We report the development and implementation of an epi-detected spectral-focusing hyperspectral stimulated Raman scattering (SRS) imaging technique for label-free biomolecular subtyping of glioblastomas (GBMs). The hyperspectral SRS imaging technique developed generates SRS image stacks (from 2800 to 3020 cm at 7 cm intervals) within 30 s through controlling the time delay between the chirped pump and Stokes beams. SRS images at representative Raman shifts (e.g., 2845, 2885, and 2935 cm) delineate the biochemical variations and morphological differences between proneural and mesenchymal subtypes of GBMs. Multivariate curve resolution (MCR) analysis on hyperspectral SRS images enables the quantification of major biomolecule distributions in mesenchymal and proneural GBMs. Further principal component analysis (PCA) and linear discriminant analysis (LDA) together with leave-one SRS spectrum-out, cross-validation (LOOCV) yields a diagnostic sensitivity of 96.7% (29/30) and specificity of 88.9% (28/36) for differentiation between mesenchymal and proneural subtypes of GBMs. This study shows great potential of applying hyperspectral SRS imaging technique developed for rapid, label-free molecular subtyping of GBMs in neurosurgery.
The dynamics of mitochondria in live cells play a pivotal role in biological events such as cell metabolism, early stage apoptosis, and cell differentiation. Triphenylphosphonium (TPP) is a commonly used mitochondria-targeting agent for mitochondrial studies. However, there has been a lack of understanding in intracellular behaviors of TPP in the course of targeting mitochondria due to the difficulty in tracking and quantifying small molecules in a biological environment. Here, we report the utility of hyperspectral stimulated Raman scattering (SRS) microscopy associated with a Raman tag synthesized for real-time visualization and quantitation of TPP dynamics within live cells at the subcellular level. With the myriad of merits offered by a synthesized aryl-diyne-based Raman tag such as excellent photostability, negligible background interferences, and a linear dependence of the SRS signal on the TPP concentration, we successfully establish a quantitative model to associate the mitochondrial membrane potential with the key pharmacokinetic parameters of TPP inside the live cells. The model reveals that reduction in the mitochondrial membrane potential leads to significant decreases in both the uptake rate and intracellular concentrations of TPP. Further, on the basis of the multiplexed SRS images concurrently highlighting the cellular proteins and lipids without further labeling, we find that the TPP uptake causes little cytotoxicity to the host cells. The bioorthogonal hyperspectral SRS microscopy imaging reveals that TPP can maintain stable affinity to mitochondria during the restructuring of mitochondrial networking, demonstrating its great potential for real-time monitoring of pharmacokinetics of small molecules associated with live biological hosts, thereby promoting the development of mitochondria-targeting imaging probes and therapies in the near future.
Antibiotics resistance developed by biofilms has posed a clinical challenge in the effective treatment of bacterial infections. However, the resistance mechanisms have not been well understood due to a lack of suitable tools for dynamic observation of the interplay between antibiotics and biofilm. In this work, with the use of rapid hyperspectral stimulated Raman scattering microscopy associated with an aryl-alkyne-based Raman tag synthesized, we investigate dynamic interactions between vancomycin and Staphylococcus aureus ( S. aureus ) biofilm to gain new insights into the resistance mechanisms of the biofilm. Methods: We utilize spectral focusing hyperspectral stimulated Raman scattering microscopy ensued with multivariate curve resolution analysis to spectrally decompose S. aureus biofilm into its major components (i.e., bacteria and extracellular polymeric substances). Concurrently, vancomycin is conjugated with aryl-alkyne Raman tag (Raman peak at 2218 cm -1 ) for in vivo tracking of its uptake into biofilm without tissue interference. Results: We find that vancomycin penetration is a non-uniform diffusion process with penetration depths limited by the preferential affinity to the cell clusters. Semi-quantitative analysis shows that the majority of vancomycin binds to the bacteria, achieving intracellular concentrations of up to 4- to 10- fold higher than the administered dosage. The diffusion constant of ~3.16 µm 2 /min based on the diffusion and antibiotic binding equations is obtained that well accounts for the antibiotic penetration into the biofilm. SRS longitudinal monitoring of antibiotic effect on the growth of biofilms shows that the antibiotics can eradicate the upper layer of the biofilm exposed to sufficient dosages, while the lower layer of the biofilm at a sub-inhibitory dose remains viable, eventually re-growing to significant bio-volume. Conclusion: The Raman-tagged hyperspectral SRS microscopy developed is a powerful imaging tool for dynamic monitoring of inhibitory effects of antibiotics on the growing biofilm in vivo , which would facilitate the formulation of new antibiotics for more effective treatments of bacterial infections in near future.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.