MEGA-FLIM: quantum technologies for megapixel time-resolved imaging and control across biological scales

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

Embryos, organs and tumours are composed of many cells, which interact with each other and communicate across distances of several cells. While it is routine to study single cells under a microscope, it is much more difficult to study collectives due to their size, light scattering properties and complexity. Fluorescence lifetime imaging (FLIM) and FRET (Forster resonance energy transfer) use the principles of energy transfer that occur when a light particle (photon) jumps from an excited fluorescent donor molecule to a nearby acceptor, thus changing the fluorescence lifetime of the donor. FLIM is used to measure close molecular interactions inside of living cells by measuring this lifetime change. We will combine physics, engineering, computation and biology to build a new light microscopic system, with extremely high spatial and temporal resolution. We call our system MEGA-FLIM and we will use it to study larger cell collectives of cells to discover how cells communicate and organise in response to both mechanical and chemical signals. MEGA-FLIM will allow much faster collection of light signals across a much larger field than previously possible. We will also develop technology to use light to control cell behaviour across these collectives using the technique called optogenetics. Our unique team of optical physicists, bioengineers and biologists is ideally placed to break down current barriers, leading to landmark discovery in each of these fields.

Why do we need a new FLIM microscope system?
Commercial systems are lacking that allow, simultaneously:
- fast acquisition (0.1 second or faster) so as to allow real-time measurements in live cells or embryos
- across a widefield area with high resolution (1 million pixels or higher), so as to allow imaging of the full cell environment and large collectives
- with high time resolution (50-100 pico seconds), so as to allow precise discrimination of lifetimes
- two-photon excitation, so as to allow precise full 3D reconstruction of cell collectives.
- widefield optogenetic activation (light-controlled cell behaviour), so as to allow study of the dynamics of collectives in the presence of complex activation stimuli that act across multiple sites.

What problems will this new system solve and what impact will it have?
-MEGA FLIM will provide a system that will allow us to interrogate living systems at molecular resolution and discover how cells collectively signal using both chemical and mechanical signals to steer when they migrate. This kind of steering allows cells to recognise each other and other cell types and to form complex patterns in 3 dimensions (like in an organ or an embryo).
-Our new system will be of great commercial interest, as it will advance capabilities in imaging and optogenetic control of cell behaviour with light.
-By building a system whereby we can discover new pathways governing how cells behave in collectives, we will gain the ability to reliably and predictably control collective cell behaviour. This discipline, known as synthetic biology, is highly desirable for medical and commercial use in building organ/tumour-on-chip systems or creating physiologically relevant systems to use in drug discovery.

Planned Impact

Embryos, organs and tumours are composed of many cells, which interact with each other and communicate across distances of several cells. While it is routine to study single cells under a microscope, it is much more difficult to study collectives due to their size, light scattering properties and complexity. Fluorescence lifetime imaging (FLIM) and FRET (Forster resonance energy transfer) use the principles of energy transfer that occur when a photon jumps from an excited fluorescent donor molecule to a nearby acceptor, thus changing the fluorescence lifetime of the donor. It is used to measure close molecular interactions inside of living cells by measuring the lifetime change. We will combine physics, engineering, computation and biology to build a new light microscopic system, with extremely high spatial and temporal resolution. We call our system MEGA-FLIM and we propose to use it to study larger cell collectives and discover how cells communicate and organise in response to both mechanical and chemical signals. MEGA-FLIM will allow much faster collection of light signals across a much larger field than previously possible. We will also develop technology to use light to control cell behaviour across these collectives using the technique of optogenetics. Our unique team of optical physicists, bioengineers and biologists is ideally placed to break down current barriers, leading to landmark discovery in each of these fields.

Why do we need a new FLIM system?
Commercial systems are lacking that allow, simultaneously:
- fast acquisition (0.1 second or faster) so as to allow real-time measurements in vivo
- across a widefield area with high resolution (1 Mpixel or higher), so as to allow imaging of the full cell environment and large collectives
- with high temporal resolution (50-100 ps), so as to allow precise discrimination of lifetimes
- two-photon excitation, so as to allow precise full 3D reconstruction of cell collectives.
- widefield optogenetic activation, so as to allow to study the dynamics of collectives in the presence of complex activation stimuli that act across multiple sites.

What problems will this new system solve and what impact will it have?
-MEGA FLIM will provide a system that will allow us to interrogate live and at molecular resolution for the first time how cells collectively signal using adhesion/mechanical sensing, G-proteins and chemical signals to steer when they migrate, to recognise each other and other cell types and to form complex patterns in 3 dimensions (like in an organ or an embryo).
-Our new system will be of great commercial interest, as it will advance capabilities in imaging and optogenetic control of cell behaviour with light.
-By building a system whereby we can discover new pathways governing how cells behave in collectives, we will gain the ability to reliably and predictably control collective cell behaviour. This discipline, known as synthetic biology, is highly desirable for medical and commercial use in building organ/tumour-on-chip systems or creating physiologically relevant systems to use in drug discovery.
-We will gain significant knowledge both in optical physics and collective cell behaviour that will impact on basic research into how organs/organisms and tumours form.

Publications

10 25 50
 
Title Ground Truth Dataset for time resolved FLIM 
Description We have created a dataset of over 1000 images to serve as ground truth to train machine learning algorithms for FLIM image analysis. We are further developing this dataset and using it to train our systems. We plan to make it available to the public as soon as we publish the first paper describing it. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2020 
Provided To Others? No  
Impact This tool is impacting our ability to develop new machine learning algorithms for FLIM image analysis. We predict that it will become useful to other groups and even to industry when we make it publicly available.