Widefield in vivo imaging system with two fluorescence and two reflectance channels, a single sCMOS detector, and shielded illumination.

Significance: Widefield microscopy of the entire dorsal part of mouse cerebral cortex enables large-scale ("mesoscopic") imaging of different aspects of neuronal activity with spectrally compatible fluorescent indicators as well as hemodynamics via oxy- and deoxyhemoglobin absorption. Versatile and cost-effective imaging systems are needed for large-scale, color-multiplexed imaging of multiple fluorescent and intrinsic contrasts. Aim: We aim to develop a system for mesoscopic imaging of two fluorescent and two reflectance channels. Approach: Excitation of red and green fluorescence is achieved through epi-illumination. Hemoglobin absorption imaging is achieved using 525- and 625-nm light-emitting diodes positioned around the objective lens. An aluminum hemisphere placed between objective and cranial window provides diffuse illumination of the brain. Signals are recorded sequentially by a single sCMOS detector. Results: We demonstrate the performance of our imaging system by recording large-scale spontaneous and stimulus-evoked neuronal, cholinergic, and hemodynamic activity in awake, head-fixed mice with a curved "crystal skull" window expressing the red calcium indicator jRGECO1a and the green acetylcholine sensor GRAB ACh 3.0 . Shielding of illumination light through the aluminum hemisphere enables concurrent recording of pupil diameter changes. Conclusions: Our widefield microscope design with a single camera can be used to acquire multiple aspects of brain physiology and is compatible with behavioral readouts of pupil diameter.

Visual stimulation drives retinotopic acetylcholine release in the mouse visual cortex.

Cholinergic signaling is involved with a variety of brain functions including learning and memory, attention, and behavioral state modulation. The spatiotemporal characteristics of neocortical acetylcholine (ACh) release in response to sensory inputs are poorly understood, but a lack of intra-region topographic organization of cholinergic projections from the basal forebrain has suggested diffuse release patterns and volume transmission. Here, we use mesoscopic imaging of fluorescent ACh sensors to show that visual stimulation results in ACh release patterns that conform to a retinotopic map of visual space in the mouse primary visual cortex, suggesting new modes of functional cholinergic signaling in cortical circuits.x.

Widefield in vivo imaging system with two fluorescence and two reflectance channels, a single sCMOS detector, and shielded illumination.

SIGNIFICANCE: Widefield microscopy of the entire dorsal part of mouse cerebral cortex enables large-scale (mesoscopic) imaging of neuronal activity with fluorescent indicators as well as hemodynamics via oxy- and deoxyhemoglobin absorption. Versatile and cost-effective imaging systems are needed for large-scale, color-multiplexed imaging of multiple fluorescent and intrinsic contrasts. AIM: Develop a system for mesoscopic imaging of two fluorescent and two reflectance channels. APPROACH: Excitation of red and green fluorescence is achieved through epi-illumination. Hemoglobin absorption imaging is achieved using 525- and 625nm LEDs positioned around the objective lens. An aluminum hemisphere placed between objective and cranial window provides diffuse illumination of the brain. Signals are recorded sequentially by a single sCMOS detector. RESULTS: We demonstrate performance of our imaging system by recording large-scale spontaneous and stimulus-evoked neuronal, cholinergic, and hemodynamic activity in awake head-fixed mice with a curved crystal skull window expressing the red calcium indicator jRGECO1a and the green acetylcholine sensor GRABACh3.0 . Shielding of illumination light through the aluminum hemisphere enables concurrent recording of pupil diameter changes. CONCLUSIONS: Our widefield microscope design with single camera can be used to acquire multiple aspects of brain physiology and is compatible with behavioral readouts of pupil diameter.

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.

A Suite of Neurophotonic Tools to Underpin the Contribution of Internal Brain States in fMRI.

Recent developments in optical microscopy, applicable for large-scale and longitudinal imaging of cortical activity in behaving animals, open unprecedented opportunities to gain a deeper understanding of neurovascular and neurometabolic coupling during different brain states. Future studies will leverage these tools to deliver foundational knowledge about brain state-dependent regulation of cerebral blood flow and metabolism as well as regulation as a function of brain maturation and aging. This knowledge is of critical importance to interpret hemodynamic signals observed with functional magnetic resonance imaging (fMRI).