NIL Projects
Multicolor (non-degenerate) 2-photo excitation
Non-degenerate 2-photon excitation (ND-2PE) uses two independently controlled pulsed laser sources of different photon energies, or color. The first beam is tuned within the standard near infrared (NIR) range of excitation wavelengths used in degenerate 2-photon microscopy. The second laser is tuned to infrared (IR) wavelengths used in 3-photon excitation (3PE). ND-2PE provides a superior alternative to 3-photon microscopy by circumventing low 3PE cross-section while taking advantage of low scattering IR illumination. Efficiency of excitation can be further increased through (1) a systematic search for the most efficient combination of excitation wavelengths for any particular fluorophore and (2) implementation of AO to optimize the overlap of the two focal spots in scattering medium. Finally, the background (“out-of-focus”) excitation can be reduced by spatial displacement of the beams. This project is focused on the development on ND-2PE microscopy instrumentation and application of this technology to achieve deep imaging in the mouse brain.
Physiological basis of non-invasive human imaging signals

For each neuronal population (e.g., excitatory and inhibitory), the current dipole moment (measured by MEG) and CBF/CMRO2 (measured by calibrated BOLD) can be expressed as convolution of the neuronal activity for neuronal population of cell type j with time-resolved weighting factor W(t) for that cell type (that can be thought of as cell-type-specific impulse response function, IRF). The weighting factors W(t) will be determined experimentally using selective optogenetic activation of specific cell types in mice. Read more on PubMed.gov.
This project combines data acquisition in mice and humans with computational modeling.
In vivo 2-photon imaging of O2 availability

Brain activity largely relies on mitochondrial oxidative metabolism to meet the energy demands. Therefore, the oxygenation level of cerebral tissue is one of the most basic physiological parameters. As such, it can be used as a biomarker in animal models of neurodegenerative disease, either to detect a departure from normal physiology or as an objective criterion for the effectiveness of treatment. However, our ability to probe microscopic availability of O2 during different levels of neuronal activity has been limited to point measurements using O2 electrodes. Recently, in collaboration with Drs. Sava Sakadzic and David Boas, we developed a new technology for in vivo measurements of pO2. This new method is based on 2-photon imaging of phosphorescent lifetime of O2-sensitive probe PtP-C343, developed by our collaborator Dr. Sergey Vinogradov, and allows measurements of intravascular or interstitial pO2 with an unprecedented spatial resolution and is well suited for imaging of pO2 changes during functional activation. We utilize this new technology to investigate dependence of pO2 changes on the location relative to the 3-D vascular network and center of neuronal response.
Check out our new paper on estimation of Cerebral Metabolic Rate of O2 (CMRO2) using this technology.
Cellular and molecular mechanisms of neurovascular communication

Mechanistic understanding of microscopic processes that govern regulation of cerebral blood flow and metabolism is a critical component for laying a solid physiological foundation for interpreting functional neuroimaging studies and a prerequisite for development of targeted treatments in neurovascular disease. A growing body of experimental evidence, including our own, indicates that while molecules produced by increased energy metabolism do have a vasoactive effect, much of the acute cerebral blood flow response in vivo under healthy conditions is driven by vasoactive messengers related to neuronal signaling. This project combines advanced in vivo 2-photon imaging technology with optogenetics and pharmacological manipulations in the mouse cortex to identify cellular players and signaling molecules in the context of functional hyperemia.
Check out our recent publication here.
Functional imaging of human iPSCs-derived neurons integrated in mouse cortex

Quantitative imaging of calcium dynamics with 2-photon resolution

Intracellular concentration of calcium ions is a key parameter in neuronal physiology, intimately related to cell’s regulatory mechanisms and gene expression. Therefore, in healthy neurons, multiple mechanisms ensure fast restoration of calcium concentration in the cytosol following dynamic signaling events. To enable imaging of these processes, we implement fluorescent lifetime imaging microscopy (FLIM) for quantitative estimation of intracellular calcium concentration in vivo and in vitro with 2-photon resolution. Further, we apply FLIM to evaluate departures from normal calcium homeostasis in mouse models of brain disease.
In vivo microscopic biomarkers of neurodegeneration

Hemodynamic modeling in realistic microvascular networks

Modeling of neuronal populations

In vivo 2-photon imaging of cellular metabolism
