Two-photon Light Field with Neuro-active Sensing for Fast Volumetric Neural Microcircuit Readout
Lead Research Organisation:
Imperial College London
Department Name: Bioengineering
Abstract
Underlying our every sensation, thought, memory, decision and action are 100 billion neurons communicating through trillions of electrical impulses each second. Over the past century, neuroscientists have explored brain function on primarily two scales, that of single neurons (i.e., by impaling them with electrodes) and that of entire brain regions (i.e. with electroencephalogram, EEG, and functional magnetic resonance imaging, fMRI). However, between these two scales lies a large knowledge gap surrounding how neurons interact in networks to process and store information, form memories and generate actions.
Over the past 10 years, geneticists have developed methods to control and read out brain cell activity with light. They can render neurons sensitive to light to activate or silence them when illuminated with certain wavelengths. In addition, neurons can be made to "glow" or become more fluorescent when active. These "optogenetic" tools make it possible to connect single-neuron properties (i.e., through electrode studies) with functions evolving on the population level (through fMRI and EEG). To achieve this, optical engineers must first overcome a key challenge: the mammalian brain severely scatters and distorts light, resulting in blurry images and thus confusion about which neuron is active. Here we propose to overcome this limitation by utilizing the "optogenetic" ability to activate individual neurons with light in rapid succession. Specifically, we will activate each neuron throughout a brain volume in turn to determine each one's "signature"; that is, the blurry, distorted light pattern it generates when active. We will then use this collection of activity signatures to rapidly and precisely determine which neuron activates and when during subsequent spontaneous activity. We will implement this "collection" approach with a three-dimensional (3D) imaging strategy called "light field." While traditional imaging captures focused images for objects lying in a single plane, "light field" captures perspectives from different angles within a single shapshot. The "light field" approach thus enables us to track neuronal activity simultaneously throughout a volume a brain tissue rather than within a single plane. This novel combination of "light field" imaging with active sensing will significantly increase the speed (10-fold) with which we can track the activity of single neurons throughout a volume. In the near future, development of faster, more sensitive cameras and sensors could increase our instrument's volume capture rates to 100-fold compared to the current state-of-the-art. Moreover, here we will, for the first time, implement "light field imaging" in "two-photon" mode. "Two-photon" is a method to excite fluorescence that is used widely in biomedical research. In contrast to the blue/green wavelengths previously used with "light field," "two-photon" utilizes near-infrared wavelengths that are far less scattered than blue and green, enabling researchers to image deep in scattering tissues. Our new two-photon light field instrument will decrease distortion and thus enable us to image deeper into the brain.
By combining targeted neural activation with 3D light-field imaging, we will overcome a key barrier to understanding how neurons interact in networks. With our new instrument, neuroscientists will at last be able to collect data on how neurons work together to process and store information, make decisions and effectuate actions. A detailed understanding of these network-level processes will inform the design of new therapies for neuronal diseases and disorders, such as Alzheimer's, in which these functions are compromised.
Over the past 10 years, geneticists have developed methods to control and read out brain cell activity with light. They can render neurons sensitive to light to activate or silence them when illuminated with certain wavelengths. In addition, neurons can be made to "glow" or become more fluorescent when active. These "optogenetic" tools make it possible to connect single-neuron properties (i.e., through electrode studies) with functions evolving on the population level (through fMRI and EEG). To achieve this, optical engineers must first overcome a key challenge: the mammalian brain severely scatters and distorts light, resulting in blurry images and thus confusion about which neuron is active. Here we propose to overcome this limitation by utilizing the "optogenetic" ability to activate individual neurons with light in rapid succession. Specifically, we will activate each neuron throughout a brain volume in turn to determine each one's "signature"; that is, the blurry, distorted light pattern it generates when active. We will then use this collection of activity signatures to rapidly and precisely determine which neuron activates and when during subsequent spontaneous activity. We will implement this "collection" approach with a three-dimensional (3D) imaging strategy called "light field." While traditional imaging captures focused images for objects lying in a single plane, "light field" captures perspectives from different angles within a single shapshot. The "light field" approach thus enables us to track neuronal activity simultaneously throughout a volume a brain tissue rather than within a single plane. This novel combination of "light field" imaging with active sensing will significantly increase the speed (10-fold) with which we can track the activity of single neurons throughout a volume. In the near future, development of faster, more sensitive cameras and sensors could increase our instrument's volume capture rates to 100-fold compared to the current state-of-the-art. Moreover, here we will, for the first time, implement "light field imaging" in "two-photon" mode. "Two-photon" is a method to excite fluorescence that is used widely in biomedical research. In contrast to the blue/green wavelengths previously used with "light field," "two-photon" utilizes near-infrared wavelengths that are far less scattered than blue and green, enabling researchers to image deep in scattering tissues. Our new two-photon light field instrument will decrease distortion and thus enable us to image deeper into the brain.
By combining targeted neural activation with 3D light-field imaging, we will overcome a key barrier to understanding how neurons interact in networks. With our new instrument, neuroscientists will at last be able to collect data on how neurons work together to process and store information, make decisions and effectuate actions. A detailed understanding of these network-level processes will inform the design of new therapies for neuronal diseases and disorders, such as Alzheimer's, in which these functions are compromised.
Technical Summary
Our goal is to increase the temporal resolution and throughput of neuronal functional fluorescence readout through a novel light field acquisition instrument and analysis strategy for highly scattering mammalian brain tissue. Light field microscopy captures both the angle and position of light emitted from a volume, enabling reconstruction of multiple perspectives and planes from single data frames. Light field enables three dimensional reconstruction of fluorescent objects without scanning, and is thus a promising technology for high speed volumetric fluorescence monitoring as demonstrated by Prevedel et al. (Nature Methods, 2014) in transparent larval zebrafish and C. elegans. Mammalian brain tissue, however, is highly scattering, precluding 3D reconstruction through standard light field algorithms. Here we propose two novel methods to mitigate scattering effects. First, we will excite neuronal fluorescence in two-photon mode using near-infrared wavelengths less scattered than the visible wavelengths used in one-photon fluorescence light field microscopes. Second, we will optogenetically evoke spiking and fluorescence transients in each neuron throughout the volume in rapid succession, recording each one's blurry, distorted light field signature. We will use this collection of signatures to segment and map subsequent spontaneous activity back onto high spatial resolution images of these neurons. Our "neuro-active sensing" acquisition and analysis approach will enable fast light field processing and will leverage signal that would otherwise be lost to scattering. We estimate that our instrument will increase the speed of mammalian live network volumetric imaging by a factor of 10 to 100, thus advancing one of Neuroscience's long-standing goals: to observe and manipulate living mammalian neural microcircuits in real time and in closed-loop.
Planned Impact
1. Economic: Our project will lead to the commercial dissemination of a turn-key microscopy and analysis system. Prime commercial partners for this include two UK companies Scientica Ltd. and Cairn Research Ltd. who have well established neurophysiology client bases (10k+ laboratories worldwide). We will ensure that the commercial design features the modularity and alignment tools necessary to simplify upgrades with future advancements in cameras and lasers.
2. Health: A long-term impact of our proposal is to improve basic understanding of neural network function critical to identifying novel, effective theraputic strategies. Neurological diseases such as dementia, epilepsy, stroke and mental health disorders cost the UK 112 billion GBP per year, accounting for medical costs, loss of productivity and forced early retirement. Treatment for diseases impacting cognition, memory, learning and motor control advance slowly and remain primitive due to a fundamental lack of understanding of how these system function. Our technology will fill critical data-gaps in animal models at the network level necessary to understand how these systems function in healthy mammals as well as those suffering from neurological disease.
3. Training the UK's cross-disciplinary technical base: A key element to the project's success will be the training and contribution of the two PDRAs who will work full time to develop, test, and refine the two-photon light field system and rapid analysis strategies. These PDRAs will receive specialized training in communicating and synthesizing effectively across signal processing, photonics, neurophysiology, and computation neuroscience disciplines. This rare cross-disclinary training will well prepare these researchers for neurotechnology development careers both in academic and industrial contexts.
4. Public enthusiasm for STEM approaches to biological research: Our hands-on exhibit at the annual Imperial College Festival and other outreach venues will generate enthusiasm for STEM approaches to brain research and encourage students to consider training in these domains. The exhibit will introduce the basic concepts of neuronal communication (action potentials and synapses) as well as the technical challenges of observing neurons as they interact in large networks (size, scattering, contrast). Key optical concepts such as diffraction, scattering, holography, and light-field photography will be demonstrated. We will show how we have applied these concepts to improve network-level neuronal imaging with videos generated from experiments.
2. Health: A long-term impact of our proposal is to improve basic understanding of neural network function critical to identifying novel, effective theraputic strategies. Neurological diseases such as dementia, epilepsy, stroke and mental health disorders cost the UK 112 billion GBP per year, accounting for medical costs, loss of productivity and forced early retirement. Treatment for diseases impacting cognition, memory, learning and motor control advance slowly and remain primitive due to a fundamental lack of understanding of how these system function. Our technology will fill critical data-gaps in animal models at the network level necessary to understand how these systems function in healthy mammals as well as those suffering from neurological disease.
3. Training the UK's cross-disciplinary technical base: A key element to the project's success will be the training and contribution of the two PDRAs who will work full time to develop, test, and refine the two-photon light field system and rapid analysis strategies. These PDRAs will receive specialized training in communicating and synthesizing effectively across signal processing, photonics, neurophysiology, and computation neuroscience disciplines. This rare cross-disclinary training will well prepare these researchers for neurotechnology development careers both in academic and industrial contexts.
4. Public enthusiasm for STEM approaches to biological research: Our hands-on exhibit at the annual Imperial College Festival and other outreach venues will generate enthusiasm for STEM approaches to brain research and encourage students to consider training in these domains. The exhibit will introduce the basic concepts of neuronal communication (action potentials and synapses) as well as the technical challenges of observing neurons as they interact in large networks (size, scattering, contrast). Key optical concepts such as diffraction, scattering, holography, and light-field photography will be demonstrated. We will show how we have applied these concepts to improve network-level neuronal imaging with videos generated from experiments.
Organisations
Publications
Howe CL
(2022)
Comparing synthetic refocusing to deconvolution for the extraction of neuronal calcium transients from light fields.
in Neurophotonics
Quicke P
(2019)
Corrigendum: Single-Neuron Level One-Photon Voltage Imaging With Sparsely Targeted Genetically Encoded Voltage Indicators.
in Frontiers in cellular neuroscience
Quicke P
(2022)
Voltage imaging reveals the dynamic electrical signatures of human breast cancer cells.
in Communications biology
Quicke P
(2023)
All-Optical Methods to Study Neuronal Function
Quicke P
(2019)
Single-Neuron Level One-Photon Voltage Imaging With Sparsely Targeted Genetically Encoded Voltage Indicators.
in Frontiers in cellular neuroscience
Quicke P.
(2019)
Calculation of high numerical aperture lightfield microscope point spread functions
in Optics InfoBase Conference Papers
Title | Learning with neurons: A musical journey |
Description | Learning with Neurons - A musical journey is an art-science project created by Dr. Foust's Neuroscience Laboratory at Imperial College London and London based harpist Irantzu Agirre. The core of this multidisciplinary production, aimed to all audiences, is the urge to share with the society the findings of Imperial College London's Neuroscience and Neuroimaging research community. Join our neuron puppets in their fantastic quest through the brain and be mesmerised by amazing movies of real neurons and live music for solo harp from different eras. |
Type Of Art | Performance (Music, Dance, Drama, etc) |
Year Produced | 2022 |
Impact | The four performances at the Great Exhibition Road Festival were well attended. Audience members asked many follow up questions about the neurons shown, the brain, and the imaging technology that was developed. |
URL | https://www.greatexhibitionroadfestival.co.uk/event/neurons-musical-journey/ |
Description | Novel imaging results: * We have demonstrated that sparsely and strongly expressed genetically encoded voltage indicators (GEVIs) can be imaged with single-neuron resolution in widefield and light-field modes. * We have discovered that the fluorescent calcium reporter CaSIR-1 can be used to readout the activity of Chronos-expressing neurons without spectral crosstalk. CaSIR-1 thus becomes a key candidate molecule for our NeuroActive Sensing strategy. * We have demonstrated for the first time that light field imaging can image neuronal electical changes (with genetically encoded voltage indicators) in four dimensions. * We have imaged CaSIR-1 loaded neurons in light field mode and have been able to track action potential-evoked calcium signals in four dimensions. We have characterized the signal-to-noise ratios and signal localization of the light field image series and compared them to standard widefield imaging. Novel image analysis techniques: * We have developed an algorithm to detect the 3D positions of neurons in a volume from a single light field frame with high accuracy and outstanding robustness to scattering. We have improved the accuracy and drastically improved the computational speed of this analysis through integration into a machine-learning framework. * We developed a method to calculate the point spread function for high numerical aperture lenses. * We developed a method to remove the artifacts seen at the native object plane during light field reconstruction. Our method outperforms classic volume reconstruction approaches (e.g. Richardson-Lucy) in terms of average computational time and image quality. * We developed optical, mathematical, machine-learning and neural network-based methods to locate neurons and track their activity in scattering brain tissue. |
Exploitation Route | * Neuroscientists can use CaSIR-1 to read out the activity of neurons sensitive to blue/green light, with out spuriously activating them. * Neuroscientists can use light field for high speed, four dimensional membrane potential imaging in neurons. * Physiologists can apply the optical, mathematical, machine-learning and neural network-based methods we developed to locate neurons and track their activity in scattering brain tissue, mitigating to some extent effects of tissue scattering. * Experts in optics and signal processes can build on our strategies for further expansions in e.g., field or view, resolution, acquisition speed, and signal processing speed, accuracy, and generalizability. |
Sectors | Healthcare |
URL | http://opticalneurophysiology.org |
Description | Capital Award support for Early Career Researchers at Imperial College London |
Amount | £600,000 (GBP) |
Funding ID | EP/S017852/1 |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Sector | Public |
Country | United Kingdom |
Start | 11/2018 |
End | 11/2020 |
Description | Imaging membrane voltage light fields: new views into the pathophysiology of cancer |
Amount | £25,000 (GBP) |
Organisation | United Kingdom Research and Innovation |
Sector | Public |
Country | United Kingdom |
Start | 04/2020 |
End | 08/2020 |
Title | Light field microscopy for imaging neuronal activity living brain tissue |
Description | This microscope prototype enables rapid volumetric imaging of fluorescent indicators of calcium and membrane potential in living brain tissue. The microscope integrates: * Widefield transmitted light microscopy * Widefield epifluorescence microscopy * Lightfield epifluorescence microscopy * Two-photon laser scanning microscopy * Two-photon temporally-focussed widefield selective volume illumination * A holographic light sculpting system for two-photon optogenetic stimulation |
Type Of Material | Improvements to research infrastructure |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | This microscope has enabled the first volumetric sub/cellular resolution imaging of neuronal membrane potential with a genetically-encoded voltage indicator. |
URL | http://opticalneurophysiology.org |
Title | 3D Localization for Light-Field Microscopy via Convolutional Sparse Coding on Epipolar Images. |
Description | We have developed an algorithm to detect the 3D positions of neurons in a volume from a single light field frame with high accuracy and outstanding robustness to scattering. |
Type Of Material | Data analysis technique |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | This model has enabled localisation of neurons in scattering tissue from light field images. More generally, it has enabled the 3D position of targets to be extracted from light field images with high accuracy. |
Title | Analysis method for removing the artifacts seen at the native object plane during light field reconstruction. |
Description | We developed a method to remove the artifacts seen at the native object plane during light field reconstruction. Our method outperforms classic volume reconstruction approaches (e.g. Richardson-Lucy) in terms of average computational time and image quality. |
Type Of Material | Data analysis technique |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | Our new method enables computationally efficient, reduced-artefact volumetric reconstruction from light field images. This work was presented at the IEEE International Conference on Acoustics, Speech and Signal Processing (ICASSP). |
Title | Calculation of high numerical aperture lightfield microscope point spread functions |
Description | We developed a method to calculate the point spread function for high numerical aperture lenses commonly used in modern light field microscopes. |
Type Of Material | Computer model/algorithm |
Year Produced | 2019 |
Provided To Others? | Yes |
Impact | This calculation has formed the basis for 3D volumetric reconstruction from light field images. |
Description | Creating a mind: an exhibit and worshop at the 2019 Imperial Festival |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Individuals and families constructed neurons out of willow and coloured tissue, arranging them in a three-dimensional network. |
Year(s) Of Engagement Activity | 2019 |
URL | https://www.greatexhibitionroadfestival.co.uk/event/creating-mind/ |
Description | Learning with Neurons: A Musical Journey performed at the Great Exhibition Road Festival 2022 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | We performed (x4) a multimedia show featuring images and videos of neurons set to live harp music interleaved with a dialogue between puppet neurons description how neurons learn in circuits and how this is enabled by new optical imaging technologies. |
Year(s) Of Engagement Activity | 2022 |
URL | https://www.greatexhibitionroadfestival.co.uk/event/neurons-musical-journey/ |
Description | Mentoring for In2Science |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | I mentored three A-level students through the In2Science Programme. We met 3 times virtually, including a virtual lab tour. In2Science placements promote social mobility and diversity in science, technology, engineering and maths."In2scienceUK empowers young people from disadvantaged backgrounds to achieve their potential through life-changing opportunities that give them insights into STEM careers and research and boosts their skills and confidence." |
Year(s) Of Engagement Activity | 2021 |
URL | https://in2scienceuk.org/ |
Description | NOVA Meet a Scientist |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | The PI led a 'Meet a Scientist' session consisting of: - One scientist talking about their area of work for 10 - 15 minutes followed by a quiz that they ask the children based on the topic they covered - 8 families on the call facilitated by a staff member from NOVA via Zoom |
Year(s) Of Engagement Activity | 2021 |
URL | https://www.novanew.org.uk/ |
Description | Spoke at Virtual British Science Week for Ricards Lodge Womens High School |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | The project PI recorded a talk for the Virtual British Science Week at Ricards Lodge Womens High School, including: - Who you are and your background - How you got into your STEM career - Who are your mentor(s) - What it's like being a woman in your field + answering questions submitted by students |
Year(s) Of Engagement Activity | 2021 |