Neurophotonic tools for microscopic measurements and manipulation: status report.

Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics' agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, this status report reviews an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion report, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed, and provide an outlook for the future directions.

All-Optical Electrophysiology in hiPSC-Derived Neurons With Synthetic Voltage Sensors.

Voltage imaging and "all-optical electrophysiology" in human induced pluripotent stem cell (hiPSC)-derived neurons have opened unprecedented opportunities for high-throughput phenotyping of activity in neurons possessing unique genetic backgrounds of individual patients. While prior all-optical electrophysiology studies relied on genetically encoded voltage indicators, here, we demonstrate an alternative protocol using a synthetic voltage sensor and genetically encoded optogenetic actuator that generate robust and reproducible results. We demonstrate the functionality of this method by measuring spontaneous and evoked activity in three independent hiPSC-derived neuronal cell lines with distinct genetic backgrounds.

Comparing the fundamental imaging depth limit of two-photon, three-photon, and non-degenerate two-photon microscopy.

We have systematically characterized the degradation of imaging quality with depth in deep brain multi-photon microscopy, utilizing our recently developed numerical model that computes wave propagation in scattering media. The signal-to-background ratio (SBR) and the resolution determined by the width of the point spread function are obtained as functions of depth. We compare the imaging quality of two-photon (2PM), three-photon (3PM), and non-degenerate two-photon microscopy (ND-2PM) for mouse brain imaging. We show that the imaging depth of 2PM and ND-2PM are fundamentally limited by the SBR, while the SBR remains approximately invariant with imaging depth for 3PM. Instead, the imaging depth of 3PM is limited by the degradation of the resolution, if there is sufficient laser power to maintain the signal level at large depth. The roles of the concentration of dye molecules, the numerical aperture of the input light, the anisotropy factor g, noise level, input laser power, and the effect of temporal broadening are also discussed.

Efficient non-degenerate two-photon excitation for fluorescence microscopy.

Non-degenerate two-photon excitation (ND-TPE) has been explored in two-photon excitation microscopy. However, a systematic study of the efficiency of ND-TPE to guide the selection of fluorophore excitation wavelengths is missing. We measured the relative non-degenerate two-photon absorption cross-section (ND-TPACS) of several commonly used fluorophores (two fluorescent proteins and three small-molecule dyes) and generated 2-dimensional ND-TPACS spectra. We observed that the shape of a ND-TPACS spectrum follows that of the corresponding degenerate two-photon absorption cross-section (D-TPACS) spectrum, but is higher in magnitude. We found that the observed enhancements are higher than theoretical predictions.

Measurement of the relative non-degenerate two-photon absorption cross-section for fluorescence microscopy.

In non-degenerate two-photon microscopy (ND-TPM), the required energy for fluorescence excitation occurs via absorption of two photons of different energies derived from two synchronized pulsed laser beams. ND-TPM is a promising imaging technology offering flexibility in the choice of the photon energy for each beam. However, a formalism to quantify the efficiency of two-photon absorption (TPA) under non-degenerate excitation, relative to the resonant degenerate excitation, is missing. Here, we derive this formalism and experimentally validate our prediction for a common fluorophore, fluorescein. An accurate quantification of non-degenerate TPA is important to optimize the choice of photon energies for each fluorophore.

Phosphorescent Pt(ii) complexes spatially arrayed in micellar polymeric nanoparticles providing dual readout for multimodal imaging.

In this paper we report phosphorescent Pt(ii) complexes as monomers which can be directly incorporated into growing polymers. Due to the amphiphilic nature of the polymers they can self-assemble into micellar nanoparticles, where the phosphorescent Pt(ii) complexes can arrange selectively in the core or shell of the nanoparticles. The complexes enable dual orthogonal imaging, made possible by the heavy metal, which enhances the contrast for these micelles in electron microscopy and facilitates spin-orbit coupling that turns on microsecond lifetime luminescence.

Deep 2-photon imaging and artifact-free optogenetics through transparent graphene microelectrode arrays.

Recent advances in optical technologies such as multi-photon microscopy and optogenetics have revolutionized our ability to record and manipulate neuronal activity. Combining optical techniques with electrical recordings is of critical importance to connect the large body of neuroscience knowledge obtained from animal models to human studies mainly relying on electrophysiological recordings of brain-scale activity. However, integration of optical modalities with electrical recordings is challenging due to generation of light-induced artifacts. Here we report a transparent graphene microelectrode technology that eliminates light-induced artifacts to enable crosstalk-free integration of 2-photon microscopy, optogenetic stimulation, and cortical recordings in the same in vivo experiment. We achieve fabrication of crack- and residue-free graphene electrode surfaces yielding high optical transmittance for 2-photon imaging down to ~ 1 mm below the cortical surface. Transparent graphene microelectrode technology offers a practical pathway to investigate neuronal activity over multiple spatial scales extending from single neurons to large neuronal populations.

Plasma Membrane is Compartmentalized by a Self-Similar Cortical Actin Meshwork.

A broad range of membrane proteins display anomalous diffusion on the cell surface. Different methods provide evidence for obstructed subdiffusion and diffusion on a fractal space, but the underlying structure inducing anomalous diffusion has never been visualized because of experimental challenges. We addressed this problem by imaging the cortical actin at high resolution while simultaneously tracking individual membrane proteins in live mammalian cells. Our data confirm that actin introduces barriers leading to compartmentalization of the plasma membrane and that membrane proteins are transiently confined within actin fences. Furthermore, superresolution imaging shows that the cortical actin is organized into a self-similar meshwork. These results present a hierarchical nanoscale picture of the plasma membrane.