Correlation properties of a spatially quasi-incoherent imaging interferometer.

The depth-gating capacity of a spatially quasi-incoherent imaging interferometer is investigated in relation to the 3D correlation properties of diffraction field laser speckles. The system exploits a phase-stepped imaging Michelson-type interferometer in which spatially quasi-incoherent illumination is generated by passing an unexpanded laser beam through a rotating diffuser. Numerical simulations and optical experiments both verify that the depth-gating capacity of the imaging interferometer scales as λ/2NAp2, where λ is the wavelength of the laser and NAp is the numerical aperture of the illumination. For a set depth gate of 150 µm, the depth-gating capacity of the interferometer is demonstrated by scanning a standard USAF target through the measurement volume. The results obtained show that an imaging tool of this kind is expected to provide useful capabilities for imaging through disturbing media and where a single wavelength is required.

Investigation of the Spatial Generation of Stimulated Raman Scattering Using Computer Simulation and Experimentation.

Stimulated Raman scattering is a phenomenon with potential use in providing real-time molecular information in three-dimensions (3D) of a sample using imaging. For precise imaging, the knowledge about the spatial generation of stimulated Raman scattering is essential. To investigate the spatial behavior in an idealized case, computer simulations and experiments were performed. For the computer simulations, diffraction theory was used for the beam propagation complemented with nonlinear phase modulation describing the interaction between the light and matter. For the experiments, a volume of ethanol was illuminated by an expanded light beam and a plane inside the volume was imaged in transmission. For generating stimulated Raman scattering, a pump beam was focused into this volume and led to a beam dump after passing the volume. The pulse duration of the two beams were 6 ns and the pump beam energy ranged from 1 to 27 mJ. The effect of increasing pump power on the spatial distribution of the Raman gain and the spatial growth of the signal at different interaction lengths between the beam and the sample was investigated. The spatial width of the region where the stimulated Raman scattering signal was generated for experiments and simulation was 0.21 and 0.09 mm, respectively. The experimental and simulation results showed that most of the stimulated Raman scattering is generated close to the pump beam focus and the maximum peak of the Stokes intensity spatially comes shortly after the peak of the pump intensity.

Impact of preprocessing methods on the Raman spectra of brain tissue.

Delineating cancer tissue while leaving functional tissue intact is crucial in brain tumor resection. Despite several available aids, surgeons are limited by preoperative or subjective tools. Raman spectroscopy is a label-free optical technique with promising indications for tumor tissue identification. To allow direct comparisons between measurements preprocessing of the Raman signal is required. There are many recognized methods for preprocessing Raman spectra; however, there is no universal standard. In this paper, six different preprocessing methods were tested on Raman spectra (n > 900) from fresh brain tissue samples (n = 34). The sample cohort included both primary brain tumors, such as adult-type diffuse gliomas and meningiomas, as well as metastases of breast cancer. Each tissue sample was classified according to the CNS WHO 2021 guidelines. The six methods include both direct and iterative polynomial fitting, mathematical morphology, signal derivative, commercial software, and a neural network. Data exploration was performed using principal component analysis, t-distributed stochastic neighbor embedding, and k-means clustering. For each of the six methods, the parameter combination that explained the most variance in the data, i.e., resulting in the highest Gap-statistic, was chosen and compared to the other five methods. Depending on the preprocessing method, the resulting clusters varied in number, size, and associated spectral features. The detected features were associated with hemoglobin, neuroglobin, carotenoid, water, and protoporphyrin, as well as proteins and lipids. However, the spectral features seen in the Raman spectra could not be unambiguously assigned to tissue labels, regardless of preprocessing method. We have illustrated that depending on the chosen preprocessing method, the spectral appearance of Raman features from brain tumor tissue can change. Therefore, we argue both for caution in comparing spectral features from different Raman studies, as well as the importance of transparency of methodology and implementation of the preprocessing. As discussed in this study, Raman spectroscopy for in vivo guidance in neurosurgery requires fast and adaptive preprocessing. On this basis, a pre-trained neural network appears to be a promising approach for the operating room.

CO2 Gasification Reactivity of Char from High-Ash Biomass.

Biomass char produced from pyrolysis processes is of great interest to be utilized as renewable solid fuels or materials. Forest byproducts and agricultural wastes are low-cost and sustainable biomass feedstocks. These biomasses generally contain high amounts of ash-forming elements, generally leading to high char reactivity. This study elaborates in detail how chemical and physical properties affect CO2 gasification rates of high-ash biomass char, and it also targets the interactions between these properties. Char produced from pine bark, forest residue, and corncobs (particle size 4-30 mm) were included, and all contained different relative compositions of ash-forming elements. Acid leaching was applied to further investigate the influence of inorganic elements in these biomasses. The char properties relevant to the gasification rate were analyzed, that is, elemental composition, specific surface area, and carbon structure. Gasification rates were measured at an isothermal condition of 800 °C with 20% (vol.) of CO2 in N2. The results showed that the inorganic content, particularly K, had a stronger effect on gasification reactivity than specific surface area and aromatic cluster size of the char. At the gasification condition utilized in this study, K could volatilize and mobilize through the char surface, resulting in high gasification reactivity. Meanwhile, the mobilization of Ca did not occur at the low temperature applied, thus resulting in its low catalytic effect. This implies that the dispersion of these inorganic elements through char particles is an important reason behind their catalytic activity. Upon leaching by diluted acetic acid, the K content of these biomasses substantially decreased, while most of the Ca remained in the biomasses. With a low K content in leached biomass char, char reactivity was determined by the active carbon surface area.

A Microfluidic System for Simultaneous Raman Spectroscopy, Patch-Clamp Electrophysiology, and Live-Cell Imaging to Study Key Cellular Events of Single Living Cells in Response to Acute Hypoxia.

The ability to sense changes in oxygen availability is fundamentally important for the survival of all aerobic organisms. However, cellular oxygen sensing mechanisms and pathologies remain incompletely understood and studies of acute oxygen sensing, in particular, have produced inconsistent results. Current methods cannot simultaneously measure the key cellular events in acute hypoxia (i.e., changes in redox state, electrophysiological properties, and mechanical responses) at controlled partial pressures of oxygen (pO2 ). The lack of such a comprehensive method essentially contributes to the discrepancies in the field. A sealed microfluidic system that combines i) Raman spectroscopy, ii) patch-clamp electrophysiology, and iii) live-cell imaging under precisely controlled pO2 have therefore been developed. Merging these modalities allows label-free and simultaneous observation of oxygen-dependent alterations in multiple cellular redox couples, membrane potential, and cellular contraction. This technique is adaptable to any cell type and allows in-depth insight into acute oxygen sensing processes underlying various physiologic and pathologic conditions.

A tactile resonance sensor for prostate cancer detection - evaluation on human prostate tissue.

Prostate cancer surgery risks erectile problems and incontinence for the patient. An instrument for guiding surgeons to avoid nerve bundle damage and ensure complete cancer removal is desirable. We present a tactile resonance sensor made of PZT ceramics, mounted in a 3D motorized translation stage for scanning and measuring tissue stiffness for detecting cancer in human prostate. The sensor may be used during surgery for guidance, scanning the prostate surface for the presence of cancer, indicating migration of cancer cells into surrounding tissue. Ten fresh prostates, obtained from patients undergoing prostate cancer surgery, were cut into 0.5 cm thick slices. Each slice was measured for tissue stiffness at about 25 different sites and compared to histology for validation cancer prediction by stiffness. The statistical analysis was based on a total of 148 sites with non-cancer and 40 sites with cancer. Using a generalized linear mixed model (GLMM), the stiffness data predicted cancer with an area under the curve of 0.74, after correcting for overfitting using bootstrap validation. Mean prostate stiffness on the logarithmic scale (p = 0.015) and standardized Z-scores (p = 0.025) were both significant predictors of cancer. This study concludes that stiffness measured by the tactile resonance sensor is a significant predictor of prostate cancer with potential for future development towards a clinical instrument for surgical guidance.

A New Approach for Evaluating Electron Transfer Dynamics by Using In Situ Resonance Raman Microscopy and Chronoamperometry in Conjunction with a Dynamic Model.

Geobacter sulfurreducens is a good candidate as a chassis organism due to its ability to form thick, conductive biofilms, enabling long-distance extracellular electron transfer (EET). Due to the complexity of EET pathways in G. sulfurreducens, a dynamic approach is required to study genetically modified EET rates in the biofilm. By coupling online resonance Raman microscopy with chronoamperometry, we were able to observe the dynamic discharge response in the biofilm's cytochromes to an increase in anode voltage. Measuring the heme redox state alongside the current allows for the fitting of a dynamic model using the current response and a subsequent validation of the model via the value of a reduced cytochrome c Raman peak. The modeled reduced cytochromes closely fitted the Raman response data from the G. sulfurreducens wild-type strain, showing the oxidation of heme groups in cytochromes until a new steady state was achieved. Furthermore, the use of a dynamic model also allows for the calculation of internal rates, such as acetate and NADH consumption rates. The Raman response of a mutant lacking OmcS showed a higher initial oxidation rate than predicted, followed by an almost linear decrease of the reduced mediators. The increased initial rate could be attributed to an increase in biofilm conductivity, previously observed in biofilms lacking OmcS. One explanation for this is that OmcS acts as a conduit between cytochromes; therefore, deleting the gene restricts the rate of electron transfer to the extracellular matrix. This could, however, be modeled assuming a linear oxidation rate of intercellular mediators.IMPORTANCE Bioelectrochemical systems can fill a vast array of application niches, due to the control of redox reactions that it offers. Although native microorganisms are preferred for applications such as bioremediation, more control is required for applications such as biosensors or biocomputing. The development of a chassis organism, in which the EET is well defined and readily controllable, is therefore essential. The combined approach in this work offers a unique way of monitoring and describing the reaction kinetics of a G. sulfurreducens biofilm, as well as offering a dynamic model that can be used in conjunction with applications such as biosensors.

Bypassing mitochondrial complex III using alternative oxidase inhibits acute pulmonary oxygen sensing.

Mitochondria play an important role in sensing both acute and chronic hypoxia in the pulmonary vasculature, but their primary oxygen-sensing mechanism and contribution to stabilization of the hypoxia-inducible factor (HIF) remains elusive. Alteration of the mitochondrial electron flux and increased superoxide release from complex III has been proposed as an essential trigger for hypoxic pulmonary vasoconstriction (HPV). We used mice expressing a tunicate alternative oxidase, AOX, which maintains electron flux when respiratory complexes III and/or IV are inhibited. Respiratory restoration by AOX prevented acute HPV and hypoxic responses of pulmonary arterial smooth muscle cells (PASMC), acute hypoxia-induced redox changes of NADH and cytochrome c, and superoxide production. In contrast, AOX did not affect the development of chronic hypoxia-induced pulmonary hypertension and HIF-1α stabilization. These results indicate that distal inhibition of the mitochondrial electron transport chain in PASMC is an essential initial step for acute but not chronic oxygen sensing.

Single-Step Preprocessing of Raman Spectra Using Convolutional Neural Networks.

Preprocessing of Raman spectra is generally done in three separate steps: (1) cosmic ray removal, (2) signal smoothing, and (3) baseline subtraction. We show that a convolutional neural network (CNN) can be trained using simulated data to handle all steps in one operation. First, synthetic spectra are created by randomly adding peaks, baseline, mixing of peaks and baseline with background noise, and cosmic rays. Second, a CNN is trained on synthetic spectra and known peaks. The results from preprocessing were generally of higher quality than what was achieved using a reference based on standardized methods (second-difference, asymmetric least squares, cross-validation). From 105 simulated observations, 91.4% predictions had smaller absolute error (RMSE), 90.3% had improved quality (SSIM), and 94.5% had reduced signal-to-noise (SNR) power. The CNN preprocessing generated reliable results on measured Raman spectra from polyethylene, paraffin and ethanol with background contamination from polystyrene. The result shows a promising proof of concept for the automated preprocessing of Raman spectra.

High-Density Plasmonic Nanoparticle Arrays Deposited on Nanoporous Anodic Alumina Templates for Optical Sensor Applications.

This study demonstrates a new, robust, and accessible deposition technique of metal nanoparticle arrays (NPAs), which uses nanoporous anodic alumina (NAA) as a template for capillary force-assisted convective colloid (40, 60, and 80 nm diameter Au) assembly. The NPA density and nanoparticle size can be independently tuned by the anodization conditions and colloid synthesis protocols. This enables production of non-touching variable-density NPAs with controllable gaps in the 20-60 nm range. The NPA nearest neighbor center distance in the present study was fixed to 100 nm by the choice of anodization protocol. The obtained Au NPAs have the resonant scattering maxima in the visible spectral range, with a refractometric sensitivity, which can be tuned by the variation of the array density. The thickness of the NAA layer in an Aluminum-NAA-NPA multilayer system enables further tuning of the resonance frequency and optimization for use with specific molecules, e.g., to avoid absorption bands. Applicability of the mentioned multilayers for colorimetric refractive index (RI) sensing is demonstrated. Their use as Surface-Enhanced Raman Scattering (SERS) substrates is tested using hemoglobin as a biological probe molecule.

On-Line Raman Spectroscopic Study of Cytochromes' Redox State of Biofilms in Microbial Fuel Cells.

Bio-electrochemical systems such as microbial fuel cells and microbial electrosynthesis cells depend on efficient electron transfer between the microorganisms and the electrodes. Understanding the mechanisms and dynamics of the electron transfer is important in order to design more efficient reactors, as well as modifying microorganisms for enhanced electricity production. Geobacter are well known for their ability to form thick biofilms and transfer electrons to the surfaces of electrodes. Currently, there are not many "on-line" systems for monitoring the activity of the biofilm and the electron transfer process without harming the biofilm. Raman microscopy was shown to be capable of providing biochemical information, i.e., the redox state of C-type cytochromes, which is integral to external electron transfer, without harming the biofilm. In the current study, a custom 3D printed flow-through cuvette was used in order to analyze the oxidation state of the C-type cytochromes of suspended cultures of three Geobacter sulfurreducens strains (PCA, KN400 and ΔpilA). It was found that the oxidation state is a good indicator of the metabolic state of the cells. Furthermore, an anaerobic fluidic system enabling in situ Raman measurements was designed and applied successfully to monitor and characterize G. sulfurreducens biofilms during electricity generation, for both a wild strain, PCA, and a mutant, ΔS. The cytochrome redox state, monitored by the Raman peak areas, could be modulated by applying different poise voltages to the electrodes. This also correlated with the modulation of current transferred from the cytochromes to the electrode. The Raman peak area changed in a predictable and reversible manner, indicating that the system could be used for analyzing the oxidation state of the proteins responsible for the electron transfer process and the kinetics thereof in-situ.

Development of a Gas-Tight Microfluidic System for Raman Sensing of Single Pulmonary Arterial Smooth Muscle Cells Under Normoxic/Hypoxic Conditions.

Acute hypoxia changes the redox-state of pulmonary arterial smooth muscle cells (PASMCs). This might influence the activity of redox-sensitive voltage-gated K⁺-channels (Kv-channels) whose inhibition initiates hypoxic pulmonary vasoconstriction (HPV). However, the molecular mechanism of how hypoxia-or the subsequent change in the cellular redox-state-inhibits Kv-channels remains elusive. For this purpose, a new multifunctional gas-tight microfluidic system was developed enabling simultaneous single-cell Raman spectroscopic studies (to sense the redox-state under normoxic/hypoxic conditions) and patch-clamp experiments (to study the Kv-channel activity). The performance of the system was tested by optically recording the O2-content and taking Raman spectra on murine PASMCs under normoxic/hypoxic conditions or in the presence of H2O2. Oxygen sensing showed that hypoxic levels in the gas-tight microfluidic system were achieved faster, more stable and significantly lower compared to a conventional open system (1.6 ± 0.2%, respectively 6.7 ± 0.7%, n = 6, p < 0.001). Raman spectra revealed that the redistribution of biomarkers (cytochromes, FeS, myoglobin and NADH) under hypoxic/normoxic conditions were improved in the gas-tight microfluidic system (p-values from 0.00% to 16.30%) compared to the open system (p-value from 0.01% to 98.42%). In conclusion, the new redox sensor holds promise for future experiments that may elucidate the role of Kv-channels during HPV.

A lab-on-a-chip for hypoxic patch clamp measurements combined with optical tweezers and spectroscopy- first investigations of single biological cells.

The response and the reaction of the brain system to hypoxia is a vital research subject that requires special instrumentation. With this research subject in focus, a new multifunctional lab-on-a-chip (LOC) system with control over the oxygen content for studies on biological cells was developed. The chip was designed to incorporate the patch clamp technique, optical tweezers and absorption spectroscopy. The performance of the LOC was tested by a series of experiments. The oxygen content within the channels of the LOC was monitored by an oxygen sensor and verified by simultaneously studying the oxygenation state of chicken red blood cells (RBCs) with absorption spectra. The chicken RBCs were manipulated optically and steered in three dimensions towards a patch-clamp micropipette in a closed microfluidic channel. The oxygen level within the channels could be changed from a normoxic value of 18% O 2 to an anoxic value of 0.0-0.5% O 2. A time series of 3 experiments were performed, showing that the spectral transfer from the oxygenated to the deoxygenated state occurred after about 227 ± 1 s and a fully developed deoxygenated spectrum was observed after 298 ± 1 s, a mean value of 3 experiments. The tightness of the chamber to oxygen diffusion was verified by stopping the flow into the channel system while continuously recording absorption spectra showing an unchanged deoxygenated state during 5400 ± 2 s. A transfer of the oxygenated absorption spectra was achieved after 426 ± 1 s when exposing the cell to normoxic buffer. This showed the long time viability of the investigated cells. Successful patching and sealing were established on a trapped RBC and the whole-cell access (Ra) and membrane (Rm) resistances were measured to be 5.033 ± 0.412 M Ω and 889.7 ± 1.74 M Ω respectively.

Optical fibre probe NIR Raman measurements in ambient light and in combination with a tactile resonance sensor for possible cancer detection.

First measurements on a combined instrument with a thin fibre optic Raman probe mounted inside a hollow tactile resonance sensor have been performed in ambient light on porcine tissue. The ambient fluorescent light was removed successfully from the spectra. The stiffness and the biomolecular composition of the tissue were analysed.

Resonance micro-Raman investigations of the rat medial preoptic nucleus: effects of a low-iron diet on the neuroglobin content.

The aim of this study was to investigate the medial preoptic nucleus (MPN) of the anterior hypothalamus by resonance Raman spectroscopy (514.5 nm) to determine if it is possible to enhance the Raman scattering of hemoproteins in fresh brain tissue slices. The resonance effect was compared with near-infrared Raman spectra. Two groups of male Sprague Dawley rats were studied, one control group on a normal diet and one group on a low-iron diet to evoke iron deficiency. Each group consisted of four rats, 38-41 days old. The diets lasted for 11, 12, and 15 days. The MPN regions of brain tissue slices were analyzed by monitoring raw and pre-processed mean data, by cluster analysis, and by deriving difference spectra from pre-processed mean spectra. Cluster analysis of the resonance Raman spectra could identify different hemoprotein groups, namely, hemoglobin (Hb) and neuroglobin (Ngb). Spectra from randomly distributed spots revealed high Hb content, whereas Ngb was evenly distributed in the MPN. The different spectra showed a decrease of the Ngb and lipid content for the animals on the low-iron diet. The Ngb decrease was approximately 20%. The data show that resonance Raman spectroscopy is well suited to study hemoproteins in fresh brain tissue.

The integration of a micropipette in a closed microfluidic chip with optical tweezers for investigations of single cells: erratum.

In July 2011 a new concept of a closed microfluidic system equipped with a fixed micropipette, optical tweezers and a UV-Vis spectrometer was presented [Biomed. Opt. Express 2, 2299 (2011)]. Figure 1 showed falsely oriented mirrors. To clarify the design of the setup, this erratum presents a correct schematic.

How to integrate a micropipette into a closed microfluidic system: absorption spectra of an optically trapped erythrocyte.

We present a new concept of integrating a micropipette within a closed microfluidic system equipped with optical tweezers and a UV-Vis spectrometer. A single red blood cell (RBC) was optically trapped and steered in three dimensions towards a micropipette that was integrated in the microfluidic system. Different oxygenation states of the RBC, triggered by altering the oxygen content in the microchannels through a pump system, were optically monitored by a UV-Vis spectrometer. The built setup is aimed to act as a multifunctional system where the biochemical content and the electrophysiological reaction of a single cell can be monitored simultaneously. The system can be used for other applications like single cell sorting, in vitro fertilization or electrophysiological experiments with precise environmental control of the gas-, and chemical content.

Optical manipulation for single-cell studies.

In the last decade optical manipulation has evolved from a field of interest for physicists to a versatile tool widely used within life sciences. This has been made possible in particular due to the development of a large variety of imaging techniques that allow detailed information to be gained from investigations of single cells. The use of multiple optical traps has high potential within single-cell analysis since parallel measurements provide good statistics. Multifunctional optical tweezers are, for instance, used to study cell heterogeneity in an ensemble, and force measurements are used to investigate the mechanical properties of individual cells. Investigations of molecular motors and forces on the single-molecule level have led to discoveries that would have been difficult to make with other techniques. Optical manipulation has prospects within the field of cell signalling and tissue engineering. When combined with microfluidic systems the chemical environment of cells can be precisely controlled. Hence the influence of pH, salt concentration, drugs and temperature can be investigated in real time. Fast advancing technical developments of automated and user-friendly optical manipulation tools and cross-disciplinary collaboration will contribute to the routinely use of optical manipulation techniques within the life sciences.

Effects of snap-freezing and near-infrared laser illumination on porcine prostate tissue as measured by Raman spectroscopy.

Most Raman spectroscopic studies on tissue are performed in vitro. To assure that the results are applicable to in vivo examinations, preparation protocols and measurement procedures of tissue for in vitro studies should preserve tissue characteristics close to the native state. This study had two aims. The first was to elucidate if photoinduced effects arise during 5 minutes' continuous illumination of tissue with an 830 nm laser at an irradiance of approximately 3 x 10(10) W/m2. The second was to investigate the effects of snap-freezing of porcine prostate tissue in liquid nitrogen and subsequent storage at -80 degrees C, by means of multivariate analysis. 830 nm laser illumination of the specified irradiance did not affect the Raman spectra. A decrease of the spectral background was observed, likely due to photobleaching of tissue fluorophores. Snap-freezing and subsequent storage at -80 degrees C gave rise to subtle but significant alterations in Raman spectra, most likely related to changes in the protein conformations.

Micro-resonance Raman study of optically trapped Escherichia coli cells overexpressing human neuroglobin.

We describe the possibility of using a microresonance Raman spectrometer combined with a microfluidic system and optical tweezers to study Escherichia coli (E. coli) overexpressing wild type (wt) neuroglobin (NGB) and its E7Leu mutant, respectively. NGB is a recently discovered heme protein and its function still is a matter of debate. So far, the protein has been studied in its purified form, and in vivo measurements on the single cell level could give more information. To study the feasibility of the combined techniques, the possibilities of the setup are investigated by taking spectra from single cells and clusters of cells. We find that the microresonance Raman technique enables studies of the wt NGB protein in a living cell under fluctuating aerobic and anaerobic conditions. E. coli cells overexpressing wt NGB are stable, and the reversible oxygenation-deoxygenation can be studied over a long period of time. Further, the experiment indicates the presence of an enzymatic system in the bacteria reducing the ferric form NGB. The study of E. coli cells overexpressing E7Leu NGB, on the other hand, gives insight into limiting factors of the setup, such as cell lysis, photoinduced chemistry, and protein concentrations.