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1.
iScience ; 23(3): 100953, 2020 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-32179477

RESUMO

In situ measurement of cellular metabolites is still a challenge in biology. Conventional methods, such as mass spectrometry or fluorescence microscopy, would either destroy the sample or introduce strong perturbations to target molecules. Here, we present multiplex stimulated Raman scattering (SRS) imaging cytometry as a label-free single-cell analysis platform with chemical specificity and high-throughput capabilities. Using SRS imaging cytometry, we studied the metabolic responses of human pancreatic cancer cells under stress by starvation and chemotherapeutic drug treatments. We unveiled protrusions containing lipid droplets as a metabolic marker for stress-resistant cancer cells. Furthermore, by spectroscopic SRS mapping, we unveiled that triglyceride in lipid droplets are used for local energy production through lipolysis, autophagy, and ß-oxidation. Our findings demonstrate the potential of targeting lipid metabolism for selective treatment of stress-resistant cancers. Collectively, these results highlight SRS imaging cytometry as a powerful label-free tool for biological discoveries with a high-throughput, high-content capacity.

2.
Nat Commun ; 11(1): 881, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060282

RESUMO

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.

3.
J Chem Phys ; 152(2): 020901, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31941290

RESUMO

Transient absorption (TA) spectroscopy has been extensively used in the study of excited state dynamics of various materials and molecules. The transition from TA spectroscopy to TA microscopy, which enables the space-resolved measurement of TA, is opening new investigations toward a more complete picture of excited state dynamics in functional materials, as well as the mapping of crucial biopigments for precision diagnosis. Here, we review the recent instrumental advancement that is pushing the limit of spatial resolution, detection sensitivity, and imaging speed. We further highlight the emerging application in materials science and life science.

4.
J Infect Dis ; 221(4): 618-626, 2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-31565732

RESUMO

BACKGROUND: Antimicrobial resistance is a significant concern to public health, and there is a pressing need to develop novel antimicrobial therapeutic modalities. METHODS: In this study, we investigated the capacity for quinine hydrochloride (Q-HCL) to enhance the antimicrobial effects of antimicrobial blue light ([aBL] 405 nm wavelength) against multidrug-resistant (MDR) Gram-negative bacteria in vitro and in vivo. RESULTS: Our findings demonstrated the significant improvement in the inactivation of MDR Pseudomonas aeruginosa and Acinetobacter baumannii (planktonic cells and biofilms) when aBL was illuminated during Q-HCL exposure. Furthermore, the addition of Q-HCL significantly potentiated the antimicrobial effects of aBL in a mouse skin abrasion infection model. In addition, combined exposure of aBL and Q-HCL did not result in any significant apoptosis when exposed to uninfected mouse skin. CONCLUSIONS: In conclusion, aBL in combination with Q-HCL may offer a novel approach for the treatment of infections caused by MDR bacteria.

5.
Light Sci Appl ; 8: 116, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31839936

RESUMO

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.

6.
J Biomed Opt ; 25(1): 1-11, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31849205

RESUMO

Hemozoin, the heme detoxification end product in malaria parasites during their growth in the red blood cells (RBCs), serves as an important marker for diagnosis and treatment target of malaria disease. However, the current method for hemozoin-targeted drug screening mainly relies on in vitro ß-hematin inhibition assays, which may lead to false-positive events due to under-representation of the real hemozoin crystal. Quantitative in situ imaging of hemozoin is highly desired for high-throughput screening of antimalarial drugs and for elucidating the mechanisms of antimalarial drugs. We present transient absorption (TA) imaging as a high-speed single-cell analysis platform with chemical selectivity to hemozoin. We first demonstrated that TA microscopy is able to identify ß-hematin, the artificial form of hemozoin, from the RBCs. We further utilized time-resolved TA imaging to in situ discern hemozoin from malaria-infected RBCs with optimized imaging conditions. Finally, we quantitatively analyzed the hemozoin amount in RBCs at different infection stages by single-shot TA imaging. These results highlight the potential of TA imaging for efficient antimalarial drug screening and drug mechanism investigation.

7.
Nano Lett ; 19(12): 8673-8682, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31726010

RESUMO

Metalenses, planar lenses realized by placing subwavelength nanostructures that locally impart lenslike phase shifts to the incident light, are promising as a replacement for refractive optics for their ultrathin, lightweight, and tailorable characteristics, especially for applications where payload is of significant importance. However, the requirement of fabricating up to billions of subwavelength structures for centimeter-scale metalenses can constrain size-scalability and mass-production for large lenses. In this Letter, we demonstrate a centimeter-scale, all-glass metalens capable of focusing and imaging at visible wavelength, using deep-ultraviolet (DUV) projection stepper lithography. Here, we show size-scalability and potential for mass-production by fabricating 45 metalenses of 1 cm diameter on a 4 in. fused-silica wafer. The lenses show diffraction-limited focusing behavior for any homogeneously polarized incidence at visible wavelengths. The metalens' performance is quantified by the Strehl ratio and the modulation transfer function (MTF), which are then compared with commercial refractive spherical and aspherical singlet lenses of similar size and focal length. We further explore the imaging capabilities of our metalens using a color-pixel sCMOS camera and scanning-imaging techniques, demonstrating potential applications for virtual reality (VR) devices or biological imaging techniques.

8.
Nat Commun ; 10(1): 5318, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31754221

RESUMO

Stimulated Raman scattering (SRS) microscopy allows for high-speed label-free chemical imaging of biomedical systems. The imaging sensitivity of SRS microscopy is limited to ~10 mM for endogenous biomolecules. Electronic pre-resonant SRS allows detection of sub-micromolar chromophores. However, label-free SRS detection of single biomolecules having extremely small Raman cross-sections (~10-30 cm2 sr-1) remains unreachable. Here, we demonstrate plasmon-enhanced stimulated Raman scattering (PESRS) microscopy with single-molecule detection sensitivity. Incorporating pico-Joule laser excitation, background subtraction, and a denoising algorithm, we obtain robust single-pixel SRS spectra exhibiting single-molecule events, verified by using two isotopologues of adenine and further confirmed by digital blinking and bleaching in the temporal domain. To demonstrate the capability of PESRS for biological applications, we utilize PESRS to map adenine released from bacteria due to starvation stress. PESRS microscopy holds the promise for ultrasensitive detection and rapid mapping of molecular events in chemical and biomedical systems.

9.
Biomed Opt Express ; 10(8): 4329-4339, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31453014

RESUMO

Thin tissue slice based histology has been used as a gold standard for disease diagnosis since over a hundred years ago. However, histopathological evaluation on two-dimensional slides suffers from large variations due to limited sampling. To improve the diagnostic accuracy, three-dimensional (3D) histology is performed through serial sectioning, staining, imaging and reconstruction of individual slices, which is highly time-consuming and labor intensive. We developed a volumetric stimulated Raman scattering (SRS) imaging method, which provides histology-like information in 3D context without the need for staining with dyes. Using a small molecule clearing agent, formamide, we performed tissue clearing within 30 min and achieved an imaging depth up to 500 µm in highly scattered tissues, including brain, kidney, liver and lung. Through a two-color SRS imaging scheme, we obtained histology-like images in cleared brain tissue slices. Our method has the potential for 3D tissue histopathology to improve the accuracy of histopathological examination.

10.
Artigo em Inglês | MEDLINE | ID: mdl-31425010

RESUMO

OBJECTIVE: Stimulated Raman projection tomography (SRPT), a recently developed label-free volumetric chemical imaging technology, has been reported to quantitatively reconstruct the distribution of chemicals in a three-dimensional (3D) complex system. The current image reconstruction scheme used in SRPT is based on a filtered back projection (FBP) algorithm that requires at least 180 angular-dependent projections to rebuild a reasonable SRPT image, resulting in a long total acquisition time. This is a big limitation for longitudinal studies on live systems. METHODS: We present a sparse-view data-based sparse reconstruction scheme, in which sparsely sampled projections at 180 degrees were used to reconstruct the volumetric information. In the scheme, the simultaneous algebra reconstruction technique (SART), combined with total variation regularization, was used for iterative reconstruction. To better describe the projection process, a pixel vertex driven model (PVDM) was developed to act as projectors, whose performance was compared with those of the distance driven model (DDM). RESULTS: We evaluated our scheme with numerical simulations and validated it for SRPT by mapping lipid contents in adipose cells. Simulation results showed that the PVDM performed better than the DDM in the case of using sparse-view data. Our scheme could maintain the quality of the reconstructed images even when the projection number was reduced to 15. The cell-based experimental results demonstrated that the proposed scheme can improve the imaging speed of the current FBP-based SRPT scheme by a factor of 9-12 without sacrificing discernible imaging details. CONCLUSION: Our proposed scheme significantly reduces the total acquisition time required for SRPT at a speed of one order of magnitude faster than the currently used scheme. This significant improvement in imaging speed would potentially promote the applicability of SRPT for imaging living organisms.

11.
Opt Lett ; 44(15): 3841-3844, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31368982

RESUMO

Intravascular photoacoustic imaging can potentially improve the identification of lipid-laden atherosclerotic plaque. Most intravascular photoacoustic endoscopes use multimode fibers, which do not allow tight focusing of photons. Recent experiments on pulse propagation in multimode graded-index fibers have shown a nonlinear improvement in beam quality. Here, we harness this nonlinear phenomenon for fiber-delivery of nanosecond laser pulses. We built a photoacoustic catheter 1.4 mm outer diameter, offering a lateral resolution as fine as 30 µm within a depth range of 2.5 mm. Such resolution is one order of magnitude better than current multimode fiber-based intravascular photoacoustic catheters. At the same time, the delivered pulse energy can reach as high as 20 µJ, which is two orders of magnitude higher than that of an optical resolution photoacoustic endoscope built with a single mode fiber. These improvements are expected to promote the biomedical application of photoacoustic endoscopes which require both high resolution and high pulse energy.

12.
Sci Adv ; 5(7): eaav7127, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31334347

RESUMO

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.

13.
Anal Chem ; 91(16): 10750-10756, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31313580

RESUMO

Vibrational spectroscopic imaging techniques, based on infrared absorption or Raman scattering, allow for noninvasive chemically specific visualization of biological systems. The infrared and Raman modalities with different selection rules provide complementary information. Specifically, infrared microscopy provides strong signals in the fingerprint region, but suffers from low spatial resolution. Raman microscopy provides submicrometer resolution, but requires a long acquisition time. We developed a system that combines the strengths of both techniques by integrating confocal Raman microspectroscopy to the recently developed mid-infrared photothermal microscopy. This hybrid system is capable of fast infrared photothermal imaging of living cells with submicrometer resolution to identify points of interest, followed by a full-spectrum Raman analysis of the identified objects. In addition, a fingerprint photothermal spectrum can be acquired by scanning the wavelengths of the infrared laser. Comprehensive vibrational fingerprint mapping of live cells, demonstrated in adipocytes and single bacteria, promises broad applications of this technology in biology and material science.

14.
J Phys Chem Lett ; 10(15): 4374-4381, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31313926

RESUMO

Voltage imaging allows mapping of the membrane potential in living cells. Yet, current intensity-based imaging approaches are limited to relative membrane potential changes, missing important information conveyed by the absolute value of the membrane voltage. This challenge arises from various factors affecting the signal intensity, such as concentration, illumination intensity, and photobleaching. Here, we demonstrate electronic preresonance hyperspectral stimulated Raman scattering (EPR-hSRS) for spectroscopic detection of the membrane voltage using a near-infrared-absorbing microbial rhodopsin expressed in E. coli. This newly developed near-infrared active microbial rhodopsin enables electronic preresonance SRS imaging at high sensitivity. By spectral profiling, we identified voltage-sensitive SRS peaks in the fingerprint region in single E. coli cells. These spectral signatures offer a new approach for quantitation of the absolute membrane voltage in living cells.


Assuntos
Rodopsinas Microbianas/química , Análise Espectral Raman/métodos , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Raios Infravermelhos , Potenciais da Membrana , Mutação , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/metabolismo , Análise de Célula Única/métodos
15.
Adv Sci (Weinh) ; 6(11): 1900030, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31179216

RESUMO

Confronted with the severe situation that the pace of resistance acquisition is faster than the clinical introduction of new antibiotics, health organizations are calling for effective approaches to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, an approach to treat MRSA through photolysis of staphyloxanthin, an antioxidant residing in the microdomain of S. aureus membrane, is reported. This photochemistry process is uncovered through transient absorption imaging and quantitated by absorption spectroscopy, Raman spectroscopy, and mass spectrometry. Photolysis of staphyloxanthin transiently elevates the membrane permeability and renders MRSA highly susceptible to hydrogen peroxide attack. Consequently, staphyloxanthin photolysis by low-level 460 nm light eradicates MRSA synergistically with hydrogen peroxide and other reactive oxygen species. The effectiveness of this synergistic therapy is well validated in MRSA planktonic culture, MRSA-infected macrophage cells, stationary-phase MRSA, persisters, S. aureus biofilms, and two mice wound infection models. Collectively, the work demonstrates that staphyloxanthin photolysis is a new therapeutic platform to treat MRSA infections.

16.
Sci Adv ; 5(5): eaav0561, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31093524

RESUMO

As a stable and accurate biomarker, glycated hemoglobin (HbA1c) is clinically used to diagnose diabetes with a threshold of 6.5% among total hemoglobin (Hb). Current methods such as boronate affinity chromatography involve complex processing of large-volume blood samples. Moreover, these methods cannot measure HbA1c fraction at single-red blood cell (RBC) level, thus unable to separate the contribution from other factors such as RBC lifetime. Here, we demonstrate a spectroscopic transient absorption imaging approach that is able to differentiate HbA1c from Hb on the basis of their distinct excited-state dynamics. HbA1c fraction inside a single RBC is derived quantitatively through phasor analysis. HbA1c fraction distribution of diabetic blood is apparently different from that of healthy blood. A mathematical model is developed to derive the long-term blood glucose concentration. Our technology provides a unique way to study heme modification and to derive clinically important information void of bloodstream glucose fluctuation.

17.
Biomed Opt Express ; 10(3): 1405-1419, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30891355

RESUMO

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.

18.
Bioconjug Chem ; 30(2): 443-453, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30395447

RESUMO

We present a robust method for loading small interfering RNA (siRNA) duplexes onto the surfaces of gold nanorods (GNRs) at high density, using near-infrared laser irradiation to trigger their intracellular release with subsequent knockdown activity. Citrate-stabilized GNRs were first coated with oleylsulfobetaine, a zwitterionic amphiphile with low cytotoxicity, which produced stable dispersions at high ionic strength. Amine-modified siRNA duplexes were converted into dithiocarbamate (DTC) ligands and adsorbed onto GNR surfaces in a single incubation step at 0.5 M NaCl, simplifying the charge screening process. The DTC anchors were effective at minimizing premature siRNA desorption and release, a common but often overlooked problem in the use of gold nanoparticles as oligonucleotide carriers. The activity of GNR-siRNA complexes was evaluated systematically against an eGFP-producing ovarian cancer cell line (SKOV-3) using folate receptor-mediated uptake. Efficient knockdown was achieved by using a femtosecond-pulsed laser source to release DTC-anchored siRNA, with essentially no contributions from spontaneous (dark) RNA desorption. GNRs coated with thiol-anchored siRNA duplexes were less effective and also permitted low levels of knockdown activity without photothermal activation. Optimized siRNA delivery conditions were applied toward the targeted knockdown of transglutaminase 2, whose expression is associated with the progression of recurrent ovarian cancer, with a reduction in activity of >80% achieved after a single pulsed laser treatment.


Assuntos
Ouro/química , Nanotubos/química , RNA Interferente Pequeno/administração & dosagem , Tiocarbamatos/química , Animais , Linhagem Celular , Humanos , Nanotubos/ultraestrutura , Oligonucleotídeos/química , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Transfecção/métodos
19.
J Cardiovasc Transl Res ; 12(3): 211-220, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30488332

RESUMO

Intravascular photoacoustic-ultrasound (IVPA-US) imaging and near-infrared spectroscopy-intravascular ultrasound (NIRS-IVUS) are two hybrid modalities that detect arterial lipid, with comparison necessary to understand the relative advantages of each. We performed in vivo and ex vivo IVPA-US imaging of the iliac arteries of Ossabaw swine with metabolic syndrome (MetS) and lean swine to investigate sensitivity for early-stage atherosclerosis. We repeated imaging ex vivo with NIRS-IVUS for comparison to IVPA-US and histology. Both modalities showed significantly greater lipid in MetS vs. lean swine, but only IVPA-US localized the lipid as perivascular. To investigate late-stage atherosclerosis, we performed ex vivo IVPA-US imaging of a human coronary artery with comparison to NIRS-IVUS and histology. Two advanced fibroatheromas were identified, with agreement between IVPA-measured lipid area and NIRS-derived lipid content. As confirmed histologically, IVPA-US has sensitivity to detect lipid content similar to NIRS-IVUS and provides additional depth resolution, enabling quantification and localization of lipid cores within plaques.

20.
J Biomed Opt ; 23(10): 1-9, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30350492

RESUMO

Stimulated Raman scattering microscopy (SRS) was deployed to quantify enamel demineralization in intact teeth. The surfaces of 15 bovine-enamel blocks were divided into four equal-areas, and chemically demineralized for 0, 8, 16, or 24 h, respectively. SRS images (spectral coverage from ∼850 to 1150 cm - 1) were obtained at 10-µm increments up to 90 µm from the surface to the dentin-enamel junction. SRS intensities of phosphate (peak: 959 cm - 1), carbonate (1070 cm - 1), and water (3250 cm - 1) were measured. The phosphate peak height was divided by the carbonate peak height to calculate the SRS-P/C-ratio, which was normalized relative to 90 µm (SRS-P/C-ratio-normalized). The water intensity against depth decay curve was fitted with exponential decay. A decay constant (SRS-water-content) was obtained. Knoop-hardness values were obtained before (SMHS) and after demineralization (SMHD). Surface microhardness-change (SMH-change) [ ( SMHD - SMHS ) / SMHS] was calculated. Depth and integrated mineral loss (ΔZ) were determined by transverse microradiography. Comparisons were made using repeated-measures of analysis of variance. For SRS-P/C-ratio-normalized, at 0-µm (surface), sound (0-h demineralization) was significantly higher than 8-h demineralization and 24-h demineralization; 16-h demineralization was significantly higher than 24-h demineralization. For SRS-water-content, 24-h demineralization was significantly higher than all other demineralization-groups; 8-h demineralization and 16-h demineralization were significantly higher than 0-h demineralization. SRS-water-content presented moderate-to-strong correlation with SMH-change and weak-to-moderate correlation with depth. These results collectively demonstrate the potential of using SRS microscopy for in-situ chemical analysis of dental caries.


Assuntos
Esmalte Dentário/diagnóstico por imagem , Microscopia Óptica não Linear/métodos , Processamento de Sinais Assistido por Computador , Desmineralização do Dente/diagnóstico por imagem , Animais , Carbonatos/química , Bovinos , Esmalte Dentário/química , Desenho de Equipamento , Dureza , Fosfatos/química , Água/química
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