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.
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.
Mid-infrared photothermal (MIP) microscopy overcomes the resolution and huge water background limits in conventional mid-infrared imaging by probing the mid-infrared absorption induced photothermal effect. However, to detect the subtle MIP signal, large probe power and lock-in detection are needed, which limit the imaging speed of current MIP systems. To overcome this limitation, we develop a single-pixel pump-probe camera that leverages the large well-depth capacity of photodiode to achieve high-speed wide-field MIP imaging. With compressive sensing applied, close to video-rate MIP imaging can be achieved, offering a powerful label-free chemical imaging tool to scrutinize the complex biological systems.
Microwaves, which have a ∼10-cm wavelength, can penetrate deeper into tissue than photons, heralding exciting deep tissue applications such as modulation or imaging via the thermoacoustic effect. Thermoacoustic conversion efficiency is however very low, even with an exogenous contrast agent. We break this low-conversion limit, using a split ring resonator to effectively collect and confine the microwaves into a submillimeter hot spot for ultrasound emission and achieve a conversion efficiency over 2000 times higher than other reported thermoacoustic contrast agents. Importantly, the frequency of emitted ultrasound can be precisely tuned and multiplexed by modulation of the microwave pulses. Such performance is inaccessible by a piezoelectric-based transducer or a photoacoustic emitter and, therefore, split ring resonators open up new opportunities to study the frequency response of cells in ultrasonic biomodulation. For applications in deep tissue localization, a split ring resonator can be used as a wireless, battery-free ultrasound beacon placed under a breast phantom.
Ultrasound has been recently explored as a new modality for neural modulation. However, one of the challenges in ultrasound neural modulation is that delivery of transcranial ultrasound would inevitably go through the skull, and eventually reach the cochlear through bone transduction. Moreover, the presence of skull will compromise ultrasound focus, resulting in poor spatial resolution. Here, we developed a miniaturized Fiber-Optoacoustic Converter (FOC), which has a diameter of 600 μm, and can convert nano-second laser pulses into omni-directional acoustic waves through the optoacoustic effect. The ball shaped FOC is composed of one ZnO /epoxy based diffusion layer and two graphite/epoxy based absorption layer. The radiofrequency spectrum of the generated US frequency ranges from 0.1-5 MHz, with multiple frequencies peaks at 0.5, 1 and 3MHz. Using this FOC system, we show that ultrasound can directly activate individual cortical neuron in vitro, and generate intracellular Ca2+ transient without neural damage. We next demonstrate that the FOC is activates neurons with a radius of 500 μm around the FOC tip, delivering superior spatial resolution. The stimulation effect is specific to neurons, but not glial cells. We also provide evidence of transient mechanical disturbance of neuronal membrane as the mechanism for FOC neural modulation. Finally, we combine FOC neural modulation with electrophysiology, and achieve direct and spatially confined neural stimulation in vivo.
Generation of ultrasound using the optoacoustic effect has received increasing attention in the field of imaging and translational medicine. However, none of the current optoacoustic converters has been used for neural modulation. Here, we developed a miniaturized Fiber-Optoacoustic Converter (FOC), which has a diameter of 600 μm, and can convert nano-second laser pulses into acoustic waves through the optoacoustic effect. The ball shaped FOC is composed of one ZnO /epoxy based diffusion layer and two graphite/epoxy based absorption layer. The radiofrequency spectrum of the generated US frequency ranges from 0.1-5 MHz, with multiple frequencies peaks at 0.5, 1 and 3MHz. Compared to traditional ultrasound transducers, the FOC system has the advantages of miniaturized size, superior spatial resolution, and produces omnidirectional propagating acoustic wave. Using this FOC system, we show that ultrasound can directly activate individual cortical neuron in vitro with a radius of 500 μm around the FOC tip, and generate intracellular Ca2+ transient without neural damage. Neural activation is the consequence of mechanical disturbance of neuronal membrane, rather than direct laser or photothermal stimulation. Finally, we combine FOC neural modulation with electrophysiology, and achieve direct and spatially confined neural stimulation in vivo. The FOC system opens new possibilities to use optoacoustic effect as a new method for precise neural modulation.
Breast-conserving surgery is a well-accepted breast cancer treatment. However, it is still challenging for the surgeon to accurately localize the tumor during the surgery. Also, the guidance provided by current methods is 1 dimensional distance information, which is indirect and not intuitive. Therefore, it creates problems on a large re-excision rate, and a prolonged surgical time. To solve these problems, we have developed a fiber-delivered optoacoustic guide (OG), which mimics the traditional localization guide wire and is preoperatively placed into tumor mass, and an augmented reality (AR) system to provide real-time visualization on the location of the tumor with sub-millimeter variance. By a nano-composite light diffusion sphere and light absorbing layer formed on the tip of an optical fiber, the OG creates an omnidirectional acoustic source inside tumor mass under pulsed laser excitation. The optoacoustic signal generated has a high dynamic range (~ 58dB) and spreads in a large apex angle of 320 degrees. Then, an acoustic radar with three ultrasound transducers is attached to the breast skin, and triangulates the location of the OG tip. With an AR system to sense the location of the acoustic radar, the relative position of the OG tip inside the tumor to the AR display is calculated and rendered. This provides direct visual feedback of the tumor location to surgeons, which will greatly ease the surgical planning during the operation and save surgical time. A proof-of-concept experiment using a tablet and a stereo-vision camera is demonstrated and 0.25 mm tracking variance is achieved.
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