cm2 indicating >99.99% inactivation. Infection by SARS-CoV-2 involves viral entry mediated by the spike glycoprotein (S), and viral reproduction, reliant on translation of its genome. We demonstrate that UVC radiation damages ribonucleic acid (RNA) and provide in-depth characterization of UVC-induced damage of the S protein. We find that UVC severely impacts SARS-CoV- 2 spike protein's ability to bind human angiotensin-converting enzyme 2 (hACE2) and this correlates with loss of native protein conformation and aromatic amino acid integrity. This report has important implications for the design and development of rapid and effective disinfection systems against the SARS-CoV-2 virus and other pathogens.

Developing a portable gas imaging camera using highly tunable active-illumination and computer vision.

We have developed a portable gas imaging camera for identifying methane leaks in real-time. The camera uses active illumination from distributed feedback InGaAs laser diodes tuned to the 1653 nm methane absorption band. An InGaAs focal plane sensor array images the active illumination. The lasers are driven off resonance every alternate frame so that computer vision can extract the gas data. A colour image is captured simultaneously and the data is superimposed to guide the operator. Image stabilisation has been employed to allow detection with a moving camera, successfully imaging leaks from mains pressure gas supplies at a range of up to 3 m and flow rates as low as 0.05 L min-1.

Ultra-low timing jitter, Ti:Al2O3 synchronization for stimulated Raman scattering and pump-probe microscopy.

SIGNIFICANCE: Stimulated Raman scattering (SRS) and pump-probe microscopy are implementations of multiphoton microscopy that acquire high-resolution, label-free images of live samples encoded with molecular contrast. Most commercial multiphoton microscopes cannot access these techniques since they require sample illumination by two temporally synchronized ultrafast pulse trains. We present a compact and robust way of synchronizing an additional Ti:sapphire laser with a conventional single-beam multiphoton microscope to realize an instrument that can acquire images with enhanced molecular specificity. AIM: A passive optical synchronization scheme for a pair of commercially available, unmodified modelocked Ti:sapphire lasers was developed. The suitability of this synchronization scheme for advanced biomedical microscopy was investigated. APPROACH: A pair of modelocked Ti:sapphire lasers were aligned in master-slave configuration. Five percent of the master laser output was used to seed the modelocking in the slave laser cavity. The timing jitter of the master and slave pulse trains was characterized using an optical autocorrelator. The synchronized output of both lasers was coupled into a laser scanning microscope and used to acquire spectral focusing SRS and pump-probe microscopy images from biological and nonbiological samples. RESULTS: A timing jitter between the modelocked pulse trains of 0.74 fs was recorded. Spectral focusing SRS allowed spectral discrimination of polystyrene and polymethyl methacrylate beads. Pump-probe microscopy was used to record excited state lifetime curves from hemoglobin in intact red blood cells. CONCLUSION: Our work demonstrates a simple and robust method of upgrading single-beam multiphoton microscopes with an additional ultrafast laser. The resulting dual-beam instrument can be used to acquire label-free images of sample structure and composition with high biochemical specificity.

Label-free optical hemogram of granulocytes enhanced by artificial neural networks.

An outstanding challenge for immunology is the classification of immune cells in a label-free fashion with high speed. For this purpose, optical techniques such as Raman spectroscopy or digital holographic microscopy have been used successfully to identify immune cell subsets. To achieve high accuracy, these techniques require a post-processing step using linear methods of multivariate processing, such as principal component analysis. Here we demonstrate for the first time a comparison between artificial neural networks and principal component analysis (PCA) to classify the key granulocyte cell lineages of neutrophils and eosinophils using both digital holographic microscopy and Raman spectroscopy. Artificial neural networks can offer advantages in terms of classification accuracy and speed over a PCA approach. We conclude that digital holographic microscopy with convolutional neural networks based analysis provides a route to a robust, stand-alone and high-throughput hemogram with a classification accuracy of 91.3 % at a throughput rate of greater than 100 cells per second.

Mapping the dynamical regimes of a SESAM mode-locked VECSEL with a long cavity using time series analysis.

The different dynamical regions of an optically-pumped SESAM mode-locked, long-cavity VECSEL system with a fundamental pulse repetition frequency of ~200 MHz are investigated. The output power, captured as 250 µs long time series using a sampling rate of 200 GSa/s, for each operating condition of the system, is analyzed to determine the dynamical state. A wavelength range of 985-995 nm and optical pump powers of 10 W-16.3 W is studied. The system produces high quality fundamental passive mode-locking (FML) over an extensive part of the parameter space, but the different dynamical regions outside of FML are the primary focus of this study. We report five types of output: CW emission, FML, mode-locking of a few modes, double pulsing, and, semi-stable 4th harmonic mode-locking. The high sampling rate of the oscilloscope, combined with the long duration of the time series analyzed, enables insight into how the structure and substructure of pulses vary systematically over thousands of round trips of the laser cavity. Higher average output power is obtained in regions characterized by semi-stable 4th harmonic mode-locking than observed for FML, raising whether such average powers might be achieved for FML. The observed dynamic transitions from fundamental mode-locking provide insights into instability challenges in developing a stable, widely tunable, low repetition rate, turn-key system; and to inform future modelling of the system.

Harnessing speckle for a sub-femtometre resolved broadband wavemeter and laser stabilization.

The accurate determination and control of the wavelength of light is fundamental to many fields of science. Speckle patterns resulting from the interference of multiple reflections in disordered media are well-known to scramble the information content of light by complex but linear processes. However, these patterns are, in fact, exceptionally rich in information about the illuminating source. We use a fibre-coupled integrating sphere to generate wavelength-dependent speckle patterns, in combination with algorithms based on the transmission matrix method and principal component analysis, to realize a broadband and sensitive wavemeter. We demonstrate sub-femtometre wavelength resolution at a centre wavelength of 780 nm, and a broad calibrated measurement range from 488 to 1,064 nm. This compares favourably to the performance of conventional wavemeters. Using this speckle wavemeter as part of a feedback loop, we stabilize a 780 nm diode laser to achieve a linewidth better than 1 MHz.

Single-longitudinal-mode ring diamond Raman laser.

We demonstrate a single-longitudinal-mode ring diamond Raman laser, pumped by a tunable single-mode Ti:sapphire laser. Two methods to achieving unidirectional operation have been demonstrated: increasing gain for one direction using a reinjecting mirror and increasing loss for one direction using sum frequency mixing in BBO. Both methods result in a stable single-longitudinal-mode operation.

Real-time imaging of methane gas leaks using a single-pixel camera.

We demonstrate a camera which can image methane gas at video rates, using only a single-pixel detector and structured illumination. The light source is an infrared laser diode operating at 1.651µm tuned to an absorption line of methane gas. The light is structured using an addressable micromirror array to pattern the laser output with a sequence of Hadamard masks. The resulting backscattered light is recorded using a single-pixel InGaAs detector which provides a measure of the correlation between the projected patterns and the gas distribution in the scene. Knowledge of this correlation and the patterns allows an image to be reconstructed of the gas in the scene. For the application of locating gas leaks the frame rate of the camera is of primary importance, which in this case is inversely proportional to the square of the linear resolution. Here we demonstrate gas imaging at ~25 fps while using 256 mask patterns (corresponding to an image resolution of 16×16). To aid the task of locating the source of the gas emission, we overlay an upsampled and smoothed image of the low-resolution gas image onto a high-resolution color image of the scene, recorded using a standard CMOS camera. We demonstrate for an illumination of only 5mW across the field-of-view imaging of a methane gas leak of ~0.2 litres/minute from a distance of ~1 metre.

Continuous-wave VECSEL Raman laser with tunable lime-yellow-orange output.

We report a compact CW KGW Raman laser with intracavity nonlinear mixing, pumped by the intracavity field of a VECSEL. By temperature tuning an intracavity LBO crystal, we obtained two separate tunable emissions bands, namely 548.5 - 566 nm for sum-frequency-generation (SFG) of the fundamental and Stokes wavelengths, and 577.5 - 596 nm for second-harmonic-generation (SHG) of the Stokes wavelength. The maximum output powers for SFG and SHG were 0.8 W @ 560 nm and 0.52 W @ 592.5 nm, with corresponding diode-to-visible optical conversion efficiencies of 4.2% and 2.9%. These preliminary results show strong potential for expanding the spectral coverage of VECSEL lasers.

Compact ultrafast semiconductor disk laser: targeting GFP based nonlinear applications in living organisms.

We present a portable ultrafast Semiconductor Disk Laser (SDL) (or vertical extended cavity surface emitting laser-VECSELs), to be used for nonlinear microscopy. The SDL is modelocked using a quantum-dot semiconductor saturable absorber mirror (SESAM), delivering an average output power of 287 mW, with 1.5 ps pulses at 500 MHz and a central wavelength of 965 nm. Specifically, despite the fact of having long pulses and high repetition rates, we demonstrate the potential of this laser for Two-Photon Excited Fluorescence (TPEF) imaging of in vivo Caenorhabditis elegans (C. elegans) expressing Green Fluorescent Protein (GFP) in a set of neuronal processes and cell bodies. Efficient TPEF imaging is achieved due to the fact that this wavelength matches the peak of the two-photon action cross section of this widely used fluorescent marker. The SDL extended versatility is shown by presenting Second Harmonic Generation images of pharynx, uterus, body wall muscles and its potential to be used to excite other different commercial dyes. Importantly this non-expensive, turn-key, compact laser system could be used as a platform to develop portable nonlinear bio-imaging devices.