Click-free imaging of carbohydrate trafficking in live cells using an azido photothermal probe.

Real-time tracking of intracellular carbohydrates remains challenging. While click chemistry allows bio-orthogonal tagging with fluorescent probes, the reaction permanently alters the target molecule and only allows a single snapshot. Here, we demonstrate click-free mid-infrared photothermal (MIP) imaging of azide-tagged carbohydrates in live cells. Leveraging the micromolar detection sensitivity for 6-azido-trehalose (TreAz) and the 300-nm spatial resolution of MIP imaging, the trehalose recycling pathway in single mycobacteria, from cytoplasmic uptake to membrane localization, is directly visualized. A peak shift of azide in MIP spectrum further uncovers interactions between TreAz and intracellular protein. MIP mapping of unreacted azide after click reaction reveals click chemistry heterogeneity within a bacterium. Broader applications of azido photothermal probes to visualize the initial steps of the Leloir pathway in yeasts and the newly synthesized glycans in mammalian cells are demonstrated.

Overtone photothermal microscopy for high-resolution and high-sensitivity vibrational imaging.

Photothermal microscopy is a highly sensitive pump-probe method for mapping nanostructures and molecules through the detection of local thermal gradients. While visible photothermal microscopy and mid-infrared photothermal microscopy techniques have been developed, they possess inherent limitations. These techniques either lack chemical specificity or encounter significant light attenuation caused by water absorption. Here, we present an overtone photothermal (OPT) microscopy technique that offers high chemical specificity, detection sensitivity, and spatial resolution by employing a visible probe for local heat detection in the C-H overtone region. We demonstrate its capability for high-fidelity chemical imaging of polymer nanostructures, depth-resolved intracellular chemical mapping of cancer cells, and imaging of multicellular C. elegans organisms and highly scattering brain tissues. By bridging the gap between visible and mid-infrared photothermal microscopy, OPT establishes a new modality for high-resolution and high-sensitivity chemical imaging. This advancement complements large-scale shortwave infrared imaging approaches, facilitating multiscale structural and chemical investigations of materials and biological metabolism.

Click-free imaging of carbohydrate trafficking in live cells using an azido photothermal probe.

Real-time tracking of intracellular carbohydrates remains challenging. While click chemistry allows bio-orthogonal tagging with fluorescent probes, the reaction permanently alters the target molecule and only allows a single snapshot. Here, we demonstrate click-free mid-infrared photothermal (MIP) imaging of azide-tagged carbohydrates in live cells. Leveraging the micromolar detection sensitivity for 6-azido-trehalose (TreAz) and the 300-nm spatial resolution of MIP imaging, the trehalose recycling pathway in single mycobacteria, from cytoplasmic uptake to membrane localization, is directly visualized. A peak shift of azide in MIP spectrum further uncovers interactions between TreAz and intracellular protein. MIP mapping of unreacted azide after click reaction reveals click chemistry heterogeneity within a bacterium. Broader applications of azido photothermal probes to visualize the initial steps of the Leloir pathway in yeasts and the newly synthesized glycans in mammalian cells are demonstrated.

High-content stimulated Raman histology of human breast cancer.

Histological examination is crucial for cancer diagnosis, however, the labor-intensive sample preparation involved in the histology impedes the speed of diagnosis. Recently developed two-color stimulated Raman histology could bypass the complex tissue processing to generates result close to hematoxylin and eosin staining, which is one of the golden standards in cancer histology. Yet, the underlying chemical features are not revealed in two-color stimulated Raman histology, compromising the effectiveness of prognostic stratification. Here, we present a high-content stimulated Raman histology (HC-SRH) platform that provides both morphological and chemical information for cancer diagnosis based on un-stained breast tissues. Methods: By utilizing both hyperspectral SRS imaging in the C-H vibration window and sparsity-penalized unmixing of overlapped spectral profiles, HC-SRH enabled high-content chemical mapping of saturated lipids, unsaturated lipids, cellular protein, extracellular matrix (ECM), and water. Spectral selective sampling was further implemented to boost the speed of HC-SRH. To show the potential for clinical use, HC-SRH using a compact fiber laser-based stimulated Raman microscope was demonstrated. Harnessing the wide and rapid tuning capability of the fiber laser, both C-H and fingerprint vibration windows were accessed. Results: HC-SRH successfully mapped unsaturated lipids, cellular protein, extracellular matrix, saturated lipid, and water in breast tissue. With these five chemical maps, HC-SRH provided distinct contrast for tissue components including duct, stroma, fat cell, necrosis, and vessel. With selective spectral sampling, the speed of HC-SRH was improved by one order of magnitude. The fiber-laser-based HC-SRH produced the same image quality in the C-H window as the state-of-the-art solid laser. In the fingerprint window, nucleic acid and solid-state ester contrast was demonstrated. Conclusions: HC-SRH provides both morphological and chemical information of tissue in a label-free manner. The chemical information detected is beyond the reach of traditional hematoxylin and eosin staining and heralds the potential of HC-SRH for biomarker discovery.

Determination of N-nitroso folic acid in folic acid and multivitamin supplements by LC-MS/MS.

Analysis of N-nitroso folic acid, a nitrosamine impurity found in folic acid, is challenging due to the complex sample matrices. Many of such supplements contain not only a variety of vitamins, including vitamin A, Bs, C, D and E, but also other ingredients such as minerals, docosahexaenoic acid (DHA), glucose syrup, sugar, and herbs. On the other hand, the strength of folic acid is typically low, ranging from 50 µg to 5 mg per unit. In this study, a highly selective and sensitive LC-MS/MS method was developed to accurately quantify N-nitroso folic acid in supplements containing folic acid. The sample was extracted by 0.1% ammonia solution: MeOH (9:1, v/v) containing 5 ng/mL of N-nitroso folic acid-d4 (Isotope internal standard). The quantification was performed by MRM in negative ionization mode. Mobile phases A and B were 0.1% formic acid in deionized water and methanol, respectively. The method was validated and found to have sufficient linearity (R2 > 0.995), accuracy (recovery 83-110%), precision (RSD 3%) and low LOD, LOQ (4 and 10 µg/g respectively, with respect to folic acid). The method was applied to the determination of N-nitroso folic acid in 40 supplements containing folic acid with different strengths and formulation. The content of N-nitroso folic acid was found to be up to 898 ng/unit (1794 µg/g with respect to folic acid). It enabled regulatory actions, such as product recall, to safeguard public health from unsafe products.

The Parkinson's drug entacapone disrupts gut microbiome homeostasis via iron sequestration.

Increasing evidence shows that many human-targeted drugs alter the gut microbiome, leading to implications for host health. However, much less is known about the mechanisms by which drugs target the microbiome and how drugs affect microbial function. Here we combined quantitative microbiome profiling, long-read metagenomics, stable isotope probing and single-cell chemical imaging to investigate the impact of two widely prescribed nervous system-targeted drugs on the gut microbiome. Ex vivo supplementation of physiologically relevant concentrations of entacapone or loxapine succinate to faecal samples significantly impacted the abundance of up to one third of the microbial species present. Importantly, we demonstrate that the impact of these drugs on microbial metabolism is much more pronounced than their impact on abundances, with low concentrations of drugs reducing the activity, but not the abundance of key microbiome members like Bacteroides, Ruminococcus or Clostridium species. We further demonstrate that entacapone impacts the microbiome due to its ability to complex and deplete available iron, and that microbial growth can be rescued by replenishing levels of microbiota-accessible iron. Remarkably, entacapone-induced iron starvation selected for iron-scavenging organisms carrying antimicrobial resistance and virulence genes. Collectively, our study unveils the impact of two under-investigated drugs on whole microbiomes and identifies metal sequestration as a mechanism of drug-induced microbiome disturbance.

Analytical methods for the detection and characterization of unapproved phosphodiesterase type 5 inhibitors (PDE-5i) used in adulteration of dietary supplements- a review.

This article is an up-to-date review of 112 unapproved phosphodiesterase type 5 inhibitors (PDE-5i) found as adulterants in sexual enhancement dietary supplements and other products from 2003 to July 2023. Seventy-five of these unapproved PDE-5i are analogues of sildenafil (67%), followed by 26 analogues of tadalafil (23%), 9 analogues of vardenafil (8%) and 2 other type of compounds (2%). The products have been formulated in various packaging, primarily in capsule, tablet, and powder forms. Common screening techniques allowing detection of such analogues include high performance or ultra-high performance liquid chromatography in tandem with ultra-violet detector (HPLC-UV or UPLC-UV) (50%) and thin-layer chromatography in tandem with ultra-violet detection (TLC-UV) (7%). Screening by mass spectrometry (MS) is relatively less common with the use of single-, triple-quadrupole or time-of-flight (TOF) mass spectrometers (9%). Meanwhile, the combined detection by UV-MS has been recorded at 10% usage. Screening by proton nuclear magnetic resonance spectroscopy (NMR) (11%) has also been applied. For compound characterization, i.e. structural elucidation, NMR spectroscopy has been preferred (100 out of 112 compounds), followed by high-resolution mass spectrometry (HRMS) (74 out of 112 compounds) and Fourier-transform infrared spectroscopy (FTIR) (44 out of 112 compounds). Over the past two decades, analytical technology has been evolving with enhanced sensitivity and resolution. Despite this, structural elucidation of the new emerging analogues in adulterated dietary supplements remains a challenge, especially when the analogues involve complex structural modification. Therefore, the above-mentioned techniques may not be adequate to characterize the analogues. Additional work involving chiroptical methods, two-dimensional (2D) NMR experiments and X-ray crystallography are likely to be required in the future.

Millimeter-deep micron-resolution vibrational imaging by shortwave infrared photothermal microscopy.

Deep-tissue chemical imaging plays a vital role in biological and medical applications. Here, we present a shortwave infrared photothermal (SWIP) microscope for millimeter-deep vibrational imaging with sub-micron lateral resolution and nanoparticle detection sensitivity. By pumping the overtone transition of carbon-hydrogen bonds and probing the subsequent photothermal lens with shortwave infrared light, SWIP can obtain chemical contrast from polymer particles located millimeter-deep in a highly scattering phantom. By fast digitization of the optically probed signal, the amplitude of the photothermal signal is shown to be 63 times larger than that of the photoacoustic signal, thus enabling highly sensitive detection of nanoscale objects. SWIP can resolve the intracellular lipids across an intact tumor spheroid and the layered structure in millimeter-thick liver, skin, brain, and breast tissues. Together, SWIP microscopy fills a gap in vibrational imaging with sub-cellular resolution and millimeter-level penetration, which heralds broad potential for life science and clinical applications.

Stimulated Raman photothermal microscopy toward ultrasensitive chemical imaging.

Stimulated Raman scattering (SRS) microscopy has shown enormous potential in revealing molecular structures, dynamics, and couplings in complex systems. However, the sensitivity of SRS is fundamentally limited to the millimolar level due to shot noise and the small modulation depth. To overcome this barrier, we revisit SRS from the perspective of energy deposition. The SRS process pumps molecules to their vibrationally excited states. The subsequent relaxation heats up the surroundings and induces refractive index changes. By probing the refractive index changes with a laser beam, we introduce stimulated Raman photothermal (SRP) microscopy, where a >500-fold boost of modulation depth is achieved. The versatile applications of SRP microscopy on viral particles, cells, and tissues are demonstrated. SRP microscopy opens a way to perform vibrational spectroscopic imaging with ultrahigh sensitivity.

High-throughput single-cell sorting by stimulated Raman-activated cell ejection.

Single-cell sorting is essential to explore cellular heterogeneity in biology and medicine. Recently developed Raman-activated cell sorting (RACS) circumvents the limitations of fluorescence-activated cell sorting, such as the cytotoxicity of labels. However, the sorting throughputs of all forms of RACS are limited by the intrinsically small cross-section of spontaneous Raman scattering. Here, we report a stimulated Raman-activated cell ejection (S-RACE) platform that enables high-throughput single-cell sorting based on high-resolution multi-channel stimulated Raman chemical imaging, in situ image decomposition, and laser-induced cell ejection. The performance of this platform was illustrated by sorting a mixture of 1 µm polymer beads, where 95% yield, 98% purity, and 14 events per second throughput were achieved. Notably, our platform allows live cell ejection, allowing for the growth of single colonies of bacteria and fungi after sorting. To further illustrate the chemical selectivity, lipid-rich Rhodotorula glutinis cells were successfully sorted from a mixture with Saccharomyces cerevisiae, confirmed by downstream quantitative PCR. Furthermore, by integrating a closed-loop feedback control circuit into the system, we realized real-time single-cell imaging and sorting, and applied this method to precisely eject regions of interest from a rat brain tissue section. The reported S-RACE platform opens exciting opportunities for a wide range of single-cell applications in biology and medicine.

Stimulated Raman Photothermal Microscopy towards Ultrasensitive Chemical Imaging.

Stimulated Raman scattering (SRS) microscopy has shown enormous potential in revealing molecular structures, dynamics and coupling in a complex system. However, the bond-detection sensitivity of SRS microscopy is fundamentally limited to milli-molar level due to the shot noise and the small modulation depth in either pump or Stokes beam4. Here, to overcome this barrier, we revisit SRS from the perspective of energy deposition. The SRS process pumps molecules to their vibrational excited states. The thereafter relaxation heats up the surrounding and induces a change in refractive index. By probing the refractive index change with a continuous wave beam, we introduce stimulated Raman photothermal (SRP) microscopy, where a >500-fold boost of modulation depth is achieved on dimethyl sulfide with conserved average power. Versatile applications of SRP microscopy on viral particles, cells, and tissues are demonstrated. With much improved signal to noise ratio compared to SRS, SRP microscopy opens a new way to perform vibrational spectroscopic imaging with ultrahigh sensitivity and minimal water absorption.

Analysis of N-nitrosodimethylamine in metformin hydrochloride products by high-resolution accurate mass gas chromatography mass spectrometry.

RATIONALE: The high resolving power of the Orbitrap mass spectrometer in a high-resolution accurate mass gas chromatography (HRAM-GC-MS) system provides greater selectivity and sensitivity for the identification and quantification of volatile analytes at low parts per billion (ppb) levels. Hence, it can be applied for the analysis of pharmaceutical impurities like N-nitrosodimethylamine (NDMA) in metformin hydrochloride products (METs). METHODS: Different METs extracted by a dichloromethane/aqueous system were analyzed by HRAM-GC-MS under softer electron ionization (EI) at 30 eV. The accurate masses of NDMA and its internal standard NDMA-d6 were analyzed by full scan and targeted selected ion monitoring modes under 60 000 and 30 000 full width at half maximum at m/z 200, respectively. Data acquisition and processing were managed by Xcalibur and Trace Finder software, respectively. RESULTS: Limits of detection (LOD) and quantification (LOQ) at 10 and 20 ng/g were achieved, which is below the allowed daily intake of 32 ng/g. The mass errors measured from experimental data were within ±2 ppm of the theoretical values over a period of a week. Sample analysis showed that 180 out of 212 samples (85%) were below LOD and 15 out of 212 samples (7 %) were within LOD and LOQ. Only 17 samples (8%) were found to be above LOQ, comprising one active pharmaceutical ingredient (API), five immediate-release METs and 11 extended-released METs. Amongst these, seven extended-release METs and one API exceeded the daily allowed intake, 32 ng/g. CONCLUSIONS: The validated method has been successfully applied for NDMA analysis in various forms of METs. The method is rather straightforward without an additional clean-up step. The scope can also be extended to other volatile impurities in finished pharmaceutical products.

Optically-generated focused ultrasound for noninvasive brain stimulation with ultrahigh precision.

High precision neuromodulation is a powerful tool to decipher neurocircuits and treat neurological diseases. Current non-invasive neuromodulation methods offer limited precision at the millimeter level. Here, we report optically-generated focused ultrasound (OFUS) for non-invasive brain stimulation with ultrahigh precision. OFUS is generated by a soft optoacoustic pad (SOAP) fabricated through embedding candle soot nanoparticles in a curved polydimethylsiloxane film. SOAP generates a transcranial ultrasound focus at 15 MHz with an ultrahigh lateral resolution of 83 µm, which is two orders of magnitude smaller than that of conventional transcranial-focused ultrasound (tFUS). Here, we show effective OFUS neurostimulation in vitro with a single ultrasound cycle. We demonstrate submillimeter transcranial stimulation of the mouse motor cortex in vivo. An acoustic energy of 0.6 mJ/cm2, four orders of magnitude less than that of tFUS, is sufficient for successful OFUS neurostimulation. OFUS offers new capabilities for neuroscience studies and disease treatments by delivering a focus with ultrahigh precision non-invasively.

Visualization of a Limonene Synthesis Metabolon Inside Living Bacteria by Hyperspectral SRS Microscopy.

Monitoring biosynthesis activity at single-cell level is key to metabolic engineering but is still difficult to achieve in a label-free manner. Using hyperspectral stimulated Raman scattering imaging in the 670-900 cm-1 region, localized limonene synthesis are visualized inside engineered Escherichia coli. The colocalization of limonene and GFP-fused limonene synthase is confirmed by co-registered stimulated Raman scattering and two-photon fluorescence images. The finding suggests a limonene synthesis metabolon with a polar distribution inside the cells. This finding expands the knowledge of de novo limonene biosynthesis in engineered bacteria and highlights the potential of SRS chemical imaging in metabolic engineering research.

SRS-FISH: A high-throughput platform linking microbiome metabolism to identity at the single-cell level.

One of the biggest challenges in microbiome research in environmental and medical samples is to better understand functional properties of microbial community members at a single-cell level. Single-cell isotope probing has become a key tool for this purpose, but the current detection methods for determination of isotope incorporation into single cells do not allow high-throughput analyses. Here, we report on the development of an imaging-based approach termed stimulated Raman scattering-two-photon fluorescence in situ hybridization (SRS-FISH) for high-throughput metabolism and identity analyses of microbial communities with single-cell resolution. SRS-FISH offers an imaging speed of 10 to 100 ms per cell, which is two to three orders of magnitude faster than achievable by state-of-the-art methods. Using this technique, we delineated metabolic responses of 30,000 individual cells to various mucosal sugars in the human gut microbiome via incorporation of deuterium from heavy water as an activity marker. Application of SRS-FISH to investigate the utilization of host-derived nutrients by two major human gut microbiome taxa revealed that response to mucosal sugars tends to be dominated by Bacteroidales, with an unexpected finding that Clostridia can outperform Bacteroidales at foraging fucose. With high sensitivity and speed, SRS-FISH will enable researchers to probe the fine-scale temporal, spatial, and individual activity patterns of microbial cells in complex communities with unprecedented detail.

Isolation and characterization of N-phenyl propoxyphenyl carbodenafil from an illegal coffee powder product.

A new carbodenafil-like compound, N-phenyl propoxyphenyl carbodenafil has been identified from an illegal coffee powder product. It was isolated using a semi-preparative liquid chromatography column. The presence of a propoxy group at the aryl alkyl ether moiety, and the direct bonding of a phenyl group to piperazine ring have been unambiguously characterized by ultra-violet (UV), Fourier transform infrared (FTIR), high-resolution mass spectrometry (HRMS) and nuclear magnet resonance (NMR) analysis.

The Moderation of Human Characteristics in the Control Mechanisms of Rumours in Social Media: The Case of Food Rumours in China.

Social networks are widely used as a fast and ubiquitous information-sharing medium. The mass spread of food rumours has seriously invaded public's healthy life and impacted food production. It can be argued that the government, companies, and the media have the responsibility to send true anti-rumour messages to reduce panic, and the risks involved in different forms of communication to the public have not been properly assessed. The manuscript develops an empirical analysis model from 683 food anti-rumour cases and 7,967 data of the users with top comments to test the influence of the strength of rumour/anti-rumour on rumour control. Furthermore, dividing the users into three categories, Leaders, Chatters, and General Public, and study the influence of human characteristics on the relationship between the strength of rumour/anti-rumour and rumour control by considering the different human characteristics as moderator variables. The results showed that anti-rumours have a significant positive impact on the control of rumours; the ambiguity of rumours has a significant negative impact on the Positive Comment Index (PCI) in rumour control. Further, the Leaders increased the overall level of PCI, but negatively adjusted the relationship between evidence and PCI; the Chatters and the General Public reduced the overall level of PCI, and Chatters weakened the relationship between the specific type of anti-rumour form and PCI while the General Public enhanced the relationship between the specific type of anti-rumour form and PCI. In the long run, the role of Leaders needs to be further improved, and the importance of the General Public is growing in the food rumour control process.

All-normal dispersion fiber laser with a bandwidth tunable fiber-based spectral filter.

We investigate numerically and experimentally an all-fiber, bandwidth tunable spectral filter comprising birefringent fibers. The spectral bandwidth tunability of the filter is based on the compensation of birefringence in polarization maintaining fibers. This unique filter allows mode-locked operation of a fiber oscillator with the ability to generate distinct laser modes with different output spectral shapes and pulse evolutions.

Isolation and characterization of N-hydroxyethyl dithio-desethyl carbodenafil from a health supplement.

A new phosphodiesterase type-5 inhibitor (PDE-5i) with thiocarbonyl and thiolactam skeleton has been identified. The unknown compound has very similar properties like dithio-desmethyl carbodenafil, which was detected alongside during the screening process. It has been isolated by a semi-preparative high performance liquid chromatography tandem ultra-violet detector (HPLC-UV). The purified compound has been characterized using Fourier-transform infrared spectroscopy (FTIR), high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy (NMR). It is named as N-hydroxyethyl dithio-desethyl carbodenafil due to attachment of a hydroxyethyl group to the heterocyclic nitrogen of dithio-desethyl carbodenafil.

Elucidation of the absolute configuration of a tadalafil analogue found as adulterant in a health supplement by mass spectrometry, chiroptical methods and NMR spectroscopy.

The chirality of a dipropylaminopretadalafil stereoisomer isolated from a health supplement has been studied. Under high resolution mass spectrometry (HRMS) study, this unknown compound seems to be one of the trans configuration tadalafil analogues i.e. (6S, 12aR) or (6R, 12aS), owing to the same precursor ion at m/z 492 with mass errors within ±2 ppm tolerance and very close retention times. Moreover, the MS2 fragmentation pattern is also very similar to the two trans isomers. Fortunately, the unknown compound can be distinguished from the two trans isomers by enantioselective separation with the use of a chiral column. Further comparison studies with a series of homologous compounds without a diketopiperazine ring on ellipticity and optical rotation support the unknown compound to be in the cis-(6R, 12aR) configuration. The nuclear magnetic resonance (NMR) in one dimensional (1D) and nuclear overhauser effect spectroscopy (NOESY) have affirmed the abovementioned configuration.