Stimulated Raman spectroscopic imaging by microsecond delay-line tuning

Liao, Chang; Huang, Kai-Chih; Hong, Wang; Chen, Andy J.; Karanja, Caroline; Wang, Pu; Eakins, Gregory; Cheng, Ji‐Xin

doi:10.1117/12.2256802

Cited by 7 publications

(10 citation statements)

References 20 publications

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“…Additional improvements may be envisioned for this technology. For example, the imaging speed can be further improved by using a multiplex SRS method, 15,16 advanced delay tuning approach, 18 or a wide-field SRS system. Secondly, harnessing the rational-designed reproducible plasmonic nanostructure fabricated by lithographic methods, our method can pave the way for reproducible and quantitative molecular imaging platform.…”

Section: Discussionmentioning

confidence: 99%

“…16 Yet, the imaging sensitivity of SRS microscopy is limited to ~10 mM for chemical bonds such as the C-H vibrations in cell membranes. 17,18 . Min and coworkers recently reported electronic pre-resonance SRS achieving sub-M-sensitivity detection for chromophores having a Raman cross-section over 10 3 or 10 4 times larger than endogenous biomolecules.…”

Section: Introductionmentioning

confidence: 99%

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Plasmon-enhanced stimulated Raman scattering microscopy with single-molecule detection sensitivity

et al. 2019

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Plasmon-enhanced stimulated Raman scattering microscopy with single-molecule detection sensitivity

et al. 2019

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“…Even higher speeds are possible, e.g. more complex, tailored, SF-CARS systems have achieved microsecond scanning, so the scan speed can be made comparable with the single-pixel dwell time [53]. Hyperspectral imaging with our ANDi fiber-based SF-CARS setup.…”

Section: Tuning Speed For Hyperspectral Imaging With Andi Fiber-basedmentioning

confidence: 99%

Multimodal spectral focusing CARS and SFG microscopy with a tailored coherent continuum from a microstructured fiber

et al. 2020

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We report a technologically novel microscopy system for bioimaging based on a 100 fs titanium:sapphire (Ti:Sa) laser pumped coherent continuum from a tailored, 9-cm long, all normal dispersion (ANDi) fiber, enabling concurrent image contrast with (a) spectral focusing coherent anti-Stokes Raman scattering (SF-CARS) (spanning 900-3200 cm −1) and (b) sum frequency generation (SFG). Both modalities were efficiently excited with power levels at the microscope focus compatible with biological samples. Moreover, using the continuum, images were recorded in the back-scattering (epi-detection) geometry, without the necessity for an expensive, computer-controlled, spatial light modulator (SLM), clearly demonstrating the strong signal levels achieved. Image contrast from the multiple modalities provided greater chemical and structural insights than imaging with any single technique in isolation. Numerical simulations supported these developments in regard to both the optimum fiber length for SC generation and the achievement of high spectral resolution in SF-CARS via careful group delay dispersion matching across the pump and Stokes pulses using just an inexpensive sequence of short glass blocks inserted into the Stokes beam. We show bio-images of mouse tissue recorded concurrently via label/stain-free contrast from multiple modalities: CARS, two-photon auto-fluorescence (TPaF) and second harmonic/sum frequency generation (SHG/ SFG). Overall, our approach delivers optimum performance in back-scattered (epi-) detection configuration, suited for thick samples, at reduced complexity and cost. The addition of this simple fiber add-on to lasers already widely used for TPF microscopy can thus extend the capabilities of a significant number of existing microscopy laboratories.

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“…It is based on a fast pulse pair-resolved wavelength-switchable Stokes laser with a synchronized pump pulse laser and a galvanometric scanner for SRS imaging of acoustically 3D-focused single cells flowing in a microfluidic channel. It achieves motion artifact-free chemical imaging of fast-flowing cells at a record high flow speed of 2 cm/s and a record high image acquisition speed of 24 klines/s with a pixel dwell time of 0.2 µs for 4-color SRS signal acquisition, which is >10 times faster than previous >3-color SRS techniques (22–26). Specifically, this pixel acquisition time of 0.2 µs for 4 colors is much shorter than the previously reported shortest value of 5 µs in single-point multiplex SRS flow cytometry (26) and even faster than that of 0.5 µs in 2-color SRS signal acquisition (27).…”

mentioning

confidence: 99%

Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering

Suzuki

Kobayashi

Wakisaka

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

156

132

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Combining the strength of flow cytometry with fluorescence imaging and digital image analysis, imaging flow cytometry is a powerful tool in diverse fields including cancer biology, immunology, drug discovery, microbiology, and metabolic engineering. It enables measurements and statistical analyses of chemical, structural, and morphological phenotypes of numerous living cells to provide systematic insights into biological processes. However, its utility is constrained by its requirement of fluorescent labeling for phenotyping. Here we present label-free chemical imaging flow cytometry to overcome the issue. It builds on a pulse pair-resolved wavelength-switchable Stokes laser for the fastest-to-date multicolor stimulated Raman scattering (SRS) microscopy of fast-flowing cells on a 3D acoustic focusing microfluidic chip, enabling an unprecedented throughput of up to ∼140 cells/s. To show its broad utility, we use the SRS imaging flow cytometry with the aid of deep learning to study the metabolic heterogeneity of microalgal cells and perform marker-free cancer detection in blood.

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Stimulated Raman spectroscopic imaging by microsecond delay-line tuning

Cited by 7 publications

References 20 publications

Plasmon-enhanced stimulated Raman scattering microscopy with single-molecule detection sensitivity

Plasmon-enhanced stimulated Raman scattering microscopy with single-molecule detection sensitivity

Multimodal spectral focusing CARS and SFG microscopy with a tailored coherent continuum from a microstructured fiber

Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering

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