Detection and time-tracking activation of a photosensitiser on live single colorectal cancer cells using Raman spectroscopy

Pablo, Julia Gala de; Chisholm, David; Ambler, Carrie A.; Peyman, Sally A.; Whiting, Andrew; Evans, Stephen D.

doi:10.1039/d0an01023e

Cited by 10 publications

(11 citation statements)

References 73 publications

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“…SRS imaging at 2923 cm –1 revealed blebbing of the UV-irradiated cells, an effect associated with cell death (Figure S10). , An SRS spectral sweep (2253–2181 cm –1 , 18 images) allowed comparison of the signal intensities at 2219 and 2232 cm –1 between live and UV-irradiated cells. We observed that the signal intensity at 2232 cm –1 was greatest in the UV-irradiated cells, indicating that these cells contained the greatest proportion of intact AM ester 4 compared to the non-irradiated cells (green dashed marker), which possessed a greater signal intensity at 2219 cm –1 .…”

Section: Resultsmentioning

confidence: 99%

Determination of Intracellular Esterase Activity Using Ratiometric Raman Sensing and Spectral Phasor Analysis

et al. 2023

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Carboxylesterases (CEs) are a class of enzymes that catalyze the hydrolysis of esters in a variety of endogenous and exogenous molecules. CEs play an important role in drug metabolism, in the onset and progression of disease, and can be harnessed for prodrug activation strategies. As such, the regulation of CEs is an important clinical and pharmaceutical consideration. Here, we report the first ratiometric sensor for CE activity using Raman spectroscopy based on a bisarylbutadiyne scaffold. The sensor was shown to be highly sensitive and specific for CE detection and had low cellular cytotoxicity. In hepatocyte cells, the ratiometric detection of esterase activity was possible, and the result was validated by multimodal imaging with standard viability stains used for fluorescence microscopy within the same cell population. In addition, we show that the detection of localized ultraviolet damage in a mixed cell population was possible using stimulated Raman scattering microscopy coupled with spectral phasor analysis. This sensor demonstrates the practical advantages of low molecular weight sensors that are detected using ratiometric Raman imaging and will have applications in drug discovery and biomedical research.

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Section: Resultsmentioning

confidence: 99%

Determination of Intracellular Esterase Activity Using Ratiometric Raman Sensing and Spectral Phasor Analysis

et al. 2023

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“…The contribution of each of these chemical species can be further discriminated with the help of multivariate methods, such as Principal Components Analysis (PCA), which decompose the dataset in new representations (loadings or eigenvectors) 48 – 50 . Such procedure is especially appealing in the case of spectroscopic data, as the extracted eigenvectors can be regarded as spectra on their own, enabling their interpretation in terms of the chemical species present 36 , 51 – 60 . While such methods are often prone to sample-to-sample variations 52 , 61 , requiring extensive pre-processing of the data sets and risking the introduction of artifacts, our surface potential modulation strategy allows acquisition of a large number of spectra locally (e.g., at a given spot and sample).…”

Section: Resultsmentioning

confidence: 99%

Surface potential modulation as a tool for mitigating challenges in SERS-based microneedle sensors

Brasiliense

Park

Berns

et al. 2022

Sci Rep

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Raman spectroscopic-based biosensing strategies are often complicated by low signal and the presence of multiple chemical species. While surface-enhanced Raman spectroscopy (SERS) nanostructured platforms are able to deliver high quality signals by focusing the electromagnetic field into a tight plasmonic hot-spot, it is not a generally applicable strategy as it often depends on the specific adsorption of the analyte of interest onto the SERS platform. This paper describes a strategy to address this challenge by using surface potential as a physical binding agent in the context of microneedle sensors. We show that the potential-dependent adsorption of different chemical species allows scrutinization of the contributions of different chemical species to the final spectrum, and that the ability to cyclically adsorb and desorb molecules from the surface enables efficient application of multivariate analysis methods. We demonstrate how the strategy can be used to mitigate potentially confounding phenomena, such as surface reactions, competitive adsorption and the presence of molecules with similar structures. In addition, this decomposition helps evaluate criteria to maximize the signal of one molecule with respect to others, offering new opportunities to enhance the measurement of analytes in the presence of interferants.

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“…This facilitates analysis of the diffusion front between the area of ROS production and the immediate surrounding area, and its time evolution. To exemplify this experimentally, we have used a ROS-generating (photosensitizing) fluorescent dye LightOx58, previously demonstrated to have localized cell distribution (de Pablo et al, 2018;Chisholm et al, 2019; in cellulo excitation/emission = 405/ 510 nm; de Pablo et al, 2018Pablo et al, , 2020 and the ROS-reporter, CellROX (Ex/Em = 633/665 nm).…”

Section: •-mentioning

confidence: 99%

“…LightOx58 was acquired from LightOx Ltd. (de Pablo et al, 2018(de Pablo et al, , 2020Chisholm et al, 2019). Working solutions were diluted from 1 mM stock solutions dissolved using dimethyl sulfoxide.…”

Section: Cell Labelingmentioning

confidence: 99%

Bullseye Analysis: A Fluorescence Microscopy Technique to Detect Local Changes in Intracellular Reactive Oxygen Species (ROS) Production

Hughes

Chisholm

Whiting

et al. 2022

Microscopy and Microanalysis

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Reactive oxygen species (ROS) are naturally produced compounds that play important roles in cell signaling, gene regulation, and biological defense, including involvement in the oxidative burst that is central to the anti-microbial actions of macrophages. However, these highly reactive, short-lived radical species also stimulate cells to undergo programmed cell death at high concentrations, as well as causing detrimental effects such as oxidation of macromolecules at more moderate levels. Imaging ROS is highly challenging, with many researchers working on the challenge over the past 10–15 years without producing a definitive method. We report a new fluorescence microscopy-based technique, Bullseye Analysis. This methodology is based on concepts provided by the FRAP (Fluorescence Recovery after Photobleaching) technique and refined to evidence the spatiotemporal production of ROS, and the subsequent consequences, on a subcellular scale. To exemplify the technique, we have used the ROS-reporter dye, CellROX, and the ROS-inducing photosensitizer, LightOx58, a potent source of ROS compared with UV irradiation alone. Further validation of the technique was carried out using differing co-stains, notably Mitotracker and JC-1.

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Detection and time-tracking activation of a photosensitiser on live single colorectal cancer cells using Raman spectroscopy

Abstract:
Raman spectroscopy has been used to observe uptake, metabolism and response of single-cells to drugs. Photodynamic therapy is based on the use of light, a photosensitiser and oxygen to destroy...

Cited by 10 publications

References 73 publications

Determination of Intracellular Esterase Activity Using Ratiometric Raman Sensing and Spectral Phasor Analysis

Determination of Intracellular Esterase Activity Using Ratiometric Raman Sensing and Spectral Phasor Analysis

Surface potential modulation as a tool for mitigating challenges in SERS-based microneedle sensors

Bullseye Analysis: A Fluorescence Microscopy Technique to Detect Local Changes in Intracellular Reactive Oxygen Species (ROS) Production

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Detection and time-tracking activation of a photosensitiser on live single colorectal cancer cells using Raman spectroscopy

Abstract: Raman spectroscopy has been used to observe uptake, metabolism and response of single-cells to drugs. Photodynamic therapy is based on the use of light, a photosensitiser and oxygen to destroy...

Cited by 10 publications

References 73 publications

Determination of Intracellular Esterase Activity Using Ratiometric Raman Sensing and Spectral Phasor Analysis

Determination of Intracellular Esterase Activity Using Ratiometric Raman Sensing and Spectral Phasor Analysis

Surface potential modulation as a tool for mitigating challenges in SERS-based microneedle sensors

Bullseye Analysis: A Fluorescence Microscopy Technique to Detect Local Changes in Intracellular Reactive Oxygen Species (ROS) Production

Contact Info

Product

Resources

About

Abstract:
Raman spectroscopy has been used to observe uptake, metabolism and response of single-cells to drugs. Photodynamic therapy is based on the use of light, a photosensitiser and oxygen to destroy...