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Switchable stimulated Raman scattering microscopy with photochromic vibrational probes.

ABSTRACT: Photochromic probes with reversible fluorescence have revolutionized the fields of single molecule spectroscopy and super-resolution microscopy, but lack sufficient chemical specificity. In contrast, Raman probes with stimulated Raman scattering (SRS) microscopy provides superb chemical resolution for super-multiplexed imaging, but are relatively inert. Here we report vibrational photochromism by engineering alkyne tagged diarylethene to realize photo-switchable SRS imaging. The narrow Raman peak of the alkyne group shifts reversibly upon photoisomerization of the conjugated diarylethene when irradiated by ultraviolet (UV) or visible light, yielding "on" or "off" SRS images taken at the photoactive Raman frequency. We demonstrated photo-rewritable patterning and encryption on thin films, painting/erasing of cells with labelled alkyne-diarylethene, as well as pulse-chase experiments of mitochondria diffusion in living cells. The design principle provides potentials for super-resolution microscopy, optical memories and switches with vibrational specificity.

SUBMITTER: Ao J

PROVIDER: S-EPMC8149663 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Switchable stimulated Raman scattering microscopy with photochromic vibrational probes.

Ao Jianpeng J Fang Xiaofeng X Miao Xianchong X Ling Jiwei J Kang Hyunchul H Park Sungnam S Wu Changfeng C Ji Minbiao M

Nature communications 20210525 1

Photochromic probes with reversible fluorescence have revolutionized the fields of single molecule spectroscopy and super-resolution microscopy, but lack sufficient chemical specificity. In contrast, Raman probes with stimulated Raman scattering (SRS) microscopy provides superb chemical resolution for super-multiplexed imaging, but are relatively inert. Here we report vibrational photochromism by engineering alkyne tagged diarylethene to realize photo-switchable SRS imaging. The narrow Raman pea ...[more]

PMID: 34035304

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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.

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Switchable stimulated Raman scattering microscopy with photochromic vibrational probes.

Publications

Switchable stimulated Raman scattering microscopy with photochromic vibrational probes.

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