Protein and lipid mass concentration measurement in tissues by stimulated Raman scattering microscopy

Oh, Seungeun; Lee, ChangHee; Yang, Wenlong; Li, Ang; Mukherjee, Avik; Basan, Markus; Ran, Chongzhao; Yin, Wei; Tabin, Clifford J.; Fu, Dan; Xie, X. Sunney; Kirschner, Marc W.

doi:10.1073/pnas.2117938119

Cited by 66 publications

(87 citation statements)

References 70 publications

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“…SRS imaging at three Raman bands can separate the contribution of proteins and lipids, at the expense of increased computational complexity and imaging throughput. 34 The second complicating factor is lipid droplets. Lipid droplets are dynamic organelles that store neutral lipids.…”

Section: ■ Conclusionmentioning

confidence: 99%

“…31−33 By unmixing the contribution of lipids, proteins, and water, we showed that SRS imaging at three Raman bands (2853, 2935, and 3420 cm −1 , corresponding to CH 3 , CH 2 , and H 2 O Raman bands, respectively) can be used to map the density of lipids and proteins in cells. 34 Importantly, the use of water SRS signal to normalize signal loss due to the ubiquitous presence of sample-induced aberration and light scattering allowed quantitative density imaging in multicellular samples and tissues. 35,36 However, a major drawback of this method is that wavelength tuning is required, and as a result, the measurement speed is very slow, on the order of minutes.…”

Section: ■ Introductionmentioning

confidence: 99%

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Quantitative Imaging of Intracellular Density with Ratiometric Stimulated Raman Scattering Microscopy

Figueroa

et al. 2022

J. Phys. Chem. B

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Cell size and density are tightly controlled in mammalian cells. They impact a wide range of physiological functions, including osmoregulation, tissue homeostasis, and growth regulation. Compared to size, density variation for a given cell type is typically much smaller, implying that cell-type-specific density plays an important role in cell function. However, little is known about how cell density affects cell function or how it is regulated. Current tools for intracellular cell density measurements are limited to either suspended cells or cells grown on 2D substrates, neither of which recapitulate the physiology of single cells in intact tissue. While optical measurements have the potential to noninvasively measure cell density in situ, 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 uses intrinsic vibrational information from intracellular macromolecules to quantify dry mass density. Moreover, water is used as an internal standard to correct for aberration and light scattering effects. We demonstrate real-time measurement of intracellular density 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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Section: ■ Conclusionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Quantitative Imaging of Intracellular Density with Ratiometric Stimulated Raman Scattering Microscopy

Figueroa

et al. 2022

J. Phys. Chem. B

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“…Stimulated Raman scattering (SRS) microscopy has demonstrated advantages of non-destructive 3D imaging with subcellular resolution in a label-free manner 7,8 . Recent work has even demonstrated quantitative mass concentration measurements of lipids, proteins, and water 9 . For label-free SRS imaging microscopy, the chemical specificity is achieved through hyperspectral imaging (HSI) or training of a deep learning model 10 .…”

Section: Introductionmentioning

confidence: 99%

Multi-Molecular Hyperspectral PRM-SRS Imaging

Shi

Zhang

et al. 2022

Preprint

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Lipids play crucial roles in many biological processes under physiological and pathological conditions. Mapping spatial distribution and examining metabolic dynamics of different lipids in cells and tissues in situ are critical for understanding aging and diseases. Commonly used imaging methods, including mass spectrometry-based technologies or labeled imaging techniques, tend to disrupt the native environment of cells/tissues and have limited spatial or spectral resolution, while traditional optical imaging techniques still lack the capacity to distinguish chemical differences between lipid subtypes. To overcome these limitations, we developed a new hyperspectral imaging platform that integrates a Penalized Reference Matching algorithm with Stimulated Raman Scattering (PRM-SRS) microscopy. With this new approach, we directly visualized and identified multiple lipid species in cells and tissues in situ with high chemical specificity and subcellular resolution. High density lipoprotein (HDL) particles containing non-esterified cholesterol was observed in the kidney, indicating that these pools of cholesterol are ectopic deposits, or have yet to be enriched. We detected a higher Cholesterol to phosphatidylethanolamine (PE) ratio inside the granule cells of hippocampal samples in old mice, suggesting altered membrane lipid synthesis and metabolism in aging brains. PRM-SRS imaging also revealed subcellular distributions of sphingosine and cardiolipin in the human brain sample. Compared with other techniques, PRM-SRS demonstrates unique advantages, including faster data processing and direct user-defined visualization with enhanced chemical specificity for distinguishing clinically relevant lipid subtypes in different organs and species. Our method has broad applications in multiplexed cell and tissue imaging.

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“…Recently, quantitative phase imaging (QPI) techniques have been employed for imaging and analyzing the dynamics of biological organisms owing to their label-free and quantitative imaging capabilities [4][5][6][7][8][9]. QPI uses the refractive index (RI) distribution of a sample as an intrinsic imaging contrast and enables three-dimensional (3D) imaging of live unlabeled biological samples [10][11][12][13]. However, unlike thin biological cells or microorganisms [11,12,14,15], the large sizes and high RI values of nematodes result in technical challenges for QPI.…”

Section: Introductionmentioning

confidence: 99%

“…QPI uses the refractive index (RI) distribution of a sample as an intrinsic imaging contrast and enables three-dimensional (3D) imaging of live unlabeled biological samples [10][11][12][13]. However, unlike thin biological cells or microorganisms [11,12,14,15], the large sizes and high RI values of nematodes result in technical challenges for QPI. Large optical phase delay variations in nematodes generate significant phase-wrapping issues, deteriorating the imaging quality of 2D QPI techniques.…”

Section: Introductionmentioning

confidence: 99%

High-fidelity optical diffraction tomography of live organisms using non-toxic tunable refractive index media

Lee

Kwak

et al. 2022

Preprint

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Optical diffraction tomography (ODT) enables the three-dimensional (3D) refractive index (RI) reconstruction. However, when the RI difference between a sample and a medium increases, effects of light scattering become significant, preventing the acquisition of high-quality and accurate RI reconstructions. Herein, we present a method for high-fidelity ODT by introducing non-toxic RI matching media. Optimally reducing the RI contrast enhances the fidelity and accuracy of 3D RI reconstruction, enabling visualization of the morphology and intra-organization of live biological samples without producing toxic effects. We validate our method using various biological organisms, including C. albicans and C. elegans.

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Protein and lipid mass concentration measurement in tissues by stimulated Raman scattering microscopy

Cited by 66 publications

References 70 publications

Quantitative Imaging of Intracellular Density with Ratiometric Stimulated Raman Scattering Microscopy

Quantitative Imaging of Intracellular Density with Ratiometric Stimulated Raman Scattering Microscopy

Multi-Molecular Hyperspectral PRM-SRS Imaging

High-fidelity optical diffraction tomography of live organisms using non-toxic tunable refractive index media

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