High-Throughput Antimicrobial Susceptibility Testing of Escherichia coli by Wide-Field Mid-Infrared Photothermal Imaging of Protein Synthesis.

Antimicrobial resistance poses great threats to global health and economics. Current gold-standard antimicrobial susceptibility testing (AST) requires extensive culture time (36-72 h) to determine susceptibility. There is an urgent need for rapid AST methods to slow down antimicrobial resistance. Here, we present a rapid AST method based on wide-field mid-infrared photothermal imaging of protein synthesis from 13C-glucose in Escherichia coli. Our wide-field approach achieved metabolic imaging for hundreds of bacteria at the single-cell resolution within seconds. The perturbed microbial protein synthesis can be probed within 1 h after antibiotic treatment in E. coli cells. The susceptibility of antibiotics with various mechanisms of action has been probed through monitoring protein synthesis, which promises great potential of the proposed platform toward clinical translation.

A rapid procedure for bacterial identification and antimicrobial susceptibility testing directly from positive blood cultures.

There is an urgent need to develop a rapid procedure that can rapidly identify and obtain antimicrobial susceptibility testing (AST) results directly from positive blood cultures. Here, we report a semi-automatic bacterial diagnosis procedure, which includes (1) a bacterial concentration process to isolate bacteria from a positive blood culture bottle (PBCB), (2) an identification process using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and (3) a rapid AST process based on stimulated Raman scattering imaging of deuterium oxide (D2O) incorporation in bacteria. A total of 105 samples were tested for bacterial identification, and a bacterial identification accuracy of 92.3% was achieved. AST takes about 2.5 h after identification. This semi-automatic procedure only takes 3.5 h, which is demonstrated to be the fastest process to obtain identification and AST results starting from PBCBs.

Quantification of Lipid Metabolism in Living Cells through the Dynamics of Lipid Droplets Measured by Stimulated Raman Scattering Imaging.

Dysregulation of lipid metabolism is associated with many diseases including cancer. Lipid droplet (LD), a ubiquitous organelle in mammalian cells, serves as a hub for lipid metabolism. Conventional assays on the measurement of lipid metabolism rely on the quantification of the lipid composition or amount. Such methods cannot distinguish LDs having different biofunctionalities in living cells, and thus could be inaccurate in measuring the instantaneous lipogenesis of the living cells. We applied label-free stimulated Raman scattering microscopy to quantify the LDs' spatial-temporal dynamics, which showed direct links to cellular lipid metabolisms and can separate LDs involved in different metabolic events. In human cancer cells, we found that changes in the maximum displacement of LDs reflected variations in cellular lipogenic activity, and changes in the average speed of LDs revealed alterations in LD size. The LD dynamics analysis allowed for more accurate measurement in the lipogenesis and LD dimensions, and can break the optical diffraction limit to detect small variation in lipid metabolism that was conventionally undetectable. By this method, we revealed changes in the lipogenic activity and LD sizes during glucose starvation of HeLa cells and transforming growth factor beta-induced epithelial-to-mesenchymal transition of SKOV-3 cells. This method opens a way to quantify lipid metabolism in living cells during cellular development and transition.

Nanocellulose-based antimicrobial aerogels with humidity-triggered release of cinnamaldehyde.

Active food packaging with controlled release behavior of volatile antimicrobials is highly desirable for enhancing the quality of fresh produce. In this study, humidity-responsive antimicrobial aerogels were developed using chitosan and dialdehyde nanocellulose, loading with cyclodextrin-cinnamaldehyde inclusion complexes (ICs) for achieving humidity-triggered release of the encapsulated antimicrobial agent. Results showed that the prepared aerogels had capable water absorption ability, which could be served as absorbent pads to take in excessive exudate from packaged fresh produce. More importantly, the accumulative release rate of cinnamaldehyde from the antimicrobial aerogels was significantly improved at RH 98 % compared to that at RH 70 %, which accordingly inactivated all the inoculated Escherichia coli, Staphylococcus aureus and Botrytis cinerea. Additionally, strawberries packaged with the antimicrobial aerogels remained in good conditions after 5 d of storage at 22 ± 1 °C. The prepared composite aerogels had the potential to extend the shelf life of fresh strawberries.

Wireless neuromodulation at submillimeter precision via a microwave split-ring resonator.

A broad spectrum of electromagnetic waves has been explored for wireless neuromodulation. Transcranial magnetic stimulation, with long wavelengths, cannot provide submillimeter spatial resolution. Visible light, with its short wavelengths, suffers from strong scattering in the deep tissue. Microwaves have centimeter-scale penetration depth and have been shown to reversibly inhibit neuronal activity. Yet, microwaves alone do not provide sufficient spatial precision to modulate target neurons without affecting surrounding tissues. Here, we report a split-ring resonator (SRR) that generates an enhanced microwave field at its gap with submillimeter spatial precision. With the SRR, microwaves at dosages below the safe exposure limit are shown to inhibit the firing of neurons within 1 mm of the SRR gap site. The microwave SRR reduced seizure activity at a low dose in both in vitro and in vivo models of epilepsy. This microwave dosage is confirmed to be biosafe via histological and biochemical assessment of brain tissue.

Influence Mechanism Analysis of the Spatial Evolution of Inter-Provincial Population Flow in China Based on Epidemic Prevention and Control.

Based on big data on migration from the Baidu Map platform, this paper divides China's epidemic prevention and control efforts into four stages. Then, the characteristics and spatial patterns of daily population flows are studied by social network analysis. Subsequently, the exponential random graph model is used to investigate the influence of dynamic characteristics of changes in the spatial structure of the interprovincial population flow network during the postepidemic period. The spatial structure of the population flow network before, during, and after the epidemic shows significantly different characteristics, with epidemic prevention and control measures playing a significant role in restricting population flows. Interprovincial population flows have a certain degree of transmissibility, but two-way flows are not obvious. In addition, for regions with a larger resident population and a higher unemployment rate, a larger population tends to flow out. For regions with higher per capita GDP, the secondary and tertiary industries account for a relatively larger proportion, and the public environment is better. The more attractive a region is to the population, the higher is the tendency towards population inflows. Moreover, the level of medical care and epidemic prevention and control have become the main influencing factors of population movement.

Video-rate Mid-infrared Photothermal Imaging by Single Pulse Photothermal Detection per Pixel.

By optically sensing the mid-infrared absorption induced photothermal effect, midinfrared photothermal (MIP) microscope enables super-resolution IR imaging and scrutinizing of biological systems in an aqueous environment. However, the speed of current lock-in based sample-scanning MIP system is limited to 1.0 millisecond or longer per pixel, which is insufficient for capturing dynamics inside living systems. Here, we report a single pulse laserscanning MIP microscope that dramatically increases the imaging speed by three orders of magnitude. We harness a lock-in free demodulation scheme which uses high-speed digitization to resolve single IR pulse induced contrast at nanosecond time scale. To realize single pulse photothermal detection at each pixel, we employ two sets of galvo mirrors for synchronized scanning of mid-infrared and probe beams to achieve an imaging line rate over 2 kHz. With video-rate imaging capability, we observed two types of distinct dynamics of lipids in living cells. Furthermore, by hyperspectral imaging, we chemically dissected a single cell wall at nanometer scale. Finally, with a uniform field of view over 200 by 200 µm 2 and 2 Hz frame rate, we mapped fat storage in free-moving C. elegans and live embryos.

Cultureless enumeration of live bacteria in urinary tract infection by single-cell Raman spectroscopy.

Urinary tract infections (UTIs) are the most common outpatient infections. Obtaining the concentration of live pathogens in the sample is crucial for the treatment. Still, the enumeration depends on urine culture and plate counting, which requires days of turn-around time (TAT). Single-cell Raman spectra combined with deuterium isotope probing (Raman-DIP) has been proven to identify the metabolic-active bacteria with high accuracy but is not able to reveal the number of live pathogens due to bacteria replication during the Raman-DIP process. In this study, we established a new approach of using sodium acetate to inhibit the replication of the pathogen and applying Raman-DIP to identify the active single cells. By combining microscopic image stitching and recognition, we could further improve the efficiency of the new method. Validation of the new method on nine artificial urine samples indicated that the exact number of live pathogens obtained with Raman-DIP is consistent with plate-counting while shortening the TAT from 18 h to within 3 h, and the potential of applying Raman-DIP for pathogen enumeration in clinics is promising.

Video-rate mid-infrared photothermal imaging by single-pulse photothermal detection per pixel.

By optically sensing absorption-induced photothermal effect, mid-infrared (IR) photothermal (MIP) microscope enables super-resolution IR imaging of biological systems in water. However, the speed of current sample-scanning MIP system is limited to milliseconds per pixel, which is insufficient for capturing living dynamics. By detecting the transient photothermal signal induced by a single IR pulse through fast digitization, we report a laser-scanning MIP microscope that increases the imaging speed by three orders of magnitude. To realize single-pulse photothermal detection, we use synchronized galvo scanning of both mid-IR and probe beams to achieve an imaging line rate of more than 2 kilohertz. With video-rate speed, we observed the dynamics of various biomolecules in living organisms at multiple scales. Furthermore, by using hyperspectral imaging, we chemically dissected the layered ultrastructure of fungal cell wall. Last, with a uniform field of view more than 200 by 200 square micrometer, we mapped fat storage in free-moving Caenorhabditis elegans and live embryos.

Bioelectrical impedance analysis for early screening of upper limb subclinical lymphedema: A case-control study.

Breast cancer-related lymphedema is a treatment-related chronic disease that causes great distress and medical burden. Early screening and precautionary measures for lymphedema could improve well-being and decrease medical costs. Herein, we used bioelectrical impedance analysis for early screening of lymphedema. We set up a verifiable standardized subclinical standard to screen subclinical lymphedema in postoperative breast cancer patients using bioelectrical impedance. The first part determined the criteria of subclinical lymphedema. Among the 424 female participants, 127 were healthy women, whereas 297 were postoperative breast cancer survivors. Subclinical standard boundaries were determined by the 95% confidence interval of the healthy women. The screening rate of patients with subclinical lymphedema was inferred by comparing the subclinical standard boundaries and the postoperative patient values. A total of 14.81-20.87% of postoperative breast cancer survivors were identified as patients with subclinical lymphedema. The second part provided the results of the verification test of this subclinical standard. The data of the verification test from 30 healthy women and 30 screened patients met the subclinical standard, and 30 breast cancer survivors with lymphedema verified the utility and feasibility of the subclinical standard. Therefore, this standard could provide a screening tool for early the identification of subclinical breast cancer survivors. Early detection helps implement personal and precise medical precautions for patients with subclinical lymphedema.

High-precision neural stimulation by a highly efficient candle soot fiber optoacoustic emitter.

Highly precise neuromodulation with a high efficacy poses great importance in neuroscience. Here we developed a candle soot fiber optoacoustic emitter (CSFOE), capable of generating a high pressure of over 10 MPa with a central frequency of 12.8 MHz, enabling highly efficient neuromodulation in vitro. The design of the fiber optoacoustic emitter, including the choice of the material and the thickness of the layered structure, was optimized in both simulations and experiments. The optoacoustic conversion efficiency of the optimized CSFOE was found to be 10 times higher than the other carbon-based fiber optoacoustic emitters. Driven by a single laser, the CSFOE can perform dual-site optoacoustic activation of neurons, confirmed by calcium (Ca2+) imaging. Our work opens potential avenues for more complex and programmed control in neural circuits using a simple design for multisite neuromodulation in vivo.

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.

The orphan nuclear receptor HNF4alpha determines PXR- and CAR-mediated xenobiotic induction of CYP3A4.

The drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4) is thought to be involved in the metabolism of nearly 50% of all the drugs currently prescribed. Alteration in the activity or expression of this enzyme seems to be a key predictor of drug responsiveness and toxicity. Currently available studies indicate that the ligand-activated nuclear receptors pregnane X receptor (PXR; NR1I2) and constitutive androstane receptor (CAR; NR1I3) regulate CYP3A4 expression. However, in cell-based reporter assays, CYP3A4 promoter activity was most pronounced in liver-derived cells and minimal or modest in non-hepatic cells, indicating that a liver-specific factor is required for physiological transcriptional response. Here we show that the orphan nuclear receptor hepatocyte nuclear factor-4alpha (HNF4alpha; HNF4A) is critically involved in the PXR- and CAR-mediated transcriptional activation of CYP3A4. We identified a specific cis-acting element in the CYP3A4 gene enhancer that confers HNF4alpha binding and thereby permits PXR- and CAR-mediated gene activation. Fetal mice with conditional deletion of Hnf4alpha had reduced or absent expression of CYP3A. Furthermore, adult mice with conditional hepatic deletion of Hnf4alpha had reduced basal and inducible expression of CYP3A. These data identify HNF4alpha as an important regulator of coordinate nuclear-receptor-mediated response to xenobiotics.

Non-genetic photoacoustic stimulation of single neurons by a tapered fiber optoacoustic emitter.

Neuromodulation at high spatial resolution poses great significance in advancing fundamental knowledge in the field of neuroscience and offering novel clinical treatments. Here, we developed a tapered fiber optoacoustic emitter (TFOE) generating an ultrasound field with a high spatial precision of 39.6 µm, enabling optoacoustic activation of single neurons or subcellular structures, such as axons and dendrites. Temporally, a single acoustic pulse of sub-microsecond converted by the TFOE from a single laser pulse of 3 ns is shown as the shortest acoustic stimuli so far for successful neuron activation. The precise ultrasound generated by the TFOE enabled the integration of the optoacoustic stimulation with highly stable patch-clamp recording on single neurons. Direct measurements of the electrical response of single neurons to acoustic stimulation, which is difficult for conventional ultrasound stimulation, have been demonstrated. By coupling TFOE with ex vivo brain slice electrophysiology, we unveil cell-type-specific responses of excitatory and inhibitory neurons to acoustic stimulation. These results demonstrate that TFOE is a non-genetic single-cell and sub-cellular modulation technology, which could shed new insights into the mechanism of ultrasound neurostimulation.

Nanosecond-resolution photothermal dynamic imaging via MHZ digitization and match filtering.

Photothermal microscopy has enabled highly sensitive label-free imaging of absorbers, from metallic nanoparticles to chemical bonds. Photothermal signals are conventionally detected via modulation of excitation beam and demodulation of probe beam using lock-in amplifier. While convenient, the wealth of thermal dynamics is not revealed. Here, we present a lock-in free, mid-infrared photothermal dynamic imaging (PDI) system by MHz digitization and match filtering at harmonics of modulation frequency. Thermal-dynamic information is acquired at nanosecond resolution within single pulse excitation. Our method not only increases the imaging speed by two orders of magnitude but also obtains four-fold enhancement of signal-to-noise ratio over lock-in counterpart, enabling high-throughput metabolism analysis at single-cell level. Moreover, by harnessing the thermal decay difference between water and biomolecules, water background is effectively separated in mid-infrared PDI of living cells. This ability to nondestructively probe chemically specific photothermal dynamics offers a valuable tool to characterize biological and material specimens.

A fiber optoacoustic emitter with controlled ultrasound frequency for cell membrane sonoporation at submillimeter spatial resolution.

Focused ultrasound has attracted great attention in minimally invasive therapeutic and mechanism studies. Frequency below 1 MHz is identified preferable for high-efficiency bio-modulation. However, the poor spatial confinement of several millimeters and large device diameter of ∼25 mm of typical sub-MHz ultrasound technology suffered from the diffraction limit, severely hindering its further applications. To address it, a fiber-based optoacoustic emitter (FOE) is developed, serving as a miniaturized ultrasound point source, with sub-millimeter confinement, composed of an optical diffusion layer and an expansion layer on an optical fiber. By modifying acoustic damping and light absorption performance, controllable frequencies in the range of 0.083 MHz-5.500 MHz are achieved and further induce cell membrane sonoporation with frequency dependent efficiency. By solving the problem of compromise between sub-MHz frequency and sub-millimeter precision via breaking the diffraction limit, the FOE shows a great potential in region-specific drug delivery, gene transfection and neurostimulation.

Optoacoustic brain stimulation at submillimeter spatial precision.

Low-intensity ultrasound is an emerging modality for neuromodulation. Yet, transcranial neuromodulation using low-frequency piezo-based transducers offers poor spatial confinement of excitation volume, often bigger than a few millimeters in diameter. In addition, the bulky size limits their implementation in a wearable setting and prevents integration with other experimental modalities. Here, we report spatially confined optoacoustic neural stimulation through a miniaturized Fiber-Optoacoustic Converter (FOC). The FOC has a diameter of 600 µm and generates omnidirectional ultrasound wave locally at the fiber tip through the optoacoustic effect. We show that the acoustic wave generated by FOC can directly activate individual cultured neurons and generate intracellular Ca2+ transients. The FOC activates neurons within a radius of 500 µm around the fiber tip, delivering superior spatial resolution over conventional piezo-based low-frequency transducers. Finally, we demonstrate direct and spatially confined neural stimulation of mouse brain and modulation of motor activity in vivo.

Ultrafast chemical imaging by widefield photothermal sensing of infrared absorption.

Infrared (IR) imaging has become a viable tool for visualizing various chemical bonds in a specimen. The performance, however, is limited in terms of spatial resolution and imaging speed. Here, instead of measuring the loss of the IR beam, we use a pulsed visible light for high-throughput, widefield sensing of the transient photothermal effect induced by absorption of single mid-IR pulses. To extract these transient signals, we built a virtual lock-in camera synchronized to the visible probe and IR light pulses with precisely controlled delays, allowing submicrosecond temporal resolution determined by the probe pulse width. Our widefield photothermal sensing microscope enabled chemical imaging at a speed up to 1250 frames/s, with high spectral fidelity, while offering submicrometer spatial resolution. With the capability of imaging living cells and nanometer-scale polymer films, widefield photothermal microscopy opens a new way for high-throughput characterization of biological and material specimens.

Bond-selective transient phase imaging via sensing of the infrared photothermal effect.

Phase-contrast microscopy converts the phase shift of light passing through a transparent specimen, e.g., a biological cell, into brightness variations in an image. This ability to observe structures without destructive fixation or staining has been widely utilized for applications in materials and life sciences. Despite these advantages, phase-contrast microscopy lacks the ability to reveal molecular information. To address this gap, we developed a bond-selective transient phase (BSTP) imaging technique that excites molecular vibrations by infrared light, resulting in a transient change in phase shift that can be detected by a diffraction phase microscope. By developing a time-gated pump-probe camera system, we demonstrate BSTP imaging of live cells at a 50 Hz frame rate with high spectral fidelity, sub-microsecond temporal resolution, and sub-micron spatial resolution. Our approach paves a new way for spectroscopic imaging investigation in biology and materials science.

Cylindrical illumination with angular coupling for whole-prostate photoacoustic tomography.

Current diagnosis of prostate cancer relies on histological analysis of tissue samples acquired by biopsy, which could benefit from real-time identification of suspicious lesions. Photoacoustic tomography has the potential to provide real-time targets for prostate biopsy guidance with chemical selectivity, but light delivered from the rectal cavity has been unable to penetrate to the anterior prostate. To overcome this barrier, a urethral device with cylindrical illumination is developed for whole-prostate imaging, and its performance as a function of angular light coupling is evaluated with a prostate-mimicking phantom.