Timely and accurate identification and antimicrobial susceptibility testing (AST) is essential to slow down the emergence of antimicrobial resistance and consequently reduce deaths due to drug-resistant infections. Here, we present multi-color fluorescence in situ hybridization (FISH) and stimulated Raman scattering (SRS) metabolic imaging to enable rapid identification and AST (iAST) of clinical samples. We have successfully developed FISH probes that utilizes oligonucleotide probes targeting the ribosomal RNA (rRNA) of bacteria strains associated with urinary tract infections (UTIs), which enables rapid and direct single-cell identification of microbes. By fast tuning between C-D and C-H vibration, we are able to use CD/(CD+CH) SRS signal ratio to quantitate the intracellular deuterium oxide (D2O) metabolism at single cell level. High-throughput, high-speed AST through robotic handling of liquid specimens and a multi-well chamber design has been demonstrated. We test the efficacy of our approach on common UTI associated bacterial isolates and clinical specimens, and demonstrate a 2 h diagnostic time, with an identification and categorical susceptible/resistant accuracy over 95%.
The imaging speed of current mid-infrared photothermal (MIP) microscope is limited to tens of seconds per frame due to the long pixel dwell time and slow sample scanning process, which is insufficient for capturing dynamics inside living systems. In this work, we developed a video-rate MIP microscope by employing a lock-in free demodulation scheme to resolve single IR pulse induced contrast. We further developed a synchronous pump-probe Galvo scanning for reaching a line rate over 2kHz. With such scheme, the system is capable of resolving chemical dynamics of various biomolecules in living organisms at multiple scales.
KEYWORDS: In vivo imaging, Mid-IR, Cancer, Signal generators, Photoacoustic spectroscopy, Magnetic resonance imaging, Infrared imaging, In vitro testing, Image resolution, Absorbance
Enzymes are vital in most physiological and biochemical processes and may function as critical biomarkers of disease. Notably, many biological events and signaling networks involve multiplex enzyme species. Thus, mapping multiple enzyme activities is significant for elucidating enzymatic functions in life and disease. Here, we report a novel technology to map the catalytic efficacy of phosphatase and caspase in live cancer cells and in C. elegans by mid-infrared photothermal (MIP) imaging of nitrile chameleons. This technology will certainly bring new insight into the roles of enzyme in biochemical activities and explore new knowledge of enzymatic activity in health and disease.
The imaging speed of the current mid-infrared photothermal (MIP) microscope is limited to tens of seconds per frame due to the long pixel dwell time and slow sample scanning process, which is insufficient for capturing dynamics inside living systems. In this work, we developed a video-rate MIP microscope by employing a lock-in free demodulation scheme to resolve single IR pulse induced contrast. We further developed a synchronous pump-probe Galvo scanning for reaching a line rate over 2.5 kHz. The system is capable of resolving chemical dynamics in living cells in a uniform imaging field of view over 300 μm.
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.
We present a novel version of mid-infrared photothermal microscopy in which thermosensitive fluorescent probes are harnessed to sense the mid-infrared photothermal effect. The fluorescence intensity can be modulated at the level of 1% per Kelvin, which is 100 times larger than the modulation of scattering intensity. In addition, fluorescence emission is free of interference, thus much improving the image quality. Moreover, fluorophores can target specific organelles or biomolecules, thus augmenting the specificity of photothermal imaging. Spectral fidelity is confirmed by fingerprinting a single bacterium. A wide-field fluorescence-detected mid-infrared photothermal microscope developed allows video-rate bond-selective imaging of biological specimens.
Spectroscopic stimulated Raman scattering (SRS) imaging has become a useful tool finding a broad range of applications. Due to the limited spectral coverage of current SRS systems, most SRS applications utilized a narrow Raman band (<300 cm-1). Multi-window SRS imaging covering C-H, C-D and fingerprint regions offers richer chemical information. In this work, we present a multi-window SRS imaging system with a rapid widely tunable fiber laser. We implemented auto-balanced detection to enhance the signal-to-noise ratio of stimulated Raman loss by 23 times. We demonstrated SRS metabolic imaging of fungi, cancer cells, and Caenorhabditis elegans across the C-H, silent and fingerprint Raman windows. Our results showcase the potential of the multi-window SRS system for a broad range of applications.
Mid-infrared photothermal microscopy (MIP) has been a promising chemical imaging technique for functionality characterization of biological and pharmaceutical specimens owing to its enhanced resolution and high-specificity. Recently developed wide-field MIP modalities drastically improved the imaging speed and thus enabled high-throughput imaging. However, the sensitivity of the wide-field MIP technique has been limited by shot-noise of background photons. Here, we demonstrate a dark-field MIP modality to allow 4-fold signal-to-noise ratio improvement. Our technique is based on selectively blocking the reflected light. Simulation and experimental results are both provided, and they are consistent with each other.
For high-throughput link of microbiome function and taxonomic identity at the single cell level, we established a stimulated Raman scattering (SRS)-fluorescence in situ hybridization (FISH) platform. SRS combined with the deuterium-based isotope probing enables chemical mapping and reveals metabolic activity of bacteria. Fluorescently tagged oligonucleotide probes identify different bacteria and are detected through two photon fluorescence (TPF) microscopy. As a proof-of-principle demonstration, we tested the platform in a mixture of two distinct gut microbiota taxa with different deuterium labeling levels. This established platform not only provides enormous potential to study microbiota in the complex environment, but also the simultaneous observation of phenotype and genotype in the general biological systems.
This work demonstrates a rapid platform that can determine the antimicrobial susceptibility testing (AST) in cation-adjusted Mueller-Hinton Broth medium, urine and blood by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) incorporation at a single bacterium level. The total AST assay time with the value of the single-cell metabolism inactivation concentration (SC-MIC) obtained is less than 2.5 h from colony to results. The SC-MIC results of 37 sets of bacterial isolate samples were systematically validated by MIC determined by the Clinical and Laboratory Standards Institute criteria, with a category agreement of 94.6% and 5.4% minor error. Furthermore, SRS imaging of D2O metabolic incorporation can rapidly determine SC-MIC directly in clinical samples for urinary tract infection or septicemia blood infection.
Mid-infrared photothermal (MIP) imaging is an emerging technique where a visible beam is deployed to sense the photothermal lensing effect induced by infrared absorption of molecules. This technology provides sub-micron spatial resolution defined by the visible probe beam. Yet, the photothermal lensing effect is a weak effect for most materials. For instance, the diffraction coefficient of poly (methyl methacrylate) changes around 0.1% per Kelvin. Thus, highly sensitive probes are desired to improve the detection sensitivity. Here, we report a fluorescence-enhanced mid-infrared photothermal (FE-MIP) microscope with high sensitivity. MIP microscopy uses a pump-probe strategy in which a mid-infrared light vibrationally excites a sample and a visible light probes the thermal effect. Instead of measuring the scattering modulated by mid-infrared absorption, we deploy thermal sensitive fluorescent dyes as the probe, and measure the modulated fluorescence intensity in both confocal mode and wide field mode. We demonstrate high imaging sensitivity and component specificity through fluorescence labeling.
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