Multiplexed measurement of molecular interactions using hyper-spectral imaging and multi-parametric detection

Lead Research Organisation: MRC Centre Cambridge
Department Name: UNLISTED

Abstract

A large number of molecules cooperate in an intricate network of interactions for the maintenance of the structural integrity, the metabolism and the function of the living cell. The spatio-temporal localization of each molecule and their propensity to interact with each other regulate the complexity of life. Among modern techniques, fluorescence microscopy is one of the most important and productive allowing to see structures and molecules in their natural biochemical environment and to probe molecular dynamics and interactions.The challenge for engineering and physics in optical microscopy is to provide tools that could offer the highest spatio-temporal resolution with the capability to decode the complex network of molecular interactions by the development of technologies and methods that, at the same time, may provide cost-effective and user-friendly instruments that could be of widespread use in the biomedical community.This project focuses on the development of a novel architecture for a spectrograph that will permit to characterize fluorescence emission (excitation and emission spectra, fluorescence anisotropy and fluorescence lifetime) in a quantitative and efficient manner. By the exploitation of Foerster resonance energy transfer and other photophysical phenomena, it is indeed possible to probe molecular interactions. The system that will be developed will offer the opportunity to image complex biochemical events with high spatial resolution retaining a comparatively high temporal resolution (1 minute) owing to the use of parallel acquisition. Typical systems make use of multiple detectors at the detriment of simplicity of use and costs or sequential acquisitions that require several minutes for the acquisition of one (multi-parametric) image. The novel architecture will offer parallel acquisition with a single detector and, by the use of a novel solid-state detector (time-gated single-photon avalanche photodiodes) and a supercontinuum light source, will provide excellent versatility of use at comparatively low costs.These instruments and the related methods of analysis will be essential for the understanding of molecular mechanisms of crucial importance for both human physiology and pathologies such as neurodegenerative diseases. In fact, neurodegenerative diseases like Alzheimer's disease and Parkinson's disease are posing an increasing economical and social threat for the modern aging society. Therefore, the developed system will be used to investigate a molecular pathway that involves the cooperation between alpha-synuclein and the protein 14-3-3. Alpha-synuclein aggregates are the main hallmark of Parkinson's disease. Furthermore, mutations in the alpha-synuclein gene are responsible for a genetic variant of Parkinson's disease. 14-3-3s exhibit a degree of homology with alpha-synuclein and both proteins share common substrates. Are 14-3-3s and alpha-synuclein concurring in the regulation of common substrates such as the protein kinase C? Which is the role of this signalling pathway in neurodegeneration? Fortunately, technologies and methods such as the one proposed in this project will provide an answer to these and other intriguing questions in the near future.

Publications

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Esposito A (2009) Quantitative fluorescence microscopy techniques. in Methods in molecular biology (Clifton, N.J.)

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Jeyasekharan AD (2010) DNA damage regulates the mobility of Brca2 within the nucleoplasm of living cells. in Proceedings of the National Academy of Sciences of the United States of America

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Kaminski Schierle GS (2011) A FRET sensor for non-invasive imaging of amyloid formation in vivo. in Chemphyschem : a European journal of chemical physics and physical chemistry

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Mauritz JM (2010) Biophotonic techniques for the study of malaria-infected red blood cells. in Medical & biological engineering & computing

Related Projects

Project Reference Relationship Related To Start End Award Value
EP/F044011/1 01/01/2009 01/11/2009 £342,082
EP/F044011/2 Transfer EP/F044011/1 02/11/2009 01/01/2012 £252,945
 
Description During this fellowship I have developed novel microscopy tool to analyse fluorescence emitted by biological samples. We have been able to enhance the biochemical resolution of microscopes and we are currently using this capability to multiplex a number of biochemical pathways within the living cells.

With these systems, we hope to decode how biochemical pathways encode for cellular decisions.
Exploitation Route We are still developing these techniques, but currently I've moved to a medical research institute. These technologies are thus applied to tumour imaging and to biochemical multiplexing routinely.
Sectors Healthcare,Other

 
Description During this fellowship I have developed novel microscopy tool to analyse fluorescence emitted by biological samples. We have been able to enhance the biochemical resolution of microscopes and we are currently using this capability to multiplex a number of biochemical pathways within the living cells. With these systems, we hope to decode how biochemical pathways encode for cellular decisions.
First Year Of Impact 2010
Sector Healthcare,Other
 
Title FluoSPAD 
Description A linear array of time-resolved SPAD to achieve fast spectrally resolved FLIM and HDIM 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact We have several systems and these are developed by David Stoppa at FBK, a collaborator of this grant. The SPAD camera is developed by the FBK team, but we have developed the microscopy system around this sensor. One system is installed at Chemical Engineering at Uni. Cambridge and another system is routinely developed at the MRC CU. Other systems are at the academic collaborator. We have demonstrated the use if this detector for FLIM detection, its use for biology and broad-band cavity ring down spectroscopy 
 
Title Hyper-dimensional imaging microscopy 
Description This is a new type of microscopy that permits the simultaneous measurements of fluorescence lifetime, spectra and anisotropy without the use of filters or analysers 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This unique system is constantly updated and used for tumour imaging and biochemical multiplexing at the MRC CU 
 
Title Spectropolarimetry 
Description This is a new type of laser scanning confocal microscope that permits the simultaneous detection of anisotropy and fluorescence spectra 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact One system is installed at the MRC CU and a second system is installed at University of Utrecht and operated to investigate biological samples