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Novel approach to real-time flash photolysis and confocal [Ca2+] imaging.

ABSTRACT: Flash photolysis of "caged" compounds using ultraviolet light is a powerful experimental technique for producing rapid changes in concentrations of bioactive signaling molecules. Studies that employ this technique have used diverse strategies for controlling the spatial and temporal application of light to the specimen. In this paper, we describe a new system for flash photolysis that delivers light from a pulsed, adjustable intensity laser through an optical fiber coupled into the epifluorescence port of a commercial confocal microscope. Photolysis is achieved with extremely brief (5 ns) pulses of ultraviolet light (355 nm) that can be synchronized with respect to confocal laser scanning. The system described also localizes the UV intensity spatially so that uncaging only occurs in defined subcellular regions; moreover, because the microscope optics are used in localization, the photolysis volume can be easily adjusted. Experiments performed on rat ventricular myocytes loaded with the Ca(2+) indicator fluo-3 and the Ca(2+) cage o-nitrophenyl ethylene glycol bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid (NP-EGTA) demonstrate the system's capabilities. Localized intracellular increases in [Ca(2+)] can trigger sarcoplasmic reticular Ca(2+) release events such as Ca(2+) sparks and, under certain conditions, regenerative Ca(2+) waves. This relatively simple and inexpensive system is, therefore, a useful tool for examining local signaling in the heart and other tissues.

SUBMITTER: Sobie EA

PROVIDER: S-EPMC2794040 | biostudies-other | 2007 Jul

REPOSITORIES: biostudies-other

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Novel approach to real-time flash photolysis and confocal [Ca2+] imaging.

Sobie Eric A EA Kao Joseph P Y JP Lederer W J WJ

Pflugers Archiv : European journal of physiology 20070224 4

Flash photolysis of "caged" compounds using ultraviolet light is a powerful experimental technique for producing rapid changes in concentrations of bioactive signaling molecules. Studies that employ this technique have used diverse strategies for controlling the spatial and temporal application of light to the specimen. In this paper, we describe a new system for flash photolysis that delivers light from a pulsed, adjustable intensity laser through an optical fiber coupled into the epifluorescen ...[more]

PMID: 17323075

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Project description:PurposeFLASH radiotherapy (FLASH-RT) is a novel irradiation modality with ultra-high dose rates (>40 Gy/s) that have shown tremendous promise for its ability to enhance normal tissue sparing while maintaining comparable tumor cell eradication toconventional radiotherapy (CONV-RT). Due to its extremely high dose rates, clinical translation of FLASH-RT is hampered by risky delivery and current limitations in dosimetric devices, which cannot accurately measure, in real time, dose at deeper tissue. This work aims to investigate ionizing radiation acoustic imaging (iRAI) as a promising image-guidance modality for real-time deep tissue dose measurements during FLASH-RT. The underlying hypothesis is that iRAI can enable mapping of dose deposition with respect to surrounding tissue with a single linear accelerator (linac) pulse precision in real time. In this work, the relationship between iRAI signal response and deposited dose was investigated as well as the feasibility of using a proof-of-concept dual-modality imaging system of ultrasound and iRAI for treatment beam co-localization with respect to underlying anatomy.MethodsTwo experimental setups were used to study the feasibility of iRAI for FLASH-RT using 6 MeV electrons from a modified Varian Clinac. First, experiments were conducted using a single element focused transducer to take a series of point measurements in a gelatin phantom, which was compared with independent dose measurements using GAFchromic film. Secondly, an ultrasound and iRAI dual-modality imaging system utilizing a phased array transducer was used to take coregistered two-dimensional (2D) iRAI signal amplitude images as well as ultrasound B-mode images, to map the dose deposition with respect to surrounding anatomy in an ex vivo rabbit liver model with a single linac pulse precision.ResultsUsing a single element transducer, iRAI measurements showed a highly linear relationship between the iRAI signal amplitude and the linac dose per pulse (r2 = 0.9998) with a repeatability precision of 1% and a dose resolution error <2.5% in a homogenous phantom when compared to GAFchromic film dose measurements. These phantom results were used to develop a calibration curve between the iRAI signal response and the delivered dose per pulse. Subsequently, a normalized depth dose curve was generated that agreed with film measurements with an RMSE of 0.0243, using correction factors to account for deviations in measurement conditions with respect to calibration. Experiments on the ex-vivo rabbit liver model demonstrated that a 2D iRAI image could be generated successfully from a single linac pulse, which was fused with the B-mode ultrasound image to provide information about the beam position with respect to surrounding anatomy in real time.ConclusionThis work demonstrates the potential of using iRAI for real-time deep tissue dosimetry in FLASH-RT. Our results show that iRAI signals are linear with dose and can accurately map the delivered radiation dose with respect to soft tissue anatomy. With its ability to measure dose for individual linac pulses at any location within surrounding soft tissue while identifying where that dose is being delivered anatomically in real time, iRAI can be an indispensable tool to enable safe and efficient clinical translation of FLASH-RT.

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Novel approach to real-time flash photolysis and confocal [Ca2+] imaging.

Publications

Novel approach to real-time flash photolysis and confocal [Ca2+] imaging.

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