CTN – Technology Resource and Development Projects

TRD #1 – Jerome Mertz – Large scale volumetric monitoring of neurovascular physiology

Two-photon microscopy (2PM) has gained enormous popularity over the years for its capacity to provide high resolution images from deep within scattering samples. However, because 2PM is a scanning technique, it is generally slow. Our goal will be to develop a 2PM that provides kilohertz-rate, multiplane imaging specifically designed for the study of neurovascular physiology. Our strategy will be based on a technique we have recently developed called “reverberation” 2PM, which combines advantages of speed, light efficiency, and depth penetration in scattering tissues. Specifically, we make use of a reverberation loop in the illumination path of our microscope that splits laser pulses into a series of beam foci, forming near-instantaneous axial scans while delivering the full illumination power to the sample. In this manner, we obtain simultaneous multiplane imaging from an arbitrary number of depths (in principle, the number of planes is limited only by laser power / tissue heating) throughout the sample. Our goal will be to develop an instrument that can simultaneously monitor cellular (neuronal and glial) activity and vascular blood flow dynamics over large volumes in the mouse cerebral cortex spanning almost a cubic millimeter. The innovation includes:

Scaling up our newly invented reverberation multiphoton microscopy, which enables conventional multiphoton microscopes to perform simultaneous multiplane and multichannel imaging, significantly increasing the information capacity of conventional multiphoton microscopes for general biomedical imaging applications.
Innovative combination of reverberation microscopy with special scan patterns to measure the speed of blood flow in ~100 capillaries every second and simultaneously measure capillary diameters and neuronal activity.

TRD #2 – Xue Han – Genetic tagging of specific types of brain cells for neurovascular investigation

This TRD aims to develop a set of genetically encoded fluorescent voltage and Ca²⁺ sensors, as well as optogenetic actuators, targeted to distinct cell types and synapses, to enable multi-color and multimodal interrogation of genetically defined cells. These genetic probes will be engineered according to end users/CPs’ specifications and specific experimental needs. The ultimate goal is to enable the capability of performing simultaneous voltage and Ca²⁺ imaging and optogenetic control of genetically defined cells, linking gene expression profiles of individual cells with their physiological functions, such as neuronal circuit computation and regulation of cerebral blood flow. Such technology will facilitate understanding of how different brain cells contribute to neuronal circuit function and neurovascular brain physiology. The innovation includes:

Novel viral vector design, named microRNA-guided neuron tag, ‘‘mAGNET,’’ for targeting of cell types based on their microRNA regulation pathways.
Novel mAGNETs – targeted to specific cell types in mice and human (tissues) – to introduce optical probes, optogenetic sensors and actuators to these cells to allow their optical interrogation
Extending mAGNET technology to measurement and interrogation of astrocytes

TRD #3 – David Boas – Speckle imaging of neurovascular physiology

Measurements of cerebral blood flow with high spatial and temporal resolution have advanced dramatically over the last 20 years with the advent of several complementary technologies including multi-photon microscopy, laser speckle contrast imaging, optical coherence tomography, and diffuse correlation spectroscopy. Collaborative projects are driving the need for further advancement of these technologies to improve the spatial coverage and depth penetration while maintaining or increasing temporal resolution and minimizing the reduction of spatial resolution. We are thus advancing methods for measuring cerebral microvascular flow dynamics and tissue dynamics associated with cellular function to impact studies of neurovascular coupling and the neurovascular unit in health and in disease. In this TRD project, we are focused on advancing technologies that exploit speckle to measure blood flow and tissue dynamics. Methods include mesoscopic interferometric laser speckle contrast imaging, microscopic optical coherence tomography of blood and cellular dynamics, as well as whole brain imaging of speckle dynamics by functional ultrasound imaging. The common theme tying these aims together is that the underlying theory of speckle dynamics is the same. The aims are complementary in that the different measurement modalities permit imaging at different length scales and depth resolution. The innovation includes:

Novel interferometric Dynamic Laser Speckle Imaging method for quantitative high-speed 3D measurements of blood flow with mesoscopic resolution.
Novel Optical Coherence Tomography method to enable more robust investigation neuronal activity.
Novel analysis of speckle dynamics applied to functional ultrasound method for quantifying microvascular blood flow speeds in the whole rodent brain with high spatiotemporal resolution.
Integration of functional ultrasound with photoacoustics to enable improved quantitation of oxygen delivery and consumption throughout the rodent brain.

TRD #4 – Maria Angela Franceschini – Assessment of human brain hemodynamics & metabolism with light

TRD4 aims to develop a comprehensive non-invasive functional optical measurement method to monitor cerebral blood flow and oxygen metabolism in humans. Non-invasive monitoring of brain physiology is an unmet need in health care, and in clinical and basic neuroscience. Near-infrared spectroscopy (NIRS) and the closely related functional NIRS (fNIRS) are established neuromonitoring and neuroimaging methodologies that enable scientists to study brain activity by non-invasively monitoring hemoglobin concentration (Hb) and oxygenation (SO₂). These biomarkers, while useful for assessing oxygen availability to the brain, are not sufficient to disentangle between vascular and neuronal contributions to functional activation and pathological conditions. Additional measures of perfusion and metabolism are possible with optical methods and need to be optimized and combined to provide a more exhaustive and robust neuromonitoring tool. In this project we aim to significantly advance diffuse correlation spectroscopy (DCS) for measuring cerebral blood flow more robustly and to combine it with multi-spectral NIRS to enable estimation of oxygen metabolism and the redox state of cytochrome c oxidase (oxCCO). The innovation includes:

Use of 1064 nm light for diffuse correlation spectroscopy (DCS) measurements of cerebral blood flow to significantly improve SNR and depth sensitivity through the opportune combination of reduced scattering and overall attenuation, increased permitted light power, increased number of photons per energy unit, and slower auto-correlation decay.
Use of an interferometric detection approach with multi-mode light collection to enable the use of non-photon counting detectors, solving the problem of the lack of 1064 nm SWIR photon counting detectors suitable for DCS, and, by using a photodiode array and developing our own custom electronics, provide a cost effective solution to expand to a larger number of DCS channels.

DCS operation to 1064 nm frees the spectra in the NIR for concurrent acquisition of hemoglobin concentration and CCO redox state changes.