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A Versatile Multichannel Instrument for Measurement of Ratiometric Fluorescence Intensity and Phosphorescence Lifetime.

ABSTRACT: Optical biosensing is being actively investigated for minimally-invasive monitoring of key biomarkers both in vitro and in vivo. However, typical benchtop instruments are not portable and are not well suited to high-throughput, real-time analysis. This paper presents a versatile multichannel instrument for measurement of emission intensity and lifetime values arising from luminescent biosensor materials. A detailed design description of the opto-electronic hardware as well as the control software is provided, elaborating a flexible, user-configurable system that may be customized or duplicated for a wide range of applications. This article presents experimental measurements that prove the in vitro and in vivo functionality of the system. Such tools may be adopted for many research and development purposes, including evaluation of new biosensor materials, and may also serve as prototypes for future miniaturized handheld or wearable devices.

SUBMITTER: Zavareh AT

PROVIDER: S-EPMC8635115 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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A Versatile Multichannel Instrument for Measurement of Ratiometric Fluorescence Intensity and Phosphorescence Lifetime.

Zavareh Amir Tofighi AT Ko Brian B Roberts Jason J Elahi Sakib S McShane Michael J MJ

IEEE access : practical innovations, open solutions 20210721

Optical biosensing is being actively investigated for minimally-invasive monitoring of key biomarkers both <i>in vitro</i> and <i>in vivo</i>. However, typical benchtop instruments are not portable and are not well suited to high-throughput, real-time analysis. This paper presents a versatile multichannel instrument for measurement of emission intensity and lifetime values arising from luminescent biosensor materials. A detailed design description of the opto-electronic hardware as well as the c ...[more]

PMID: 34858770

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Project description:Cytoplasmic viscosity is a crucial parameter in determining rates of diffusion-limited reactions. Changes in viscosity are associated with several diseases, whilst nuclear viscosity determines gene integrity, regulation and expression. Yet how drugs including DNA-damaging agents affect viscosity is unknown. We demonstrate the use of a platinum complex, Pt[L]Cl, that localizes efficiently mostly in the nucleus as a probe for nuclear viscosity. The phosphorescence lifetime of Pt[L]Cl is sensitive to viscosity and provides an excellent tool to investigate the impact of DNA damage. We show using Fluorescence Lifetime Imaging (FLIM) that the lifetime of both green and red fluorescent proteins (FP) are also sensitive to changes in cellular viscosity and refractive index. However, Pt[L]Cl proved to be a more sensitive viscosity probe, by virtue of microsecond phosphorescence lifetime versus nanosecond fluorescence lifetime of FP, hence greater sensitivity to bimolecular reactions. DNA damage was inflicted by either a two-photon excitation, one-photon excitation microbeam and X-rays. DNA damage of live cells causes significant increase in the lifetime of either Pt[L]Cl (HeLa cells, 12.5-14.1 µs) or intracellularly expressed mCherry (HEK293 cells, 1.54-1.67 ns), but a decrease in fluorescence lifetime of GFP from 2.65 to 2.29 ns (in V15B cells). These values represent a viscosity change from 8.59 to 20.56 cP as well as significant changes in the refractive index (RI), according to independent calibration. Interestingly DNA damage localized to a submicron region following a laser microbeam induction showed a whole cell viscosity change, with those in the nucleus being greater than the cytoplasm. We also found evidence of a by-stander effect, whereby adjacent un-irradiated cells also showed nuclear viscosity change. Finally, an increase in viscosity following DNA damage was also observed in bacterial cells with an over-expressed mNeonGreen FP, evidenced by the change in its lifetime from 2.8 to 2.4 ns.

Dataset Information

A Versatile Multichannel Instrument for Measurement of Ratiometric Fluorescence Intensity and Phosphorescence Lifetime.

Publications

A Versatile Multichannel Instrument for Measurement of Ratiometric Fluorescence Intensity and Phosphorescence Lifetime.

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