Label-Free Digital Quantification of Lipid Droplets in Single Cells by Stimulated Raman Microscopy on a Microfluidic Platform

In this paper, the implementation and main realization issues of a stimulated Raman scattering (SRS) microscope, obtained by the integration of a femtosecond SRS spectroscopy set-up with an optical microscope equipped with a scanning unit, are described. In order to characterize capabilities and limitations of SRS microscope, the optical resolution is measured and validation tests on polystyrene beads imaging are reported. In addition, taking account of strength of SRS vibrational contrast, label-free imaging of small biological structures, as such as lipid droplets (LDs), in adipocyte cells is carried out. The visualization of small LDs, with dimensions comparable to the optical lateral resolution of our system, is demonstrated.

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3D reconstruction of lipid droplets in single cells by stimulated raman imaging

20th Italian National Conference on Photonic Technologies (Fotonica 2018) ◽

10.1049/cp.2018.1667 ◽

2018 ◽

Author(s):

M.A. Ferrara ◽

R. Ranjan ◽

A. Filograna ◽

A. D'Arco ◽

C. Valente ◽

...

Keyword(s):

3D Reconstruction ◽

Lipid Droplets ◽

Single Cells ◽

Raman Imaging ◽

Stimulated Raman

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Three-dimensional label-free imaging throughout adipocyte differentiation by stimulated Raman microscopy

PLoS ONE ◽

10.1371/journal.pone.0216811 ◽

2019 ◽

Vol 14 (5) ◽

pp. e0216811 ◽

Cited By ~ 4

Author(s):

Maria Antonietta Ferrara ◽

Angela Filograna ◽

Rajeev Ranjan ◽

Daniela Corda ◽

Carmen Valente ◽

...

Keyword(s):

Adipocyte Differentiation ◽

Three Dimensional ◽

Raman Microscopy ◽

Label Free ◽

Stimulated Raman

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Label-free imaging of biomolecules in food products using stimulated Raman microscopy

Journal of Biomedical Optics ◽

10.1117/1.3516591 ◽

2011 ◽

Vol 16 (2) ◽

pp. 021118 ◽

Cited By ~ 32

Author(s):

Maarten B. J. Roeffaers ◽

Xu Zhang ◽

Christian W. Freudiger ◽

Brian G. Saar ◽

Marjolein van Ruijven ◽

...

Keyword(s):

Food Products ◽

Raman Microscopy ◽

Label Free ◽

Stimulated Raman

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Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy

Science ◽

10.1126/science.1165758 ◽

2008 ◽

Vol 322 (5909) ◽

pp. 1857-1861 ◽

Cited By ~ 1241

Author(s):

Christian W. Freudiger ◽

Wei Min ◽

Brian G. Saar ◽

Sijia Lu ◽

Gary R. Holtom ◽

...

Keyword(s):

Raman Scattering ◽

Biomedical Imaging ◽

Stimulated Raman Scattering ◽

Three Dimensional ◽

High Sensitivity ◽

Raman Microscopy ◽

Label Free ◽

Omega 3 ◽

Low Sensitivity ◽

Stimulated Raman

Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. Here we report a three-dimensional multiphoton vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS imaging is significantly greater than that of spontaneous Raman microscopy, which is achieved by implementing high-frequency (megahertz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-free and readily interpretable chemical contrast. We show a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast, and monitoring drug delivery through the epidermis.

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Novel strategies of Raman imaging for monitoring intracellular retinoid metabolism in cancer cells

10.1101/2020.05.05.078410 ◽

2020 ◽

Author(s):

Halina Abramczyk ◽

Anna Imiela ◽

Jakub Surmacki

Keyword(s):

Cancer Cells ◽

Conformational Changes ◽

Lipid Droplets ◽

Binding Proteins ◽

Single Cells ◽

Raman Imaging ◽

Label Free ◽

Aberrant Expression ◽

Retinoid Metabolism ◽

Biochemical Imaging

AbstractWe developed a label-free Raman method for whole-cell biochemical imaging to detect molecular processes that occur in normal and cancer brain cells due to retinol transport in human cancers at the level of isolated organelles. Our approach allows the creation of biochemical maps of lipid droplets, mitochondria and nuclei in single cells. The maps were capable of discriminating triglycerides (TAG) from retinyl ester (RE) in lipid droplets (LD), providing an excellent tool to monitor intracellular retinoid metabolism. We detected spectral changes that arose in proteins and lipids due to retinoid metabolism in human cell lines of normal astrocytes and high-grade cancer cells of glioblastoma as well as in human medulloblastoma and glioblastoma tissue. Raman imaging is an effective tool for monitoring of retinoids and retinol binding proteins involved in carcinogenesis, as monitored by the unique spectral signatures of vibrations. We found two functionally distinct lipid droplets: TAG-LD, for energy storage, and RE-LD, for regulating the level of apo-CRBP1 in cytosol. Raman polarization measurements revealed the occurrence of conformational changes affecting discrete regions of proteins associated with retinol binding. Aberrant expression of retinoids and retinol binding proteins in human tumours were localized in lipid droplets, mitochondria and nuclei according Raman imaging.

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Utilizing Stimulated Raman Scattering Microscopy To Study Intracellular Distribution of Label-Free Ponatinib in Live Cells

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.9b01546 ◽

2019 ◽

Vol 63 (5) ◽

pp. 2028-2034 ◽

Cited By ~ 4

Author(s):

Kristel Sepp ◽

Martin Lee ◽

Marie T. J. Bluntzer ◽

G. Vignir Helgason ◽

Alison N. Hulme ◽

...

Keyword(s):

Raman Scattering ◽

Stimulated Raman Scattering ◽

Intracellular Distribution ◽

Live Cells ◽

Label Free ◽

Stimulated Raman

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3D phonon microscopy with sub-micron axial-resolution

Scientific Reports ◽

10.1038/s41598-021-82639-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Richard J. Smith ◽

Fernando Pérez-Cota ◽

Leonel Marques ◽

Matt Clark

Keyword(s):

Signal To Noise Ratio ◽

Single Cells ◽

Optical Resolution ◽

Acquisition Time ◽

Label Free ◽

Axial Resolution ◽

Force Microscopy ◽

Atomic Force ◽

Accuracy And Precision ◽

Good Agreement

AbstractBrillouin light scattering (BLS) is an emerging method for cell imaging and characterisation. It allows elasticity-related contrast, optical resolution and label-free operation. Phonon microscopy detects BLS from laser generated coherent phonon fields to offer an attractive route for imaging since, at GHz frequencies, the phonon wavelength is sub-optical. Using phonon fields to image single cells is challenging as the signal to noise ratio and acquisition time are often poor. However, recent advances in the instrumentation have enabled imaging of fixed and living cells. This work presents the first experimental characterisation of phonon-based axial resolution provided by the response to a sharp edge. The obtained axial resolution is up to 10 times higher than that of the optical system used to take the measurements. Validation of the results are obtained with various polymer objects, which are in good agreement with those obtained using atomic force microscopy. Edge localisation, and hence profilometry, of a phantom boundary is measured with accuracy and precision of approximately 60 nm and 100 nm respectively. Finally, 3D imaging of fixed cells in culture medium is demonstrated.

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