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Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session E

Poster R9 - Label-free Metabolic Nanoscopy by Ultrasensitive Reweighted Visible Stimulated Raman Scattering

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  We introduce Ultrasensitive Reweighted Visible Stimulated Raman Scattering (URV-SRS), a label-free nanoscopy technique achieving 86- nm resolution for studying cell metabolism. This method employs highly chirped spectral focusing visible SRS to improve the detection limit of SRS by over 20 fold, and introduces a self-supervised denoiser that suppresses noise by over 7.2 dB. The 50-fold sensitivity increase allows subsequent resolution enhancement via reweighting the high spatial frequencies previously overwhelmed by noise. URV-SRS enables the investigation of metabolic nanostructures during virus replication in mammalian cells and detailed analysis of fatty acid synthesis in engineered bacteria, shedding light on nanoscale spatial metabolomics.

Materials and

Methods:  The setup for URV-SRS is described below. Two synchronized femtosecond pulses (906 nm and 1045 nm) generated by the same laser source (Insight X3, Spectra-Physics, USA) are first frequency-doubled by nonlinear crystals to produce visible pump (453 nm) and Stokes (522 nm) beams. The visible beams are then chirped by high-dispersion glass rods (SF57, Schott, Switzerland) to temporally separate different wavelength components by means of spectral focusing. By tuning the temporal delay between the two pulses, the beating frequency is continuously changed to generate a Raman spectrum. Using 45 cm rods for pump and 60 cm rods for Stokes, the pulse duration has been elongated to 4 picoseconds. The extensive chirping of the pulses effectively separates Raman modes in the frequency domain, leading to a superior sub-10 cm-1 spectral resolution in the CH region. The significantly reduced peak pulse energy effectively addresses nonlinear photodamage on cells, allowing for sufficient power on the sample to enable high-sensitivity imaging. Computationally, we introduced a self-supervised learning-based denoiser to effectively reduce non-independent SRS noise, resulting in a 50-fold increase in detection sensitivity compared to near-infrared SRS. Additionally, we applied Fourier reweighting to enhance sub-100 nm spatial frequencies previously masked by noise, pushing the resolution to 86 nm, as validated by Fourier Ring Correlation.

Results, Conclusions, and Discussions: We used 30 nm AuNP and measured the pump-probe transient absorption, which shares the same OTF as SRS. The results show that the resolution is enhanced from 132 nm to 86 nm, demonstrating the nanoscopic resolving capability of the system. We further tested the biocompatibility of the system by imaging SKOV3 mammalian cancer cells on our setup. As shown in Fig. 2a-b, we performed 2-color VFR-SRS imaging at 2930 cm-1 and 2850 cm-1, which represent CH3 and CH3 vibrational modes, respectively. All major metabolites, including protein and lipids, contribute to 2930 cm-1, while lipids have a significant contribution at 2850 cm-1. Lipid droplets, nuclear membranes, and cytoskeletal compartments are clearly depicted in the images, which demonstrate the rich chemical contrast and resolving capability of the system.

Acknowledgements (Optional):