Imaging Drug Uptake by Bioorthogonal Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy in tandem with bioorthogonal Raman labelling strategies is set to revolutionise the direct visualisation of intracellular drug uptake. Rational evaluation of a series of Raman-active labels has allowed the identification of highly active labels which have minimal perturbation on the biological efficacy of the parent drug. Drug uptake has been correlated with markers of cellular composition and cell cycle status, and mapped across intracellular structures using dual-colour and multi-modal imaging. The minimal phototoxicity and low photobleaching associated with SRS microscopy has enabled real-time imaging in live cells. These studies demonstrate the potential for SRS microscopy in the drug development process

Ratiometric sensing of fluoride ions using Raman spectroscopy

Ratiometric Raman spectroscopy represents a novel sensing approach for the detection of fluoride anions based on alkyne desilylation chemistry. This method enables rapid, anion selective and highly sensitive detection of fluoride in a simple paper-based assay format using a portable Raman spectrometer

Label-free imaging of lipid droplets in prostate cells using stimulated Raman scattering microscopy and multivariate analysis

Hyperspectral stimulated Raman scattering (SRS) microscopy is a powerful imaging modality for the analysis of biological systems. Here, we report the application of k-means cluster analysis (KMCA) of multi-wavelength SRS images in the high wavenumber region of the Raman spectrum, as a robust and reliable method for the segmentation of cellular organelles based on the intrinsic SRS spectrum. KMCA has been applied to the study of the endogenous lipid biochemistry of prostate cancer and prostate healthy cell models, whilst the corresponding SRS spectrum of the lipid droplet cluster enabled direct comparison of their composition. The application of KMCA in visualising the lipid droplet (LD) content of prostate cell models following inhibition of de novo lipid synthesis (DNL) using the acetyl-coA carboxylase inhibitor, 5-(tetradecyloxy)-2-furoic acid (TOFA) is demonstrated. This method identified a reliance of prostate cancer cell models upon DNL for metabolic requirements, with a significant reduction in the cellular lipid droplet content after treatment with TOFA, which was not observed in normal prostate cell models. SRS imaging combined with KMCA is a robust method for investigating drug-cell interactions in a label-free manner

Label-free cytometric evaluation of mitosis via stimulated Raman scattering microscopy and spectral phasor analysis

Hyperspectral stimulated Raman scattering (SRS) microscopy is a robust imaging tool for the analysis of biological systems. Here we present a unique perspective, a label-free spatiotemporal map of mitosis by integrating hyperspectral SRS microscopy with advanced chemometrics to assess the intrinsic biomolecular properties of an essential process of mammalian life. The application of spectral phasor analysis to multi-wavelength SRS images in the high-wavenumber (HWN) region of the Raman spectrum enabled the segmentation of subcellular organelles based on innate SRS spectra. Traditional imaging of DNA is primarily reliant on using fluorescent probes or stains which can affect the biophysical properties of the cell, here we demonstrate the label-free visualization of nuclear dynamics during mitosis coupled with an evaluation of its spectral profile in a rapid and reproducible manner. These results provide a snapshot of the cell division cycle and chemical variability between intracellular compartments in single cell models, which is central to understanding the molecular foundations of these fundamental biological processes. The evaluation of HWN images by phasor analysis also facilitated the differentiation between cells in separate phases of the cell cycle based solely on their nuclear SRS spectral signal, which offers an interesting label-free approach in combination with flow cytometry. Therefore this study demonstrates that SRS microscopy combined with spectral phasor analysis is a valuable method for detailed optical fingerprinting at the subcellular level

A Palette of Minimally Tagged Sucrose Analogues for Real‐Time Raman Imaging of Intracellular Plant Metabolism

Sucrose is the main saccharide used for long-distance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real-time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small (≈50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne-tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real-time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy

Spectral fingerprinting of cellular lipid droplets using stimulated Raman scattering microscopy and chemometric analysis

Hyperspectral stimulated Raman scattering (SRS) microscopy is a powerful method for direct visualisation and compositional analysis of cellular lipid droplets. Here we report the application of spectral phasor analysis as a convenient method for the segmentation of lipid droplets using the hyperspectral SRS spectrum in the high wavenumber and fingerprint region of the spectrum. Spectral phasor analysis was shown to discriminate six fatty acids based on vibrational spectroscopic features in solution. The methodology was then applied to studying fatty acid metabolism and storage in a mammalian cancer cell model and during drug-induced steatosis in a hepatocellular carcinoma cell model. The accumulation of fatty acids into cellular lipid droplets was shown to vary as a function of the degree of unsaturation, whilst in a model of drug-induced steatosis, the detection of increased saturated fatty acid esters was observed. Taking advantage of the fingerprint and high wavenumber regions of the SRS spectrum has yielded a greater insight into lipid droplet composition in a cellular context. This approach will find application in the label-free profiling of intracellular lipids in complex disease models

Spectral fingerprinting of cellular lipid droplets using stimulated Raman scattering microscopy and chemometric analysis.

peer reviewedHyperspectral stimulated Raman scattering (SRS) microscopy is a powerful method for direct visualisation and compositional analysis of cellular lipid droplets. Here we report the application of spectral phasor analysis as a convenient method for the segmentation of lipid droplets using the hyperspectral SRS spectrum in the high wavenumber and fingerprint region of the spectrum. Spectral phasor analysis was shown to discriminate six fatty acids based on vibrational spectroscopic features in solution. The methodology was then applied to studying fatty acid metabolism and storage in a mammalian cancer cell model and during drug-induced steatosis in a hepatocellular carcinoma cell model. The accumulation of fatty acids into cellular lipid droplets was shown to vary as a function of the degree of unsaturation, whilst in a model of drug-induced steatosis, the detection of increased saturated fatty acid esters was observed. Taking advantage of the fingerprint and high wavenumber regions of the SRS spectrum has yielded a greater insight into lipid droplet composition in a cellular context. This approach will find application in the label-free profiling of intracellular lipids in complex disease models

A new class of ratiometric small molecule intracellular pH sensors for Raman microscopy

Intracellular pH (pHi) homeostasis is intertwined with a myriad of normal cellular behaviors as well as pathological processes. As such, small molecule probes for the measurement of pHi are invaluable tools for chemical biology, facilitating the study of the role of pH in cellular function and disease. The field of small molecule pHi sensors has traditionally been dominated with probes based on fluorescent scaffolds. In this study, a series of low molecular weight (<260) oligoyne compounds have been developed which exhibit pH sensitive alkyne stretching frequencies (νalkyne) in Raman spectroscopy. The modular design of the compounds enabled tuneability of their pKa(H) through simple structural modification, such that continuous pH sensitivity is achieved over the range 2-10. Alkyne stretching bands reside in the 'cell-silent' region of the Raman spectrum (1800-2600 cm-1) and are readily detectable in a cellular environment with subcellular spatial resolution. This enabled the application of a pH sensitive oligoyne compound to the ratiometric sensing of pHi in prostate cancer (PC3) cells in response to drug treatment. We propose that probes based on Alkyne Tag Raman Imaging offer an entirely new platform for the sensing of pHi, complementary to fluorescence microscopy

Raman spectroscopy in prostate cancer : techniques, applications and advancements

Optical techniques are widely used tools in the visualisation of biological species within complex matrices, including biopsies, tissue resections and biofluids. Raman spectroscopy is an emerging analytical approach that probes the molecular signature of endogenous cellular biomolecules under biocompatible conditions with high spatial resolution. Applications of Raman spectroscopy in prostate cancer include biopsy analysis, assessment of surgical margins and monitoring of treatment efficacy. The advent of advanced Raman imaging techniques, such as stimulated Raman scattering, is creating opportunities for real-time in situ evaluation of prostate cancer. This review provides a focus on the recent preclinical and clinical achievements in implementing Raman-based techniques, highlighting remaining challenges for clinical applications. The research and clinical results achieved through in vivo and ex vivo Raman spectroscopy illustrate areas where these evolving technologies can be best translated into clinical practice

Raman imaging of nanocarriers for drug delivery

The efficacy of pharmaceutical agents can be greatly improved through nanocarrier delivery. Encapsulation of pharmaceutical agents into a nanocarrier can enhance their bioavailability and biocompatibility, whilst also facilitating targeted drug delivery to specific locations within the body. However, detailed understanding of the in vivo activity of the nanocarrier-drug conjugate is required prior to regulatory approval as a safe and effective treatment strategy. A comprehensive understanding of how nanocarriers travel to, and interact with, the intended target is required in order to optimize the dosing strategy, reduce potential off-target effects, and unwanted toxic effects. Raman spectroscopy has received much interest as a mechanism for label-free, non-invasive imaging of nanocarrier modes of action in vivo. Advanced Raman imaging techniques, including coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), are paving the way for rigorous evaluation of nanocarrier activity at the single-cell level. This review focuses on the development of Raman imaging techniques to study organic nanocarrier delivery in cells and tissues