Metformin Preconditioning Augments Cardiac Perfusion and Performance in a Large Animal Model of Chronic Coronary Artery Disease.

OBJECTIVE: To test the efficacy of metformin (MET) during the induction of coronary ischemia on myocardial performance in a large animal model of coronary artery disease (CAD) and metabolic syndrome (MS), with or without concomitant extracellular vesicular (EV) therapy. BACKGROUND: Although surgical and endovascular revascularization are durably efficacious for many patients with CAD, up to one-third are poor candidates for standard therapies. For these patients, many of whom have comorbid MS, adjunctive strategies are needed. EV therapy has shown promise in this context, but its efficacy is attenuated by MS. We investigated whether MET pretreatment could ameliorate therapeutic decrements associated with MS. METHODS: Yorkshire swine (n = 29) were provided a high-fat diet to induce MS, whereupon an ameroid constrictor was placed to induce CAD. Animals were initiated on 1000 mg oral MET or placebo; all then underwent repeat thoracotomy for intramyocardial injection of EVs or saline. Swine were maintained for 5 weeks before the acquisition of functional and perfusion data immediately before terminal myocardial harvest. Immunoblotting and immunofluorescence were performed on the most ischemic tissue from all groups. RESULTS: Regardless of EV administration, animals that received MET exhibited significantly improved ejection fraction, cardiac index, and contractility at rest and during rapid myocardial pacing, improved perfusion to the most ischemic myocardial region at rest and during pacing, and markedly reduced apoptosis. CONCLUSIONS: MET administration reduced apoptotic cell death, improved perfusion, and augmented both intrinsic and load-dependent myocardial performance in a highly translatable large animal model of chronic myocardial ischemia and MS.

A high-density microfluidic bioreactor for the automated manufacturing of CAR T cells.

The manufacturing of autologous chimaeric antigen receptor (CAR) T cells largely relies either on fed-batch and manual processes that often lack environmental monitoring and control or on bioreactors that cannot be easily scaled out to meet patient demands. Here we show that human primary T cells can be activated, transduced and expanded to high densities in a 2 ml automated closed-system microfluidic bioreactor to produce viable anti-CD19 CAR T cells (specifically, more than 60 million CAR T cells from donor cells derived from patients with lymphoma and more than 200 million CAR T cells from healthy donors). The in vitro secretion of cytokines, the short-term cytotoxic activity and the long-term persistence and proliferation of the cell products, as well as their in vivo anti-leukaemic activity, were comparable to those of T cells produced in a gas-permeable well. The manufacturing-process intensification enabled by the miniaturized perfusable bioreactor may facilitate the analysis of the growth and metabolic states of CAR T cells during ex vivo culture, the high-throughput optimization of cell-manufacturing processes and the scale out of cell-therapy manufacturing.

Precise and High-Throughput Delivery of Micronutrients in Plants Enabled by Pollen-Inspired Spiny and Biodegradable Microcapsules.

Decarbonizing food production and mitigating agriculture's environmental impact require new technologies for precise delivery of fertilizers and pesticides to plants. The cuticle, a waxy barrier that protects the surface of leaves, causes 60%-90% runoff of fertilizers and pesticides, leading to the wastage of intensive resources, soil depletion, and water bodies pollution. Solutions to mitigate runoff include adding chemicals (e.g., surfactants) to decrease surface tension and enhance cuticles' permeability but have low efficacy. In this study, vapor-induced synergistic differentiation (VISDi) is used to nanomanufacture echinate pollen-like, high payload content (≈50 wt%) microcapsules decorated with robust spines that mechanically disrupt the cuticle and adhere to the leaf. VISDi induces a core-shell structure in the spines, enabling the release of agrochemicals from the microparticles' body into the leaf. As proof of concept, precise and highthroughput delivery of iron fertilizer in Fe-deficient spinach plants is demonstrated. Spray of spiny microparticles improves leaf adhesion by mechanical interlocking, reduces wash-off by an ≈12.5 fold, and enhances chlorophyll content by ≈7.3 times compared to the application of spherical counterparts. Together, these results show that spiny microparticles can mitigate agricultural runoff and provide a high-throughput tool for precise plant drug delivery.

Harnessing Raman spectroscopy for the analysis of plant diversity.

Here, we explore the application of Raman spectroscopy for the assessment of plant biodiversity. Raman spectra from 11 vascular plant species commonly found in forest ecosystems, specifically angiosperms (both monocots and eudicots) and pteridophytes (ferns), were acquired in vivo and in situ using a Raman leaf-clip. We achieved an overall accuracy of 91% for correct classification of a species within a plant group and identified lignin Raman spectral features as a useful discriminator for classification. The results demonstrate the potential of Raman spectroscopy in contributing to plant biodiversity assessment.

Effects of Storage Temperatures on Nitrogen Assimilation and Remobilization during Post-Harvest Senescence of Pak Choi.

In the agricultural industry, the post-harvest leafy vegetable quality and shelf life significantly influence market value and consumer acceptability. This study examined the effects of different storage temperatures on leaf senescence, nitrogen assimilation, and remobilization in Pak Choi (Brassica rapa subsp. chinensis). Mature Pak Choi plants were harvested and stored at two different temperatures, 4 °C and 25 °C. Senescence was tracked via chlorophyll content and leaf yellowing. Concurrently, alterations in the total nitrogen, nitrate, and protein content were quantified on days 0, 3, 6, and 9 in old, mid, and young leaves of Pak Choi plants. As expected, 4 °C alleviated chlorophyll degradation and delayed senescence of Pak Choi compared to 25 °C. Total nitrogen and protein contents were inversely correlated, while the nitrate content remained nearly constant across leaf groups at 25 °C. Additionally, the transcript levels of genes involved in nitrogen assimilation and remobilization revealed key candidate genes that were differentially expressed between 4 °C and 25 °C, which might be targeted to extend the shelf life of the leafy vegetables. Thus, this study provides pivotal insights into the molecular and physiological responses of Pak Choi to post-harvest storage conditions.

On-site growth of perovskite nanocrystal arrays for integrated nanodevices.

Despite remarkable progress in the development of halide perovskite materials and devices, their integration into nanoscale optoelectronics has been hindered by a lack of control over nanoscale patterning. Owing to their tendency to degrade rapidly, perovskites suffer from chemical incompatibility with conventional lithographic processes. Here, we present an alternative, bottom-up approach for precise and scalable formation of perovskite nanocrystal arrays with deterministic control over size, number, and position. In our approach, localized growth and positioning is guided using topographical templates of controlled surface wettability through which nanoscale forces are engineered to achieve sub-lithographic resolutions. With this technique, we demonstrate deterministic arrays of CsPbBr3 nanocrystals with tunable dimensions down to <50 nm and positional accuracy <50 nm. Versatile, scalable, and compatible with device integration processes, we then use our technique to demonstrate arrays of nanoscale light-emitting diodes, highlighting the new opportunities that this platform offers for perovskites' integration into on-chip nanodevices.

Patterned Arteriole-Scale Vessels Enhance Engraftment, Perfusion, and Vessel Branching Hierarchy of Engineered Human Myocardium for Heart Regeneration.

Heart regeneration after myocardial infarction (MI) using human stem cell-derived cardiomyocytes (CMs) is rapidly accelerating with large animal and human clinical trials. However, vascularization methods to support the engraftment, survival, and development of implanted CMs in the ischemic environment of the infarcted heart remain a key and timely challenge. To this end, we developed a dual remuscularization-revascularization therapy that is evaluated in a rat model of ischemia-reperfusion MI. This study details the differentiation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for engineering cardiac tissue containing patterned engineered vessels 400 µm in diameter. Vascularized engineered human myocardial tissues (vEHMs) are cultured in static conditions or perfused in vitro prior to implantation and evaluated after two weeks. Immunohistochemical staining indicates improved engraftment of hiPSC-CMs in in vitro-perfused vEHMs with greater expression of SMA+ vessels and evidence of inosculation. Three-dimensional vascular reconstructions reveal less tortuous and larger intra-implant vessels, as well as an improved branching hierarchy in in vitro-perfused vEHMs relative to non-perfused controls. Exploratory RNA sequencing of explanted vEHMs supports the hypothesis that co-revascularization impacts hiPSC-CM development in vivo. Our approach provides a strong foundation to enhance vEHM integration, develop hierarchical vascular perfusion, and maximize hiPSC-CM engraftment for future regenerative therapy.

Nanosecond pulsed CMOS LED for all-silicon time-of-flight ranging.

Light detection and ranging (LIDAR) is a widely used technique for measuring distance. With recent advancements in integrated photonics, there is a growing interest in miniaturizing LIDAR systems through on-chip photonic devices, but a LIDAR light source compatible with current integrated circuit technology remains elusive. In this letter, we report a pulsed CMOS LED based on native Si, which spectrally overlaps with Si detectors' responsivity and can produce optical pulses as short as 1.6 ns. A LIDAR prototype is built by incorporating this LED and a Si single-photon avalanche diode (SPAD). By utilizing time-correlated single-photon counting (TCSPC) to measure the time-of-flight (ToF) of reflected optical pulses, our LIDAR successfully estimated the distance of targets located approximately 30 cm away with sub-centimeter resolution, approaching the Cramér-Rao lower bound set by the pulse width and instrument jitter. Additionally, our LIDAR is capable of generating depth images of natural targets. This all-Si LIDAR demonstrates the feasibility of integrated distance sensors on a single photonic chip.

One Billion hiPSC-Cardiomyocytes: Upscaling Engineered Cardiac Tissues to Create High Cell Density Therapies for Clinical Translation in Heart Regeneration.

Despite the overwhelming use of cellularized therapeutics in cardiac regenerative engineering, approaches to biomanufacture engineered cardiac tissues (ECTs) at clinical scale remain limited. This study aims to evaluate the impact of critical biomanufacturing decisions-namely cell dose, hydrogel composition, and size-on ECT formation and function-through the lens of clinical translation. ECTs were fabricated by mixing human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) and human cardiac fibroblasts into a collagen hydrogel to engineer meso-(3 × 9 mm), macro- (8 × 12 mm), and mega-ECTs (65 × 75 mm). Meso-ECTs exhibited a hiPSC-CM dose-dependent response in structure and mechanics, with high-density ECTs displaying reduced elastic modulus, collagen organization, prestrain development, and active stress generation. Scaling up, cell-dense macro-ECTs were able to follow point stimulation pacing without arrhythmogenesis. Finally, we successfully fabricated a mega-ECT at clinical scale containing 1 billion hiPSC-CMs for implantation in a swine model of chronic myocardial ischemia to demonstrate the technical feasibility of biomanufacturing, surgical implantation, and engraftment. Through this iterative process, we define the impact of manufacturing variables on ECT formation and function as well as identify challenges that must still be overcome to successfully accelerate ECT clinical translation.

A sub-wavelength Si LED integrated in a CMOS platform.

A nanoscale on-chip light source with high intensity is desired for various applications in integrated photonics systems. However, it is challenging to realize such an emitter using materials and fabrication processes compatible with the standard integrated circuit technology. In this letter, we report an electrically driven Si light-emitting diode with sub-wavelength emission area fabricated in an open-foundry microelectronics complementary metal-oxide-semiconductor platform. The light-emitting diode emission spectrum is centered around 1100 nm and the emission area is smaller than 0.14 µm2 (~[Formula: see text] nm). This light-emitting diode has high spatial intensity of >50 mW/cm2 which is comparable with state-of-the-art Si-based emitters with much larger emission areas. Due to sub-wavelength confinement, the emission exhibits a high degree of spatial coherence, which is demonstrated by incorporating the light-emitting diode into a compact lensless in-line holographic microscope. This centimeter-scale, all-silicon microscope utilizes a single emitter to simultaneously illuminate ~9.5 million pixels of a complementary metal-oxide-semiconductor imager.

Rapid Detection and Quantification of Plant Innate Immunity Response Using Raman Spectroscopy.

We have developed a rapid Raman spectroscopy-based method for the detection and quantification of early innate immunity responses in Arabidopsis and Choy Sum plants. Arabidopsis plants challenged with flg22 and elf18 elicitors could be differentiated from mock-treated plants by their Raman spectral fingerprints. From the difference Raman spectrum and the value of p at each Raman shift, we derived the Elicitor Response Index (ERI) as a quantitative measure of the response whereby a higher ERI value indicates a more significant elicitor-induced immune response. Among various Raman spectral bands contributing toward the ERI value, the most significant changes were observed in those associated with carotenoids and proteins. To validate these results, we investigated several characterized Arabidopsis pattern-triggered immunity (PTI) mutants. Compared to wild type (WT), positive regulatory mutants had ERI values close to zero, whereas negative regulatory mutants at early time points had higher ERI values. Similar to elicitor treatments, we derived an analogous Infection Response Index (IRI) as a quantitative measure to detect the early PTI response in Arabidopsis and Choy Sum plants infected with bacterial pathogens. The Raman spectral bands contributing toward a high IRI value were largely identical to the ERI Raman spectral bands. Raman spectroscopy is a convenient tool for rapid screening for Arabidopsis PTI mutants and may be suitable for the noninvasive and early diagnosis of pathogen-infected crop plants.

Computational polarized Raman microscopy on sub-surface nanostructures with sub-diffraction-limit resolution.

Raman microscopy with resolution below the diffraction limit is demonstrated on sub-surface nanostructures. Unlike most other modalities for nanoscale measurements, our approach is able to image nanostructures buried several microns below the sample surface while still extracting details about the chemistry, strain, and temperature of the nanostructures. In this work, we demonstrate that combining polarized Raman microscopy adjusted to optimize edge enhancement effects and nanostructure contrast with fast computational deconvolution methods can improve the spatial resolution while preserving the flexibility of Raman microscopy. The cosine transform method demonstrated here enables significant computational speed-up from O(N3) to O(Nlog N) - resulting in computation times that are significantly below the image acquisition time. CMOS poly-Si nanostructures buried below 0.3 - 6 µm of complex dielectrics are used to quantify the performance of the instrument and the algorithm. The relative errors of the feature sizes, the relative chemical concentrations and the fill factors of the deconvoluted images are all approximately 10% compared with the ground truth. For the smallest poly-Si feature of 230 nm, the absolute error is approximately 25 nm.

Low-power swept-source Raman spectroscopy.

'Molecular fingerprinting' with Raman spectroscopy can address important problems-from ensuring our food safety, detecting dangerous substances, to supporting disease diagnosis and management. However, the broad adoption of Raman spectroscopy demands low-cost, portable instruments that are sensitive and use lasers that are safe for human eye and skin. This is currently not possible with existing Raman spectroscopy approaches. Portability has been achieved with dispersive Raman spectrometers, however, fundamental entropic limits to light collection both limits sensitivity and demands high-power lasers and cooled expensive detectors. Here, we demonstrate a swept-source Raman spectrometer that improves light collection efficiency by up to 1000× compared to portable dispersive spectrometers. We demonstrate high detection sensitivity with only 1.5 mW average excitation power and an uncooled amplified silicon photodiode. The low optical power requirement allowed us to utilize miniature chip-scale MEMS-tunable lasers with close to eye-safe optical powers for excitation. We characterize the dynamic range and spectral characteristics of this Raman spectrometer in detail, and use it for fingerprinting of different molecular species consumed everyday including analgesic tablets, nutrients in vegetables, and contaminated alcohol. By moving the complexity of Raman spectroscopy from bulky spectrometers to chip-scale light sources, and by replacing expensive cooled detectors with low-cost uncooled alternatives, this swept-source Raman spectroscopy technique could make molecular fingerprinting more accessible.

A predictive in vitro risk assessment platform for pro-arrhythmic toxicity using human 3D cardiac microtissues.

Cardiotoxicity of pharmaceutical drugs, industrial chemicals, and environmental toxicants can be severe, even life threatening, which necessitates a thorough evaluation of the human response to chemical compounds. Predicting risks for arrhythmia and sudden cardiac death accurately is critical for defining safety profiles. Currently available approaches have limitations including a focus on single select ion channels, the use of non-human species in vitro and in vivo, and limited direct physiological translation. We have advanced the robustness and reproducibility of in vitro platforms for assessing pro-arrhythmic cardiotoxicity using human induced pluripotent stem cell-derived cardiomyocytes and human cardiac fibroblasts in 3-dimensional microtissues. Using automated algorithms and statistical analyses of eight comprehensive evaluation metrics of cardiac action potentials, we demonstrate that tissue-engineered human cardiac microtissues respond appropriately to physiological stimuli and effectively differentiate between high-risk and low-risk compounds exhibiting blockade of the hERG channel (E4031 and ranolazine, respectively). Further, we show that the environmental endocrine disrupting chemical bisphenol-A (BPA) causes acute and sensitive disruption of human action potentials in the nanomolar range. Thus, this novel human 3D in vitro pro-arrhythmic risk assessment platform addresses critical needs in cardiotoxicity testing for both environmental and pharmaceutical compounds and can be leveraged to establish safe human exposure levels.

Tissues with Patterned Vessels or Protein Release Induce Vascular Chemotaxis in an In Vitro Platform.

Engineered tissues designed for translational applications in regenerative medicine require vascular networks to deliver oxygen and nutrients rapidly to the implanted cells. A limiting factor of in vivo translation is the rapid and successful inosculation, or connection, of host and implanted vascular networks and subsequent perfusion of the implant. An approach gaining favor in vascular tissue engineering is to provide instructive cues from the engineered tissue to enhance host vascular penetration and connection with the implant. Here, we use a novel in vitro platform based on the aortic ring assay to evaluate the impact of patterned, endothelialized vessels or growth factor release from engineered constructs on preinosculative vascular cell outgrowth from surrogate host tissue in a controlled, defined environment, and introduce robust tools for evaluating vascular morphogenesis and chemotaxis. We demonstrate the creation of engineered vessels at the arteriole scale, which develop basement membrane, exhibit tight junctions, and actively sprout into the surrounding bulk hydrogel. Vessel-containing constructs are co-cultured adjacent to rodent aortic rings, and the resulting heterocellular outgrowth is quantified. Cells originating from the aortic ring migrate preferentially toward constructs containing engineered vessels with 1.5-fold faster outgrowth kinetics, 2.5-fold increased cellular density, and 1.6-fold greater network formation versus control (no endothelial cells and growth factor-reduced culture medium). Growth factor release from constructs with nonendothelialized channels and in reduced factor medium equivalently stimulates sustained vascular outgrowth distance, cellular density, and network formation, akin to engineered vessels in endothelial growth medium 2 (EGM-2) medium. In conclusion, we show that three-dimensional endothelialized patterned vessels or growth factor release stimulate a robust, host-derived vascular cell chemotactic response at early time points critical for instructive angiogenic cues. Further, we developed robust, unbiased tools to quantify metrics of vascular morphogenesis and preinosculative heterocellular outgrowth from rat aortic rings and demonstrated the utility of our complex, controlled environment, heterocellular in vitro platform. Impact statement Using a novel in vitro platform, we show that engineered constructs with patterned vessels or angiogenic growth factor release, two methods of instructing host revascularization responses, equivalently improve early host-derived vascular outgrowth. Our platform leverages the aortic ring assay in a tissue engineering context to study preinosculative vascular cell chemotaxis from surrogate host vascular cells in response to paracrine cues from co-cultured engineered tissues using robust, open-source quantification tools. Our accessible and flexible platform enables translationally focused studies in revascularization using implantable therapeutics containing prepatterned vessels with greater environmental control than in vivo studies to advance vascular tissue engineering.

Risky Riding and Its Correlates in Two-Wheeler Riding Young Men: Pillion Riders' Perspective.

BACKGROUND: Risky riding is one of the major contributing factors in road fatalities. The present study aimed to explore the risky riding behaviors and its correlates in two-wheeler riding young men, as ascertained from pillion riders' perspective. MATERIALS AND METHODS: A survey that captured perspective of pillion riders about two-wheeler riding young men with whom they used to pillion ride most frequently was administered on 115 subjects. The survey consisted of items pertaining to risky riding, perceived impact of negative emotion on riding, expression of negative emotion on roads in response to frustrating situations, road traffic accidents, and pillions' strategies to reduce anger/stress in their two-wheeler riders. RESULTS: Two-wheeler riders who comprised young men were categorized into two groups: (i) high-risk riding group (n = 54 [48%]) and (ii) low-risk riding group (n = 61 [52%]) based on the subjective report of risky riding behaviors by their pillion riders. The results showed that negative emotions were perceived to have adverse influence on riding in persons with high-risky riding. Pillion riders reported that two-wheeler riding young men with high-risky riding expressed more aggressive behaviors (verbal and nonverbal) while riding in response to frustrating situations and also experienced near misses and minor accidents more frequently than their counterparts. Pillion riders reported utilizing various strategies to regulate emotions and behaviors of two-wheeler riders. CONCLUSIONS: The present study highlights assessing risky riding and their correlates from pillion riders' perspective and strengthening their positive influence on two-wheeler riding. It has significant implications in minimizing risky behaviors on roads and enhancing road safety.

Species-independent analytical tools for next-generation agriculture.

Innovative approaches are urgently required to alleviate the growing pressure on agriculture to meet the rising demand for food. A key challenge for plant biology is to bridge the notable knowledge gap between our detailed understanding of model plants grown under laboratory conditions and the agriculturally important crops cultivated in fields or production facilities. This Perspective highlights the recent development of new analytical tools that are rapid and non-destructive and provide tissue-, cell- or organelle-specific information on living plants in real time, with the potential to extend across multiple species in field applications. We evaluate the utility of engineered plant nanosensors and portable Raman spectroscopy to detect biotic and abiotic stresses, monitor plant hormonal signalling as well as characterize the soil, phytobiome and crop health in a non- or minimally invasive manner. We propose leveraging these tools to bridge the aforementioned fundamental gap with new synthesis and integration of expertise from plant biology, engineering and data science. Lastly, we assess the economic potential and discuss implementation strategies that will ensure the acceptance and successful integration of these modern tools in future farming practices in traditional as well as urban agriculture.

Impact of laser frequency noise on high-extinction optical modulation.

In present literature on integrated modulation and filtering, limitations in the extinction ratio are dominantly attributed to a combination of imbalance in interfering wave amplitude, instability of control signals, stray light (e.g., in the cladding), or amplified spontaneous emission from optical amplifiers. Here we show that the existence of optical frequency noise in single longitudinal mode lasers presents an additional limit to the extinction ratio of optical modulators. A simple frequency-domain model is used to describe a linear optical system's response in the presence of frequency noise, and an intuitive picture is given for systems with arbitrary sampling time. Understanding the influence of frequency noise will help guide the design choices of device and system engineers and offer a path toward even higher-extinction optical modulators.

Portable Raman leaf-clip sensor for rapid detection of plant stress.

Precision agriculture requires new technologies for rapid diagnosis of plant stresses, such as nutrient deficiency and drought, before the onset of visible symptoms and subsequent yield loss. Here, we demonstrate a portable Raman probe that clips around a leaf for rapid, in vivo spectral analysis of plant metabolites including carotenoids and nitrates. We use the leaf-clip Raman sensor for early diagnosis of nitrogen deficiency of the model plant Arabidopsis thaliana as well as two important vegetable crops, Pak Choi (Brassica rapa chinensis) and Choy Sum (Brassica rapa var. parachinensis). In vivo measurements using the portable leaf-clip Raman sensor under full-light growth conditions were consistent with those obtained with a benchtop Raman spectrometer measurements on leaf-sections under laboratory conditions. The portable leaf-clip Raman sensor offers farmers and plant scientists a new precision agriculture tool for early diagnosis and real-time monitoring of plant stresses in field conditions.

Rapid metabolite response in leaf blade and petiole as a marker for shade avoidance syndrome.

BACKGROUND: Shade avoidance syndrome (SAS) commonly occurs in plants experiencing vegetative shade, causing morphological and physiological changes that are detrimental to plant health and consequently crop yield. As the effects of SAS on plants are irreversible, early detection of SAS in plants is critical for sustainable agriculture. However, conventional methods to assess SAS are restricted to observing for morphological changes and checking the expression of shade-induced genes after homogenization of plant tissues, which makes it difficult to detect SAS early. RESULTS: Using the model plant Arabidopsis thaliana, we introduced the use of Raman spectroscopy to measure shade-induced changes of metabolites in vivo. Raman spectroscopy detected a decrease in carotenoid contents in leaf blades and petioles of plants with SAS, which were induced by low Red:Far-red light ratio or high density conditions. Moreover, by measuring the carotenoid Raman peaks, we were able to show that the reduction in carotenoid content under shade was mediated by phytochrome signaling. Carotenoid Raman peaks showed more remarkable response to SAS in petioles than leaf blades of plants, which greatly corresponded to their morphological response under shade or high plant density. Most importantly, carotenoid content decreased shortly after shade induction but before the occurrence of visible morphological changes. We demonstrated this finding to be similar in other plant species. Comprehensive testing of Brassica vegetables showed that carotenoid content decreased during SAS, in both shade and high density conditions. Likewise, carotenoid content responded quickly to shade, in a manner similar to Arabidopsis plants. CONCLUSIONS: In various plant species tested in this study, quantification of carotenoid Raman peaks correlate to the severity of SAS. Moreover, short-term exposure to shade can induce the carotenoid Raman peaks to decrease. These findings highlight the carotenoid Raman peaks as a biomarker for early diagnosis of SAS in plants.