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Spatial light-modulated stimulated Raman scattering (SLM-SRS) microscopy for rapid multiplexed vibrational imaging.

ABSTRACT: High speed imaging is pre-requisite for monitoring of dynamic processes in biological events. Here we report the development of a unique spatial light-modulated stimulated Raman scattering (SLM-SRS) microscopy that tailors the broadband excitation beam with sparse-sampling masks designed for rapid multiplexed vibrational imaging to monitor real-time cancer treatment effects and in vivo transport of drug solvent. Methods: We design an optimal mask pattern that enables selection of predominant windows in SRS spectrum for collective excitation at the highest possible peak power, thus providing an improved signal-to-noise ratio (SNR) without compromise of chemical specificity. The mask pattern generated is applied to the broad excitation beam using a flexible spatial light modulator. The SLM module further offers complementary function whereby rapid scanning of SRS spectrum can be facilitated prior to the mask generation, thereby making the SLM-SRS system a stand-alone imaging platform. Results: We demonstrate that SLM-SRS microscopy permits rapid multiplexed SRS imaging of polystyrene and polymethyl methacrylate beads in Brownian motion in dimethyl sulfoxide (DMSO) at 70 ms intervals without motion artiacts. We further apply SLM-SRS to monitor the therapeautic effect of mild alkaline solution on cancer cells, which shows immediate apoptotic response. Finally, we visualize in vivo penetration of DMSO into the plant tissue and evaluate acute toxicity of DMSO on cellulose and proteins within the tissue. Conclusion: We develop novel SLM-SRS microscopy and affirm its broad applicability for rapid monitoring of dynamic biological processes at the subcellular and molecular level.

SUBMITTER: Bae K

PROVIDER: S-EPMC6929623 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Spatial light-modulated stimulated Raman scattering (SLM-SRS) microscopy for rapid multiplexed vibrational imaging.

Bae Kideog K Zheng Wei W Huang Zhiwei Z

Theranostics 20200101 1

High speed imaging is pre-requisite for monitoring of dynamic processes in biological events. Here we report the development of a unique spatial light-modulated stimulated Raman scattering (SLM-SRS) microscopy that tailors the broadband excitation beam with sparse-sampling masks designed for rapid multiplexed vibrational imaging to monitor real-time cancer treatment effects and <i>in vivo</i> transport of drug solvent. <b>Methods:</b> We design an optimal mask pattern that enables selection of p ...[more]

PMID: 31903122

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Project description:Traditionally, molecules are analyzed in a test tube. Taking biochemistry as an example, the majority of our knowledge about cellular content comes from analysis of fixed cells or tissue homogenates using tools such as immunoblotting and liquid chromatography-mass spectrometry. These tools can indicate the presence of molecules but do not provide information on their location or interaction with each other in real time, restricting our understanding of the functions of the molecule under study. For real-time imaging of labeled molecules in live cells, fluorescence microscopy is the tool of choice. Fluorescent labels, however, are too bulky for small molecules such as fatty acids, amino acids, and cholesterol. These challenges highlight a critical need for development of chemical imaging platforms that allow in situ or in vivo analysis of molecules. Vibrational spectroscopy based on spontaneous Raman scattering is widely used for label-free analysis of chemical content in cells and tissues. However, the Raman process is a weak effect, limiting its application for fast chemical imaging of a living system. With high imaging speed and 3D spatial resolution, coherent Raman scattering microscopy is enabling a new approach for real-time vibrational imaging of single cells in a living system. In most experiments, coherent Raman processes involve two excitation fields denoted as pump at ωp and Stokes at ωs. When the beating frequency between the pump and Stokes fields (ωp - ωs) is resonant with a Raman-active molecular vibration, four major coherent Raman scattering processes occur simultaneously, namely, coherent anti-Stokes Raman scattering (CARS) at (ωp - ωs) + ωp, coherent Stokes Raman scattering (CSRS) at ωs - (ωp - ωs), stimulated Raman gain (SRG) at ωs, and stimulated Raman loss (SRL) at ωp. In SRG, the Stokes beam experiences a gain in intensity, whereas in SRL, the pump beam experiences a loss. Both SRG and SRL belong to stimulated Raman scattering (SRS), in which the energy difference between the pump and Stokes fields is transferred to the molecule for vibrational excitation. The SRS signal appears at the same wavelengths as the excitation fields and is commonly extracted through a phase-sensitive detection scheme. The detected intensity change because of a Raman transition is proportional to Im[χ(3)]IpIs, where χ(3) represents the third-order nonlinear susceptibility, Ip and Is stand for the intensity of the pump and Stokes fields. In this Account, we discuss the most recent advances in the technical development and enabling applications of SRS microscopy. Compared to CARS, the SRS contrast is free of nonresonant background. Moreover, the SRS intensity is linearly proportional to the density of target molecules in focus. For single-frequency imaging, an SRS microscope offers a speed that is ∼1000 times faster than a line-scan Raman microscope and 10,000 times faster than a point-scan Raman microscope. It is important to emphasize that SRS and spontaneous Raman scattering are complementary to each other. Spontaneous Raman spectroscopy covers the entire window of molecular vibrations, which allows extraction of subtleties via multivariate analysis. SRS offers the speed advantage by focusing on either a single Raman band or a defined spectral window of target molecules. Integrating single-frequency SRS imaging and spontaneous Raman spectroscopy on a single platform allows quantitative compositional analysis of objects inside single live cells.

Dataset Information

Spatial light-modulated stimulated Raman scattering (SLM-SRS) microscopy for rapid multiplexed vibrational imaging.

Publications

Spatial light-modulated stimulated Raman scattering (SLM-SRS) microscopy for rapid multiplexed vibrational imaging.

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