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Single-molecule imaging with SPAD array detection

Project description

Advancing single-molecule imaging

Conventional optical microscopes achieve a maximum resolution of 250 nm, posing significant limitations to structural biology efforts. The main goal of the EU-funded SM-SPAD project is to develop a 3D single-molecule fluorescence lifetime imaging technique to visualise macromolecules smaller than 100 nm. The proposed approach circumvents size restrictions by stochastically switching on single fluorescent molecules and determining their position in the image plane. SM-SPAD scientists will improve this technique through a combination of technologies that offer very high spatial and temporal resolution perfectly suitable for studying complex biological samples. Application of this technique in neuroscience will enable the localisation of large protein complexes responsible for the development of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s.

Objective

The resolution limit of about 250 nm in conventional optical microscopes is problematic in the study of structural biology, since proteins, macromolecules and nuclear acids are typically much smaller than 100 nm. Single-molecule localization microscopy is able to circumvent this limit by sequentially and stochastically switching on/activating single fluorescent molecules and determining their position in the image plane. E.g. MINFLUX demonstrated a 3D resolution of 6 nm. However, the point-detection system in MINFLUX does not allow directly recording the image of the fluorescent molecules in the image plane, which is required for the localization, thus a rather complex and slow beam-scanning approach is required. Secondly, the technique does not leverage the fluorescence lifetime information, which can provide nanometer-scale information on the structure of interest, e.g. via Förster resonance energy transfer. In this project, both limitations will be tackled:

The main goal of the SM-SPAD project is to develop a 3D single-molecule fluorescence lifetime imaging technique for structural biology. The goal will be reached by combining the concept of MINFLUX with three new ideas: (i) 3D motionless structured illumination and structured detection for improved spatial resolution in all three dimensions; (ii) fluorescence antibunching analysis to speed up the data acquisition by enabling simultaneous localization of several active molecules; (iii) SM level fluorescence lifetime analysis to extract the maximum amount of information from the sample.

The proposed molecular-scale imaging technique, with very high spatial and temporal resolution, is perfectly suited for the study of complex biological samples. SM-SPAD is, in particular, promising in the field of neuroscience research, in which the organization of large protein complexes are of high interest, as they may play a crucial role in the development of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s .

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Programme(s)

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Topic(s)

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Funding Scheme

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MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)

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Call for proposal

Procedure for inviting applicants to submit project proposals, with the aim of receiving EU funding.

(opens in new window) H2020-MSCA-IF-2019

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Coordinator

FONDAZIONE ISTITUTO ITALIANO DI TECNOLOGIA
Net EU contribution

Net EU financial contribution. The sum of money that the participant receives, deducted by the EU contribution to its linked third party. It considers the distribution of the EU financial contribution between direct beneficiaries of the project and other types of participants, like third-party participants.

€ 171 473,28
Address
VIA MOREGO 30
16163 GENOVA
Italy

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Region
Nord-Ovest Liguria Genova
Activity type
Research Organisations
Links
Total cost

The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.

€ 171 473,28
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