This PDF file contains the front matter associated with SPIE Proceedings Volume 11957 including the Title Page, Copyright information, and Table of Contents.

Raman spectroscopy (RS) has the ability to retrieve in a non-invasive way molecular information from cells and tissues, allowing the detection of various biological responses. It often relies on multivariate analysis to detect the changes of interest. We show here how regularized methods can improve the accuracy and stability of detection in the context of single-cell measurements for the detection of inflammation, or different phenotypes. We then use protein synthesis as a case study to assess which Raman bands are the most significant, and find that small bands outside of the main Raman regions are often more accurate for detection.

Tuberculosis (TB) is a deadly disease that causes 1.4 million deaths per year. It is crucial to diagnose and isolate TB patients in the early stages to control this contagious disease. The current gold standard test for TB detection is the culture of a patients' sputum. Although the culture test provides fair accuracy, it takes days to weeks to produce results and needs trained laboratory personals. Here, we demonstrate an optical sensor that uses Raman spectroscopy to detect TB via sputum samples. We collected and prepared sputum samples of potential TB patients in a controlled environment, conducted culture tests, and labeled them as TB-positive and TB-negative samples. We then acquired Raman spectrums of the prepared samples using a 785 nm laser spectroscopy setup, employed principal component analysis (PCA) on the spectroscopic data, and found TB-related unique features which form the basis of TB diagnosis. To evaluate the sensor performance, we tested 40 TB patients, of which 17 were TB-positive and 23 TB-negative. The sensor judiciously classified the two groups with an accuracy of 95%. The proposed sensor is a step towards a rapid, non-invasive, technician-free, and portable TB diagnostic point-of-care device.

Raman spectroscopy is a non-invasive vibrational technique that yields the biochemical signature of bone, and this can be done transcutaneously using spatially offset Raman spectroscopy. The percentage of bone signal detected will increase with further source-detector offsets, but the overall signal will be decreased. In recent work, our work suggests that 3 mm is an optimal offset for detecting bone signal for phalanges and 5 mm for measuring metacarpals. The objective of this work is to create and validate a SORS instrument that collects offsets at 0, 3, and 6 mm offsets simultaneously. By conducting simulations with an optical design software, we were able to optimize the imaging throughput for each offset location. Preliminary data from a cadaver specimen suggests we collect good quality data from offsets 0, 3, and 6 mm from both metacarpals and phalanges. Future work will work on validating this instrument as a valid tool to perform bone quality assessment.

One major obstacle hampering the utilization of human induced pluripotent stem cells (hiPS) derived cells from bench to bedside is the safety concern of residual undifferentiated hiPS cells. To ensure the clinical use of differentiated cells, a method which can monitor the regenerative processes and assess the cell population without harming and modifying those cells is very critical in this field. Raman microscopy has emerged as a powerful tool in label-free observation and discrimination of cell types without external labels. Cell proliferation, differentiation, and maturation all trigger molecular changes that can be detected via Raman, enabling non-destructive characterization of cell and tissue constructs. By using Raman microscopy, we are aiming at establishing a non-invasive and quantitative evaluation method to monitor hepatic differentiation. In the future, this technique would be an invaluable tool in the quality control and safety assessment of hiPS-derived cell products.

The Oral Squamous Cell carcinoma (OSCC) is one of the most common and aggressive oral malignancies. Despite all significant advances in medicine, five-year survival rate is still low. This study aims to develop a full scheme for diagnosing oral cancer in early stages by using Raman spectroscopy. Patients undergoing biopsy or histopathological examination will be enrolled in this study. Ex vivo measurement will be carried out using saliva specimens and in vivo analysis will involve measurements taken on healthy and malignant tissue. In the future, this optical diagnostic approach using Raman spectroscopy and SERS can help in improving diagnostic accuracy and the survival rate by affecting the treatment outcome via early stage detection of oral cancer.

SISSI-Bio is the Chemical and Life Sciences branch of the infrared beamline SISSI (Synchrotron Infrared Source for Spectroscopy and Imaging) at Elettra-Sincrotrone, Trieste, Italy. The laboratory has always been kept up-to-date with the latest equipment for Fourier Transform InfraRed (FTIR) analysis and currently hosts three endstations: one for spectroscopy, one for microscopy and one for nanospectroscopy. Although the synchrotron radiation allows for measurements at the diffraction limit over the full IR spectral range, all three end stations can alternatively be operated with benchtop sources, increasing the usage time of the setups beyond the infrared synchrotron radiation availability. In this contribution, the SISSI-Bio equipment will be presented in an integrated manner to highlight the multipurpose capabilities of the laboratory, emphasizing the opportunities for facility users and collaborators to perform cutting-edge scientific experiments. Selected examples, focusing on multi-scale and correlative analyses will be presented, highlighting topics such as cell and tissue analysis for FTIR hyperspectral histology and cytology, strategies for bio-specimen measurements in physiological conditions, environmental science and cultural heritage. The short-term development plans to incur in the field of biophysics in the far-IR and THz region will be also introduced.

The real-time polymerase chain reaction (RT-PCR) analysis using nasal swab samples is the gold standard approach for COVID-19 diagnosis. However, due to the high false-negative rate at lower viral loads and complex test procedure, PCR is not suitable for fast mass screening. Therefore, the need for a highly sensitive and rapid detection system based on easily collected fluids such as saliva during the pandemic has emerged. In this study, we present a surface-enhanced Raman spectroscopy (SERS) metasurface optimized with genetic algorithm (GA) to detect SARS-CoV-2 directly using unprocessed saliva samples. During the GA optimization, the electromagnetic field profiles were used to calculate the field enhancement of each structure and the fitness values to determine the performance of the generated substrates. The obtained design was fabricated using electron beam lithography, and the simulation results were compared with the test results using methylene blue fluorescence dye. After the performance of the system was validated, the SERS substrate was tested with inactivated SARS-CoV-2 virus for virus detection, viral load analysis, cross-reactivity, and variant detection using machine learning models. After the inactivated virus tests are completed, with 36 PCR positive and 33 negative clinical samples, we were able to detect the SARS-CoV-2 positive samples from Raman spectra with 95.2% sensitivity and specificity.

Stimulated Raman scattering (SRS) microscopy allows for sensitive molecular vibrational imaging and is opening up a variety of biomedical applications. To fully utilize the capability of SRS, we developed a multicolor SRS microscope, which is capable of acquiring SRS images at the video rate, while the wavenumber can be changed in a frame-by-frame manner. Also our system is integrated with a confocal fluorescence microscope. I will introduce various vibrational imaging applications such as label-free imaging, supermultiplex imaging, metabolic imaging, and so on. We also describe functional Raman probes, which will further broaden the applications of SRS microscopy.

Photoswitchable fluorescence is a powerful technique to realize super-resolution imaging, highlighting, and optical storage, while its multiplexing capability is limited. Raman scattering is attracting attention because it generates narrowband vibrational signatures, which are potentially useful for highly multiplexed detection of different constituents. Here, we demonstrate photoswitchable SRS spectroscopy and microscopy assisted by photochromic molecules. The narrowband Raman signatures can be switched with full reversibility by applying the irradiation of UV or visible light. The switching speed under low power irradiation is evaluated as fast as within 1 microsecond which is compatible with a conventional point-scan microscope. The demonstration of live-cell imaging suggests the good compatibility to living systems and satisfying sensitivity of this method. We anticipate that photoswitchable SRS imaging will be a powerful foundation for super-multiplex super-resolution imaging.

In surgery, peripheral nerves should be preserved as much as possible to suppress the dysfunction and improve the quality of life after surgery. However, it is difficult to distinguish colorless, transparent, and thin nerves from other tissues. We had developed a coherent anti-Stokes Raman scattering (CARS) rigid endoscope to visualize nerves in a label-free manner. CARS allows for imaging without staining based on the information of molecular vibrations. In the conference, we show near real-time nerve visualization using CARS endoscopy and deep learning. We demonstrate that the image taken at 1.6 s/image satisfies the segmentation quality required for medical images.

Raman spectroscopy is a hugely informative tool with a plethora of applications from biomedicine to analytical chemistry. Potentially, the technique could improve liver transplantation success rates through investigating Raman signals associated with metabolic changes prior to transplant rejection. However, studying biological systems is challenging since background fluorescence dominates the weak Raman signal. Thus, there is a need to improve signal-to-noise and Raman-tofluorescence ratios and drive down spectral acquisition times. Pulsed lasers combined with time-resolving single photon avalanche diode (SPAD) detection systems have been shown to enhance Raman and fluorescence discrimination. We report significant advances in time-correlated single photon counting (TCSPC) Raman spectroscopy using a laser exhibiting up to 200 W peak power and 40 MHz repetition rates in combination with a 512 spectral channel, 16.5 gigaevent/s throughput SPAD histogramming line sensor. Using a diamond sample, we report 0.4 MHz Raman count rates, millisecond spectral acquisition times, and signal-to-noise ratios of over 200. We demonstrate simultaneous, singleexposure acquisition of Raman and fluorescence signals in sesame oil. Time-based Raman-fluorescence discrimination techniques are subject to fluorescence signal tail influences from previous pulses, and data obtained with laser periods of 25 ns and 50 ns are presented. We achieved optimised Raman-to-fluorescence ratios through adjustment of histogram bin positions in 63 ps increments. Achieving high count rates while discriminating fluorescence from Raman signals unlocks the potential of combined Raman/fluorescence lifetime spectroscopy for biomedical imaging applications.

Mid-infrared dispersion spectroscopy is a novel alternative approach to classical absorption spectroscopy for qualitative and quantitative analysis of liquid-phase samples focused on broadband refractive index variation sensing originating from IR absorption. We present the redesigned and improved version of an external cavity-quantum cascade laser-based MachZehnder interferometer setup dedicated for refractive index sensing of liquids, which outperforms classic absorption spectroscopy. The refined version of the setup features greater compactness, a new dual-channel transmission cell and a hysteresis-free piezo-actuator for phase locked interferometric detection. Moreover, a new routine for fast and almost simultaneous acquisition of real and imaginary part of the complex refractive index (i.e., dispersion and absorption spectra) was introduced for mutual validation of the spectra. Dispersion spectra at sample temperatures ranging from 15 to 90°C can be recorded as the setup shows a stable noise-floor over that temperature range. Introduction of a hysteresis-free piezoactuator to the system enabled fast spectral acquisition at constant sensitivity with speed rates of 100 cm^-1 /s, long-term stability and allowed to improve the reproducibility, robustness, and limits of detection of the method. We compare the performance of the refined setup with the previously demonstrated version by comparing the figures of merit for univariate glucose detection. In this context, the dispersion and absorption spectra of glucose were acquired and assessed. The achieved limit of detection for dispersion sensing was 5 times lower when compared to previous version and ~2 times lower than for classic absorption sensing at 5 times shorter spectra acquisition times. In summary, the improvements in the instrumentation for dispersion spectroscopy have improved the sensitivity, reliability, and quality of the method. The achieved results set a basis for further extension of the range of application presented for this technique.

Early-stage cancer detection is challenging due to the lack of associated oral-tissue clinical features and absence of changes on conventional cellular-imaging, serological and histopathological exams. By using a molecular-sensitive optical technique such as Fourier-transform infrared (FT-IR) spectroscopy, disease-specific biochemical changes can be detected non-destructively, non-invasively and with small sample volumes. In this study, we have used FT-IR spectroscopy to analyze saliva samples of control, smoker, and occasional smoker groups in the fingerprint region (900cm-1 to 1800cm-1). Saliva-sample classification was performed with a neural network algorithm and leave-one-out validation. Correctly classified instances were 72.7% for the control group, 65.5% for occasional smokers and 75% for smokers.

We have developed a vertical flow (VF) method to improve the generation and collection efficiency of Raman scattering signals. The VF method enhances the Raman signal up to 90 times [Li and Hiramatsu, Anal. Chem. 2019, 91, 9806]. The VF method enabled an online Raman measurement of HPLC eluate (LC-Raman) [Lo and Hiramatsu, Anal. Chem. 2020, 92, 14601]. A programmable pump of the HPLC system is available to gradually change the pH value of the solvent by changing the mixing fraction of two solvents. In this study, we used the programmable pump combined with the Raman spectroscopy to probe the denaturation of bovine serum albumin (BSA) change in the range of pH 7 – 9. The vertical flow method allowed us to record 8300 spectra in 30 min. The obtained 2D Raman data having the spectral and temporal axes was analyzed with singular value decomposition and reconstruction method. We found that four temporal and spectral components predominate the 2D Raman data, namely, (i) the pH-independent Raman signal of BSA, (ii) that due to the denaturation, (iii) the ionization of phosphate in the buffer, and (iv) pH-dependent background. The sufficiently large number of the data points enabled precise determination of the pK_a values of the denaturation process of BSA in the range of pH 7 – 9 and detailed analysis of the structural change in the denaturation.

External cavity-quantum cascade laser (EC-QCL) based mid-infrared (IR) spectroscopy is an emerging technology for analyzing proteins in aqueous solutions. Higher sensitivity and larger applicable optical path lengths compared to conventional Fourier-transform IR (FTIR) spectroscopy open a wide range of possible applications, including near realtime protein monitoring from complex downstream operations. In this work, an EC-QCL based mid-IR spectrometer was coupled to a preparative liquid chromatography (LC) system. The large optical path length (25 μm) and the broad tuning range of the laser (1350-1750 cm^-1 ) allowed robust spectra acquisition in the most important wavenumber range for protein secondary structure determination. A model system based on size exclusion chromatography (SEC) and three different proteins was employed to demonstrate the advantages of LCQCL-IR coupling. The recorded spectra showed distinct amide I and II bands across the chromatographic run. Mid-IR spectra, extracted from the three chromatographic peak maxima showed features typical for the secondary structures of the exhibited proteins with high comparability to off-line reference spectra. Band positions and maxima of mid-IR absorbances were compared to a conventional UV detector, revealing excellent agreement of peak shapes and maxima. This work demonstrates that laser-based mid-IR spectroscopy offers the significant advantage of providing almost realtime information about protein secondary structure, which typically has to be obtained by laborious and time-consuming offline analysis. Consequently, coupling of LC and laser-based mid-IR spectroscopy holds high potential for replacing conventional off-line methods for monitoring proteins in complex biotechnological processes.