Super-resolution SRS microscopy with A-PoD.

Stimulated Raman scattering (SRS) offers the ability to image metabolic dynamics with high signal-to-noise ratio. However, its spatial resolution is limited by the numerical aperture of the imaging objective and the scattering cross-section of molecules. To achieve super-resolved SRS imaging, we developed a deconvolution algorithm, adaptive moment estimation (Adam) optimization-based pointillism deconvolution (A-PoD) and demonstrated a spatial resolution of lower than 59 nm on the membrane of a single lipid droplet (LD). We applied A-PoD to spatially correlated multiphoton fluorescence imaging and deuterium oxide (D2O)-probed SRS (DO-SRS) imaging from diverse samples to compare nanoscopic distributions of proteins and lipids in cells and subcellular organelles. We successfully differentiated newly synthesized lipids in LDs using A-PoD-coupled DO-SRS. The A-PoD-enhanced DO-SRS imaging method was also applied to reveal metabolic changes in brain samples from Drosophila on different diets. This new approach allows us to quantitatively measure the nanoscopic colocalization of biomolecules and metabolic dynamics in organelles.

A high-throughput technique to map cell images to cell positions using a 3D imaging flow cytometer.

We develop a high-throughput technique to relate positions of individual cells to their three-dimensional (3D) imaging features with single-cell resolution. The technique is particularly suitable for nonadherent cells where existing spatial biology methodologies relating cell properties to their positions in a solid tissue do not apply. Our design consists of two parts, as follows: recording 3D cell images at high throughput (500 to 1,000 cells/s) using a custom 3D imaging flow cytometer (3D-IFC) and dispensing cells in a first-in-first-out (FIFO) manner using a robotic cell placement platform (CPP). To prevent errors due to violations of the FIFO principle, we invented a method that uses marker beads and DNA sequencing software to detect errors. Experiments with human cancer cell lines demonstrate the feasibility of mapping 3D side scattering and fluorescent images, as well as two-dimensional (2D) transmission images of cells to their locations on the membrane filter for around 100,000 cells in less than 10 min. While the current work uses our specially designed 3D imaging flow cytometer to produce 3D cell images, our methodology can support other imaging modalities. The technology and method form a bridge between single-cell image analysis and single-cell molecular analysis.

NLRC4-mediated pyroptosis was involved in coagulation disorders of acute pancreatitis.

BACKGROUND: Acute pancreatitis (AP) is a potentially lethal acute disease highly involved in coagulation disorders. Pyroptosis has been reported to exacerbate coagulation disorders, yet this implication has not been illustrated completely in AP. METHODS: RNA sequencing data of peripheral blood of AP patients were downloaded from the Gene Expression Omnibus database. Gene set variation analysis and single sample gene set enrichment analysis were used to calculate the enrichment score of coagulation-related signatures and pyroptosis. Spearman and Pearson correlation analysis was used for correlation analysis. Peripheral blood samples and related clinical parameters were collected from patients with AP and healthy individuals. A severe AP (SAP) model of mice was established using caerulein and lipopolysaccharide. Enzyme-linked immunosorbent assay, chemiluminescence immunoassay and immunohistochemical analysis were employed to detect the level of coagulation indicators and pyroptosis markers in serum and pancreas tissues. Additionally, we evaluated the effect of pyroptosis inhibition and NLRC4 silence on the function of human umbilical vein endothelial cells (HUVECs). RESULTS: Coagulation disorders were significantly positively correlated to the severity of AP, and they could be a predictor for AP severity. Further analyses indicated that six genes-DOCK9, GATA3, FCER1G, NLRC4, C1QB and C1QC-may be involved in coagulation disorders of AP. Among them, NLRC4 was positively related to pyroptosis that had a positive association with most coagulation-related signatures. Data from patients showed that NLRC4 and other pyroptosis markers, including IL-1ß, IL-18, caspase1 and GSDMD, were significant correlation to AP severity. In addition, NLRC4 was positively associated with coagulation indicators in AP patients. Data from mice showed that NLRC4 was increased in the pancreas tissues of SAP mice. Treatment with a pyroptosis inhibitor effectively alleviated SAP and coagulation disorders in mice. Finally, inhibiting pyroptosis or silencing NLRC4 could relieve endothelial dysfunction in HUVECs. CONCLUSIONS: NLRC4-mediated pyroptosis damages the function of endothelial cells and thereby exacerbates coagulation disorders of AP. Inhibiting pyroptosis could improve coagulation function and alleviate AP.

The effects of combined acupuncture anesthesia and serratus anterior plane block with ropivacaine on postoperative analgesia in patients undergoing chest surgery.

OBJECTIVE: To investigate the effects of combined acupuncture anesthesia and ropivacaine on postoperative analgesia and neuro-related factors in patients undergoing chest surgery. METHODS: The analgesic drug dosage, postoperative PCIA pressing times, VAS scores at rest and during activity at 6 h (T1), 12 h (T2), 18 h (T3), and 24 h (T4) postoperatively. RESULTS: The analgesic drug dosage and postoperative PCIA pressing times were lower in the observation group than in the control group (p < 0.05). The VAS scores at T1-T4 postoperatively were lower in the observation group than in the control group (p < 0.05). The SAS scores at T1-T4 postoperatively were lower in the observation group than in the control group (p < 0.05). The levels of IL-6 and IL-10 on postoperative day 1 were higher than those on preoperative day 1 in both groups, with a smaller change in the observation group (p < 0.05). The levels of S100ß protein on postoperative day 1 were higher than those on preoperative day 1 in both groups, while the BDNF levels were lower, with a smaller change in the observation group (p < 0.05). There was no significant difference in the incidence of adverse reactions between the control group (11.36%) and the observation group (15.56%) (p > 0.05). CONCLUSION: Combined acupuncture anesthesia and ropivacaine can effectively improve postoperative analgesia and agitation in patients undergoing chest surgery, reduce the dosage of analgesic drugs, regulate the levels of inflammatory factors and neurotrophic factors in patients, and do not increase the risk of adverse reactions related to patients.

Self-Assembled Homopolymeric Spherulites from Small Molecules in Solution.

Polymeric spherulites are typically formed by melt crystallization: spherulitic growth in solution is rare and requires complex polymers and dilute solutions. Here, we report the mild and unique formation of luminescent spherulites at room temperature via the simple molecule benzene-1,4-dithiol (BDT). Specifically, BDT polymerized into oligomers (PBDT) via disulfide bonds and assembled into uniform supramolecular nanoparticles in aqueous buffer; these nanoparticles were then dissolved back into PBDT in a good solvent (i.e., dimethylformamide) and underwent chain elongation to form spherulites (rPBDT) in 10 min. The spherulite geometry was modulated by changing the PBDT concentration and reaction time. Due to the step-growth polymerization and reorganization of PBDT, these spherulites not only exhibited robust structure but also showed broad clusterization-triggered emission. The biocompatibility and efficient cellular uptake of the spherulites further underscore their value as traceable drug carriers. This system provides a new pathway for designing versatile superstructures with value for hierarchical assembly of small molecules into a complicated biological system.

Mid-infrared metabolic imaging with vibrational probes.

Understanding metabolism is indispensable in unraveling the mechanistic basis of many physiological and pathological processes. However, in situ metabolic imaging tools are still lacking. Here we introduce a framework for mid-infrared (MIR) metabolic imaging by coupling the emerging high-information-throughput MIR microscopy with specifically designed IR-active vibrational probes. We present three categories of small vibrational tags including azide bond, 13C-edited carbonyl bond and deuterium-labeled probes to interrogate various metabolic activities in cells, small organisms and mice. Two MIR imaging platforms are implemented including broadband Fourier transform infrared microscopy and discrete frequency infrared microscopy with a newly incorporated spectral region (2,000-2,300 cm-1). Our technique is uniquely suited to metabolic imaging with high information throughput. In particular, we performed single-cell metabolic profiling including heterogeneity characterization, and large-area metabolic imaging at tissue or organ level with rich spectral information.

Alzheimer's-Associated Upregulation of Mitochondria-Associated ER Membranes After Traumatic Brain Injury.

Traumatic brain injury (TBI) can lead to neurodegenerative diseases such as Alzheimer's disease (AD) through mechanisms that remain incompletely characterized. Similar to AD, TBI models present with cellular metabolic alterations and modulated cleavage of amyloid precursor protein (APP). Specifically, AD and TBI tissues display increases in amyloid-ß as well as its precursor, the APP C-terminal fragment of 99 a.a. (C99). Our recent data in cell models of AD indicate that C99, due to its affinity for cholesterol, induces the formation of transient lipid raft domains in the ER known as mitochondria-associated endoplasmic reticulum (ER) membranes ("MAM" domains). The formation of these domains recruits and activates specific lipid metabolic enzymes that regulate cellular cholesterol trafficking and sphingolipid turnover. Increased C99 levels in AD cell models promote MAM formation and significantly modulate cellular lipid homeostasis. Here, these phenotypes were recapitulated in the controlled cortical impact (CCI) model of TBI in adult mice. Specifically, the injured cortex and hippocampus displayed significant increases in C99 and MAM activity, as measured by phospholipid synthesis, sphingomyelinase activity and cholesterol turnover. In addition, our cell type-specific lipidomics analyses revealed significant changes in microglial lipid composition that are consistent with the observed alterations in MAM-resident enzymes. Altogether, we propose that alterations in the regulation of MAM and relevant lipid metabolic pathways could contribute to the epidemiological connection between TBI and AD.

Phospholipase D and phosphatidic acid mediate regulation in the biosynthesis of spermidine and ganoderic acids by activating GlMyb in Ganoderma lucidum under heat stress.

Polyamines are essential for all kinds of organisms and take part in the regulation of multiple biological processes. Our previous study revealed that heat stress promoted the conversion of putrescine to spermidine and subsequently promoted the accumulation of ganoderic acids (GAs). However, how heat stress affects polyamine homeostasis remains unclear. To explore the underlying mechanism by which heat stress promoted spermidine biosynthesis, we assessed the effects of signalling molecules that respond to heat stress on spermidine biosynthesis. Our data suggested that heat stress-induced spermidine biosynthesis and GAs accumulation via a phospholipase D (PLD)-mediated phosphatidic acid (PA) signal. Further research revealed that the transcription factor GlMyb promoted spermidine biosynthesis via regulating spermidine synthase genes (spds1 and spds2) expression by directly bonding to their promoters and it responded to heat stress and PA signal. In summary, heat stress activated GlMyb by PLD-mediated PA signalling and subsequently induced the expression of spds1 and spds2 to promote the biosynthesis of spermidine and the accumulation of GAs. Our findings firstly reveal a detailed mechanism by which heat signalling regulates polyamine homeostasis by PLD-mediated PA signal in fungi and provide a greater understanding of how organisms alter polyamine levels in response to environmental changes.

GCN4 Enhances the Transcriptional Regulation of AreA by Interacting with SKO1 To Mediate Nitrogen Utilization in Ganoderma lucidum.

Fungi utilize a wide range of nitrogen to adapt their metabolism. The transcription factor GCN4 has a pivotal role in nitrogen metabolism. However, the mechanism by which GCN4 regulates nitrogen utilization in Ganoderma lucidum is not well understood. In this study, we found that GCN4 physically interacts with SKO1, a bZIP (basic leucine zipper) transcription factor. GCN4 cooperated with SKO1 to positively regulate nitrogen utilization, especially for the expression of areA. Electrophoretic mobility shift assays (EMSA) indicate that GCN4 directly binds to the areA promoter region. Further affinity analysis through biolayer interferometry (BLI) experiments and surface plasmon resonance (SPR) confirmed that GCN4 specifically binds to the promoter region of areA with a strong binding affinity to activate the transcription of areA. In contrast, SKO1 showed no specified binding effect on the areA promoter. However, SKO1 activates the expression of the areA by forming a complex with GCN4, which exhibits a 14.2-fold-higher affinity than GCN4 alone. Furthermore, the presence of SKO1 promotes the stability of GCN4 protein. Accordingly, our study found that the transcription factor SKO1 enhances the transcriptional activity of GCN4 on its target gene areA by interacting with GCN4. Our study illustrates a specific regulatory mechanism for the involvement of GCN4 and SKO1 in nitrogen utilization, which provides innovative insight into the regulation of nitrogen utilization in fungi. IMPORTANCE Nitrogen is an essential nutrient for cell growth and proliferation. Limitations of nitrogen availability in organisms elicit a series of rapid transcriptional reprogramming mechanisms, which involve the participation of many transcription factors. However, the specific mechanism of coordination between different transcription factors regulating nitrogen metabolism has not been explored. Our study revealed that GCN4 interacts with SKO1 and that they are both involved in regulating nitrogen utilization by affecting the transcription level of areA. We also found that GCN4 activates transcription by directly binding to the promoter recognition region of areA. SKO1 facilitates the transcription of areA by GCN4 by forming a more stable complex with GCN4. Our study deepens our understanding of the regulatory network of nitrogen metabolism and demonstrates a further level of regulation for transcription factors.

Dioscin ameliorates inflammatory bowel disease by up-regulating miR-125a-5p to regulate macrophage polarization.

PURPOSE: Dioscin has been proven to have anti-cancer, anti-inflammatory, and anti-infection roles. However, the role of Dioscin in inflammatory bowel disease (IBD) and its related mechanisms is unclear and needs further study. METHODS: The colitis model in mice was established. After Dioscin (20, 40, or 80 mg/kg) treatment, the colon length was measured by a ruler. Histopathology, inflammatory cytokines, gut permeability, tight junction proteins, macrophage infiltration, macrophage polarization, and miR-125a-5p level were detected by hematoxylin-eosin staining, enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction (qRT-PCR), FITC-dextran, Western blot, and flow cytometry. In vitro experiments, after RAW264.7 cells induced by lipopolysaccharide (LPS)/interleukin-4 (IL-4), were treated with Dioscin and miR-125a-5p inhibitor, miR-125a-5p level, cell vitality, inflammatory cytokines, and M1/M2 marker genes were measured by qRT-PCR and MTT assay. RESULTS: Dioscin (20, 40, or 80 mg/kg) relieved DSS-triggered colitis and restrained the serum and colon of pro-inflammatory cytokines expression. Meanwhile, different concentrations' Dioscin weakened M1 macrophage polarization but facilitated tight junction protein expressions, M2 macrophage polarization, and miR-125a-5p level in colitic mice. Moreover, miR-125a-5p inhibitor reversed the modulation of Dioscin on miR-125a-5p expression, cell vitality, and inflammatory cytokines in lipopolysaccharide (LPS)-induced RAW264.7 cells. We further discovered that Dioscin restrained M1 marker gene (CD16) expression while intensifying M2 marker genes (CD206 and Arginase-1) expressions in vitro, which was reversed by miR-125a-5p inhibitor. CONCLUSION: Dioscin modulated macrophage polarization by increasing miR-125a-5p, thereby improving the intestinal epithelial barrier function and reducing IBD.

Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy.

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy.

DO-SRS imaging of metabolic dynamics in aging Drosophila.

Emerging studies have shown that lipid metabolism plays an important role in aging. High resolution in situ imaging of lipid metabolic dynamics inside cells and tissues affords a novel and potent approach for understanding many biological processes such as aging. Here we established a new optical imaging platform that combines D2O-probed stimulated Raman scattering (DO-SRS) imaging microscopy and a Drosophila model to directly visualize metabolic activities in situ during aging. The sub-cellular spatial distribution of de novo lipogenesis in the fat body was quantitatively imaged and examined. We discovered a dramatic decrease in lipid turnover in 35-day-old flies. Decreases in protein turnover occurred earlier than lipids (25-day vs. 35-day), and there are many proteins localized on the cell and lipid droplet membrane. This suggests that protein metabolism may act as a prerequisite for lipid metabolism during aging. This alteration of maintenance of protein turnover indicates disrupted lipid metabolism. We further found a significantly higher lipid turnover rate in large LDs, indicating more active metabolism in large LDs, suggesting that large and small LDs play different roles in metabolism to maintain cellular homeostasis. This is the first study that directly visualizes spatiotemporal alterations of lipid (and protein) metabolism in Drosophila during the aging process. Our study not only demonstrates a new imaging platform for studying lipid metabolism, but also unravels the important interconnections between lipid metabolism and aging.

Background-free imaging of chemical bonds by a simple and robust frequency-modulated stimulated Raman scattering microscopy.

Being able to image chemical bonds with high sensitivity and speed, stimulated Raman scattering (SRS) microscopy has made a major impact in biomedical optics. However, it is well known that the standard SRS microscopy suffers from various backgrounds, limiting the achievable contrast, quantification and sensitivity. While many frequency-modulation (FM) SRS schemes have been demonstrated to retrieve the sharp vibrational contrast, they often require customized laser systems and/or complicated laser pulse shaping or introduce additional noise, thereby hindering wide adoption. Herein we report a simple but robust strategy for FM-SRS microscopy based on a popular commercial laser system and regular optics. Harnessing self-phase modulation induced self-balanced spectral splitting of picosecond Stokes beam propagating in standard single-mode silica fibers, a high-performance FM-SRS system is constructed without introducing any additional signal noise. Our strategy enables adaptive spectral resolution for background-free SRS imaging of Raman modes with different linewidths. The generality of our method is demonstrated on a variety of Raman modes with effective suppressing of backgrounds including non-resonant cross phase modulation and electronic background from two-photon absorption or pump-probe process. As such, our method is promising to be adopted by the SRS microscopy community for background-free chemical imaging.

Enhanced stimulated raman scattering of solvent due to anharmonic energy transfer from resonance raman solute molecules.

A new nonlinear optical process, named enhanced stimulated Raman scattering (ESRS), is reported for the first time from resonance Raman in ß-carotene-methanol solution. It is well known that absorption decreases the efficiency of the nonlinear optical and laser processes; however, we observed enhanced stimulated Raman peaks at the first and second Stokes from methanol solvent at 2834 cm-1 with the addition of ß-carotene solutes. This enhanced SRS effect in methanol is attributed to the resonance Raman (RR) process in ß-carotene, which creates a significant number of vibrations from RR and the excess vibrations are transferred to methanol from anharmonic vibrational interactions between the ß-carotene solutes and the methanol solvent, and consequently leads to the increased Raman gain.

Comparison of continuous wave versus picosecond SRS and the resonance SRS effect.

Stimulated Raman scattering (SRS) is a powerful optical technique for probing the vibrational states of molecules in biological tissues and provides greater signal intensities than when using spontaneous Raman scattering. In this study, we examined the use of continuous wave (cw) and picosecond (ps) laser excitations to generate SRS signals in pure methanol, a carotene-methanol solution, acetone, and brain tissue samples. The cw-SRS system, which utilized two cw lasers, produced better signal-to-noise (S/N) than the conventional ps-SRS system, suggesting that the cw-SRS system is an efficient and cost-effective approach for studying SRS in complex systems like the brain. The cw-SRS approach will reduce the size of the SRS system, allowing for stimulated Raman gain/loss microscopy. In addition, we showed that there exists a resonance SRS (RSRS) effect from the carotene-methanol solution and brain tissue samples using cw laser excitations. The RSRS effect will further improve the signal-to-noise and may be utilized as an enhanced, label-free SRS microscopic tool for the study of biological tissues.

Explanation of the competition between O- and E-wave induced stimulated Raman and supercontinuum in calcite under ultrafast laser excitation.

Key optical properties of calcite were measured to unravel the difference between stimulated Raman scattering (SRS) and self-phase modulation (SPM) for the supercontinuum (SC) for ordinary (O) wave and extraordinary (E) wave. These properties are group velocity dispersion, walk-off, spontaneous Raman spectra and cross section, optical 1086cm-1 phonon linewidth, nonlinear susceptibility (χ3), steady-state and transient SRS, and SC caused from SPM. These are investigated for O-waves and E-waves from a 2.7 cm thick calcite crystal. Using 390 fs pulses (∼0.8µJ pulse energy) at 517 nm, the O-wave produced a stronger sharp SRS peak at 1086cm-1 and a weaker SC spectrum in the visible range than the E-wave. The salient difference found between the O- and E-waves for SRS and SPM in calcite is attributed to the larger Raman cross section and the size of nonlinear susceptibility (χ3) for O-waves as compared to E-waves.

Metabolic activity induces membrane phase separation in endoplasmic reticulum.

Membrane phase behavior has been well characterized in model membranes in vitro under thermodynamic equilibrium state. However, the widely observed differences between biological membranes and their in vitro counterparts are placing more emphasis on nonequilibrium factors, including influx and efflux of lipid molecules. The endoplasmic reticulum (ER) is the largest cellular membrane system and also the most metabolically active organelle responsible for lipid synthesis. However, how the nonequilibrium metabolic activity modulates ER membrane phase has not been investigated. Here, we studied the phase behavior of functional ER in the context of lipid metabolism. Utilizing advanced vibrational imaging technique, that is, stimulated Raman scattering microscopy, we discovered that metabolism of palmitate, a prevalent saturated fatty acid (SFA), could drive solid-like domain separation from the presumably uniformly fluidic ER membrane, a previously unknown phenomenon. The potential of various fatty acids to induce solid phase can be predicted by the transition temperatures of their major metabolites. Interplay between saturated and unsaturated fatty acids is also observed. Hence, our study sheds light on cellular membrane biophysics by underscoring the nonequilibrium metabolic status of living cell.

Dual-view holographic three-dimensional display using a single spatial light modulator and a directional light-guide plate composed of pixelated gratings.

In this paper, a dual-view holographic three-dimensional (3D) display using a single spatial light modulator (SLM) and a directional light-guide plate (DLGP) is proposed and implemented. The SLM is used to load the phase-only hologram calculated from two different 3D scenes for optical holographic reconstruction, and the DLGP composed of pixelated gratings with different periods and orientation angles is employed to guide the reconstructed images into two completely separated viewing zones, where different reconstructed perspectives in each viewing zone will form a stereoscopic 3D image. Furthermore, an experimental verification system for the proposed dual-view holographic 3D display is constructed, and the experimental results demonstrate that the proposed system can successfully present different 3D images in the left and right viewing zones simultaneously, verifying the feasibility of the proposed dual-view holographic 3D display.

Binocular holographic three-dimensional display using a single spatial light modulator and a grating.

In this paper, a binocular holographic three-dimensional (3D) display system combining a single spatial light modulator (SLM) and a grating is proposed and implemented. A synthetic phase-only hologram of the left and right 3D perspective images of an object is calculated by the layer-based Fresnel diffraction method according to the depth information, and uploaded onto the SLM for holographic 3D reconstruction with correct depth cues. The grating is designed and fabricated to guide the reconstructed left and right 3D perspective images to the corresponding eyes. Optical experiments demonstrate that the proposed system can successfully present binocular holographic 3D images with both the accommodation effect and binocular parallax, which enables observation free of the accommodation-vergence conflict and visual fatigue problem.

Deep transmission of Laguerre-Gaussian vortex beams through turbid scattering media.

Light scattering and transmission of Gaussian (G) and Laguerre-Gaussian (LG) vortex beams with different orbital angular momentum (L) in various turbid media were investigated. Transmittance was measured with varied ratios of sample thickness (z) to scattering mean free path (ls) of turbid media, z/ls. In the ballistic region, the LG and G beams were found to have no significant difference on transmittance, while in the diffusive region, the LG beams showed a higher received signal than the G beams, and the LG beams with higher L values showed a higher received signal than those with lower L values. The transition points from ballistic to diffusive regions for different scattering media were determined. This newly observed transmittance difference of LG and G beams may be used for deep target detection in turbid media through LG beam imaging.