Project Details

The goal of the Nemonic Project (Next generation Multiphoton Neuroimaging Consortium) is to develop and widely disseminate state-of-the-art technology for multiphoton imaging and associated techniques to the neuroscience community, and to advance emerging technology for future innovation in multiphoton neuroimaging. Ultimately, the activities of the Nemonic project will enable new experiments and accelerate discoveries in neuroscience.

Development

1a. Extending multiphoton calcium imaging to large animals

Most two-photon imaging systems are retro-fitted microscopes for slides or dishes of cells. These are only somewhat sufficient for rodents, and far from optimal for larger animals, especially for repeated, chronic imaging for time-lapse studies. We are creating new instrumentation for chronic two-photon imaging in larger animals (monkeys and ferrets). We aim to develop a new microscope objective with a long working distance that requires no water immersion, imaging chambers suitable for chronic experiments, and strategies to track and correct brain movements to reduce imaging artifacts. (Kristina Nielsen; Johns Hopkins University, JHU)

1b. Extending multiphoton glutamate imaging to large animals

Three-photon imaging uses ultrafast pulses of light centered at wavelengths > 1 micron. Most off-the-shelf optics are not optimized for that application. Moreover, glutamate imaging requires relatively high time resolution (compared to calcium imaging). Appropriate instrumentation is not currently available. We are setting up a three-photon laser and optics to explore the parameters for three-photon imaging of calcium and glutamate in cats. We are optimizing the optics and testing three-photon imaging quality. We will perform brain imaging experiments in cats using the large field-of-view multiphoton imaging systems developed in the Smith lab. (Prakash Kara; University of Minnesota, UMN)

1c. Increasing throughput for multiphoton imaging in behaving animals

The biggest bottleneck for multiphoton imaging of neural activity in behaving mice is the weeks-to-months of daily hands-on training of animals. We are designing, constructing, and optimizing automated behavior rigs for training mice on behavior tasks (currently, two alternative, forced-choice visual discrimination tasks) in the home cages of mice. (Michael Goard; University of California Santa Barbara, UCSB)

1d. Pushing the limits of multiplexed imaging beams in multiphoton imaging

Temporally multiplexed imaging beams can increase the time resolution of imaging spatially distant regions. However, the instrumentation can be complex, and the practical limits of temporally multiplexed beams are unclear. We are developing an advanced multi-area two-photon microscope that is exploring the limits of temporally multiplexed beams. We are designing and constructing the microscope system, ensuring that its technical specifications enables large-scale sub-cellular resolution population-level imaging across the mouse cortex, and performing test case biological experiments using the system. (Jerry Chen; Boston University, BU)

1e. Integrating subcortical and cortical multiphoton imaging

Multiphoton imaging through implanted GRIN lenses has been transformative for studying heterogeneous cell populations deep (> 1 mm) in the brain. However, these applications have almost exclusively examined one brain region at a time. We are developing tools and techniques for imaging a cortical area simultaneously with a subcortical brain region, through a GRIN lens. This technology will enable new studies into how cortical and subcortical areas interact during behavior. (Garret Stuber; University of Washington, UW)

Dissemination

2a. Workshops

To train the neuroscience and engineering communities on the technology developed in this project, we hold a series of educational workshops. In these workshops, the attendees learn about the technology developed in this project, as well as multiphoton imaging more generally. They also learn about the technological headroom that is left to explore. We help engineers and neurobiologists to communicate using a key basis set of terminology. We also help all parties understand the biological demands and the technical limitations and tradeoffs of multiphoton imaging technology.

2b. Resource Sharing

Shared resources are necessary to not only disseminate but also sustain the technology developed here. We will share technical data (e.g., lens prescriptions, tolerancing analysis, and mechanical designs), computer code/software, and practical tips for applications. We will also pursue commercialization options to facilitate replication of the instrumentation we develop.

Advancement

3a. Miniaturizing multiphoton imaging instrumentation

Multiphoton imaging instrumentation is typically quite large (~ meter length scale). Applying the technology to animals typically requires head-fixation. Early attempts at miniaturization have enabled tethered movement, but the imaging limitations (e.g., field-of-view) and movement restriction have prevented these approaches from realizing the full potential. A key goal is to develop new technology to enable untethered multiphoton imaging and stimulation in awake animals. We are using our expertise in integrated photonics to build a miniaturized two-photon microscope which can be mounted on the head of the animal for in vivo neuroimaging. An auxiliary goal is to develop a relatively cheap two-photon microscope thereby lowering the barrier to entry for performing two-photon experiments, making it accessible to a larger population of researchers. (John Bowers and Luke Theogarajan; University of California Santa Barbara, UCSB)

3b. Superresolution multiphoton imaging technology

Multiphoton imaging can resolve subcellular structures, but further resolution gains could reveal important dynamics of synaptic and intracellular proteins. In this component, a key goal is to develop linear and nonlinear imaging modalities that are capable of enhanced resolution that use extended excitation sources but are compatible with scattering specimens (i.e., single element detection, rather than imaging detectors). Further, the technology should be compatible with existing imaging platforms and consequently could be readily adopted by researchers seeking to extend the capability of their imaging systems. Finally, we are pushing the efficiency of this enhanced resolution modality to produce optimized image rates using minimal excitation light. (Jeff Squier; Colorado School of Mines, CSOM)

3c. Advancement meetings

To help develop the technological headroom for multiphoton imaging in neuroscience, we will host a series of high-level technical meetings on emerging technology that is relevant to multiphoton imaging. We hope that new ideas and collaborations will emerge from these meetings.