KEYWORDS: In vivo imaging, Light sources and illumination, Confocal microscopy, Signal to noise ratio, Optical imaging, Neuroimaging, Crosstalk, Brain, Tissues, Tissue optics
Advancements in genetically encoded voltage indicators (GEVIs) have made it possible to measure cellular membrane potential changes optically. But performing GEVI imaging in vivo remains highly challenging due to factors such as low GEVI concentrations, modest signal dynamic range, tissue scattering, out-of-focus fluorescence. To address these challenges, we developed a microscopy technique that take advatanges from both widefield targeted illumination and confocal background rejection, enabling high SNR low crosstalk GEVI imaging across millimeter fields-of-view, at supra-kilohertz frame rates, over extended durations, and at high penetration depths. We demonstrate our technique under a variety of imaging conditions across multiple brain regions and with different classes of GEVIs.
Recent improvements in genetically encoded voltage indicators have enabled optical imaging of action potentials and subthreshold membrane voltage from single neurons in the mammalian brain. However, most current voltage imaging techniques can only simultaneously sample a few cell, limited either by strong background or small field-of-view. We show that, both theoretically and experimentally, targeted illumination with a widefield microscopy can significantly improve voltage imaging performance by improving signal contrast and reducing background cross-contamination. With such improvements, we demonstrated large-scale voltage imaging with fully genetically encoded voltage indicator SomArchon from tens of neurons over a large anatomical area, while maintaining signal contrast over a prolonged recording duration of several continuous minutes.
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, in this article we review 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 article, 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.
We present a single-shot multiplane widefield imaging strategy using a z-splitter prism, which can be assembled from off-the-self components and only requires a single camera. We further introduce a novel extended-volume 3D deconvolution strategy to suppress far-out-of-focus fluorescence background to significantly improve the contrast of our recorded images, conferring to our system a capacity for quasi optical sectioning. By swapping in different z-splitter configurations, we can prioritize high speed or large 3D field-of-view imaging depending on the application of interest. Moreover, our system can be readily applied to a variety of imaging modalities in addition to fluorescence, such as phase-contrast and darkfield imaging, making it a versatile tool for a wide range of biological or biomedical imaging applications.
Ultrasound has been recently explored as a new modality for neural modulation. However, one of the challenges in ultrasound neural modulation is that delivery of transcranial ultrasound would inevitably go through the skull, and eventually reach the cochlear through bone transduction. Moreover, the presence of skull will compromise ultrasound focus, resulting in poor spatial resolution. Here, we developed a miniaturized Fiber-Optoacoustic Converter (FOC), which has a diameter of 600 μm, and can convert nano-second laser pulses into omni-directional acoustic waves through the optoacoustic effect. The ball shaped FOC is composed of one ZnO /epoxy based diffusion layer and two graphite/epoxy based absorption layer. The radiofrequency spectrum of the generated US frequency ranges from 0.1-5 MHz, with multiple frequencies peaks at 0.5, 1 and 3MHz. Using this FOC system, we show that ultrasound can directly activate individual cortical neuron in vitro, and generate intracellular Ca2+ transient without neural damage. We next demonstrate that the FOC is activates neurons with a radius of 500 μm around the FOC tip, delivering superior spatial resolution. The stimulation effect is specific to neurons, but not glial cells. We also provide evidence of transient mechanical disturbance of neuronal membrane as the mechanism for FOC neural modulation. Finally, we combine FOC neural modulation with electrophysiology, and achieve direct and spatially confined neural stimulation in vivo.
Generation of ultrasound using the optoacoustic effect has received increasing attention in the field of imaging and translational medicine. However, none of the current optoacoustic converters has been used for neural modulation. Here, we developed a miniaturized Fiber-Optoacoustic Converter (FOC), which has a diameter of 600 μm, and can convert nano-second laser pulses into acoustic waves through the optoacoustic effect. The ball shaped FOC is composed of one ZnO /epoxy based diffusion layer and two graphite/epoxy based absorption layer. The radiofrequency spectrum of the generated US frequency ranges from 0.1-5 MHz, with multiple frequencies peaks at 0.5, 1 and 3MHz. Compared to traditional ultrasound transducers, the FOC system has the advantages of miniaturized size, superior spatial resolution, and produces omnidirectional propagating acoustic wave. Using this FOC system, we show that ultrasound can directly activate individual cortical neuron in vitro with a radius of 500 μm around the FOC tip, and generate intracellular Ca2+ transient without neural damage. Neural activation is the consequence of mechanical disturbance of neuronal membrane, rather than direct laser or photothermal stimulation. Finally, we combine FOC neural modulation with electrophysiology, and achieve direct and spatially confined neural stimulation in vivo. The FOC system opens new possibilities to use optoacoustic effect as a new method for precise neural modulation.
Fast, volumetric imaging over large scales has been a long-standing challenge in biological microscopy. To address this issue, we developed a variant of confocal microscopy that provides simultaneous multiplane imaging over large field of view and at video rate. Our apparatus, called multi-Z confocal microscopy, differs from a conventional confocal microscope in both its illumination and detection parts. First, axially elongated illumination is achieved by under filling the back aperture of the microscope objective. The resulting low NA provides an axial extent of the illumination of the order of 100 µm. The light from the sample is then collected by the same objective, now taking advantage of the full NA which ensures high collection efficiency, and send to the detection unit. The latter is comprised by four reflecting pinholes axially distributed in the image plane such that they are conjugated to different depths within the sample. Each detection channel spans a probe volume at a different depth and volumetric imaging is obtained by simply combining the four channels.
In our current configuration, each imaging plane covers a field of view of 1.2 mm and the distance between two planes is equal to 25 µm. In other words, we image 1200x1200x100 µm3 at 30Hz. We first demonstrated the applicability of our technique by imaging entire C. elegans in vivo with a cellular resolution. Secondly we applied our technique to image multiple layers of neurons in mouse brain. We were able to record the activity of 550 neurons, with 100-150 neurons present in each imaging plane.
Jacob Bernstein, Xue Han, Michael Henninger, Emily Ko, Xiaofeng Qian, Giovanni Talei Franzesi, Jackie McConnell, Patrick Stern, Robert Desimone, Edward Boyden
Many neural disorders are associated with aberrant activity in specific cell types or neural projection pathways
embedded within the densely-wired, heterogeneous matter of the brain. An ideal therapy would permit correction of
activity just in specific target neurons, while leaving other neurons unaltered. Recently our lab revealed that the
naturally-occurring light-activated proteins channelrhodopsin-2 (ChR2) and halorhodopsin (Halo/NpHR) can, when
genetically expressed in neurons, enable them to be safely, precisely, and reversibly activated and silenced by pulses
of blue and yellow light, respectively. We here describe the ability to make specific neurons in the brain light-sensitive,
using a viral approach. We also reveal the design and construction of a scalable, fully-implantable optical
prosthetic capable of delivering light of appropriate intensity and wavelength to targeted neurons at arbitrary 3-D
locations within the brain, enabling activation and silencing of specific neuron types at multiple locations. Finally,
we demonstrate control of neural activity in the cortex of the non-human primate, a key step in the translation of
such technology for human clinical use. Systems for optical targeting of specific neural circuit elements may enable
a new generation of high-precision therapies for brain disorders.
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