Broadband hyperspectral stimulated Raman scattering microscopy with a parabolic fiber amplifier source

Figueroa, Benjamin; Fu, Walter; Nguyen, Tai; Shin, Kseniya; Manifold, Bryce; Wise, Frank W.; Fu, Dan

doi:10.1364/boe.9.006116

Cited by 50 publications

(40 citation statements)

References 73 publications

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“…Currently, the spectral coverage of 200 cm −1 is due to the spectral bandwidth of the laser sources. However, since the delay range is freely tunable, if combined with broadband lasers by fiber amplification 54 or supercontinuum laser sources, the scheme can potentially be used to obtain the entire fingerprint SRS spectrum within 20 µs. In addition, the high-speed delay scanning scheme can be applied to a broad range of modalities requiring a long delay scan, such as transient absorption spectroscopy and impulsive SRS imaging.…”

Section: Discussionmentioning

confidence: 99%

Microsecond fingerprint stimulated Raman spectroscopic imaging by ultrafast tuning and spatial-spectral learning

et al. 2021

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Label-free vibrational imaging by stimulated Raman scattering (SRS) provides unprecedented insight into real-time chemical distributions. Specifically, SRS in the fingerprint region (400–1800 cm−1) can resolve multiple chemicals in a complex bio-environment. However, due to the intrinsic weak Raman cross-sections and the lack of ultrafast spectral acquisition schemes with high spectral fidelity, SRS in the fingerprint region is not viable for studying living cells or large-scale tissue samples. Here, we report a fingerprint spectroscopic SRS platform that acquires a distortion-free SRS spectrum at 10 cm−1 spectral resolution within 20 µs using a polygon scanner. Meanwhile, we significantly improve the signal-to-noise ratio by employing a spatial-spectral residual learning network, reaching a level comparable to that with 100 times integration. Collectively, our system enables high-speed vibrational spectroscopic imaging of multiple biomolecules in samples ranging from a single live microbe to a tissue slice.

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Section: Discussionmentioning

confidence: 99%

Microsecond fingerprint stimulated Raman spectroscopic imaging by ultrafast tuning and spatial-spectral learning

et al. 2021

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“…Broadband rea-time ratiometric SRS imaging: A femtosecond dual-beam laser system (Insight DS + from Spectra-Physics) was used for hyperspectral SRS imaging based on an orthogonal modulation spectral focusing scheme as described in our earlier publication. 39 Briefly, we used a parabolic fiber amplifier to increase the Stokes laser bandwidth from 6 nm to ~40 nm, 40 increasing the bandwidth from the original laser system of ~200 cm to ~500 cm -1 , which allows us to acquire both C-H region at 2935 cm -1 and the lower shoulder of the Raman spectral signature of water around 3190 cm -1 . We then employed an orthogonal modulation technique on our Stokes laser for simultaneous dual-band imaging, which was previously used for real-time microscale thermal imaging with SRS.…”

Section: Methodsmentioning

confidence: 99%

Quantitative imaging of intracellular density with ratiometric stimulated Raman scattering microscopy

Figueroa

et al. 2021

Preprint

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Cell size and density impact a wide range of physiological functions, including tissue homeostasis, growth regulation, and osmoregulation. Both are tightly regulated in mammalian cells. In comparison, density variation of a given cell type is much smaller than cell size, indicating that maintenance of cell type-specific density is important for cell function. Despite this importance, little is known about how cell density affects cell function and how it is controlled. Current tools for intracellular cell density measurements are limited either to suspended cells or cells growing on 2D substrates, neither of which recapitulate the physiology of single cells in intact tissue. While optical measurements have the potential to measure cell density in situ and noninvasively, light scattering in multicellular systems prevents direct quantification. Here, we introduce an intracellular density imaging technique based on ratiometric stimulated Raman scattering microscopy (rSRS). It quantifies intracellular drymass density through vibrational imaging of macromolecules. Moreover, water is used as an internal standard to correct for aberration and light scattering. We demonstrate real-time measurement of intracellular density quantification and show that density is tightly regulated across different cell types and can be used to differentiate cell types as well as cell states. We further demonstrate dynamic imaging of density change in response to osmotic challenge as well as intracellular density imaging of a 3D tumor spheroid. Our technique has the potential for imaging intracellular density in intact tissue and understanding density regulation and its role in tissue homeostasis.

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“…The Stokes pulse was modulated at 20 MHz using an electro-optic modulator (EOM) and split into two separate arms, one of which was then delayed by 12.5 ns (1/80 MHz) relative to the other. The two arms were recombined using a 50:50 beamsplitter and stretched to 2 ps using a grating-based pulse stretcher 30,31 . The tunable source was centered at 798 nm, and the pulses were dispersed using 60 cm of glass rods.…”

Section: Methodsmentioning

confidence: 99%

“…The tunable source was centered at 798 nm, and the pulses were dispersed using 60 cm of glass rods. The average spectral width is estimated to be 300 cm −1 31 . The pump and Stokes beams were then combined with a dichroic mirror and overlapped temporally using a delay line in the tunable arm.…”

Section: Methodsmentioning

confidence: 99%

Intraoperative assessment of skull base tumors using stimulated Raman scattering microscopy

Shin

Francis

Hill

et al. 2019

Sci Rep

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Intraoperative consultations, used to guide tumor resection, can present histopathological findings that are challenging to interpret due to artefacts from tissue cryosectioning and conventional staining. Stimulated Raman histology (SRH), a label-free imaging technique for unprocessed biospecimens, has demonstrated promise in a limited subset of tumors. Here, we target unexplored skull base tumors using a fast simultaneous two-channel stimulated Raman scattering (SRS) imaging technique and a new pseudo-hematoxylin and eosin (H&E) recoloring methodology. To quantitatively evaluate the efficacy of our approach, we use modularized assessment of diagnostic accuracy beyond cancer/non-cancer determination and neuropathologist confidence for SRH images contrasted to H&E-stained frozen and formalin-fixed paraffin-embedded (FFPE) tissue sections. Our results reveal that SRH is effective for establishing a diagnosis using fresh tissue in most cases with 87% accuracy relative to H&E-stained FFPE sections. Further analysis of discrepant case interpretation suggests that pseudo-H&E recoloring underutilizes the rich chemical information offered by SRS imaging, and an improved diagnosis can be achieved if full SRS information is used. In summary, our findings show that pseudo-H&E recolored SRS images in combination with lipid and protein chemical information can maximize the use of SRS during intraoperative pathologic consultation with implications for tissue preservation and augmented diagnostic utility.

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Broadband hyperspectral stimulated Raman scattering microscopy with a parabolic fiber amplifier source

Cited by 50 publications

References 73 publications

Microsecond fingerprint stimulated Raman spectroscopic imaging by ultrafast tuning and spatial-spectral learning

Microsecond fingerprint stimulated Raman spectroscopic imaging by ultrafast tuning and spatial-spectral learning

Quantitative imaging of intracellular density with ratiometric stimulated Raman scattering microscopy

Intraoperative assessment of skull base tumors using stimulated Raman scattering microscopy

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