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Measurements of three-photon action cross-sections for fluorescein (dissolved in water, pH ∼11.5) are presented in the excitation wavelength range from 1154 to 1500 nm in ∼50 nm steps. The excitation source is a femtosecond wavelength tunable non-collinear optical parametric amplifier, which has been spectrally filtered with 50 nm full width at half maximum band pass filters. Cube-law power dependance is confirmed at the measurement wavelengths. The three-photon excitation spectrum is found to differ from both the one- and two-photon excitation spectra. The three-photon action cross-section at 1154 nm is more than an order of magnitude larger than those at 1450 and 1500 nm (approximately three times the wavelength of the one-photon excitation peak), which possibly indicates the presence of resonance enhancement. 

Abstract Understanding the brain requires understanding neurons’ functional responses to the circuit architecture shaping them. Here we introduce the MICrONS functional connectomics dataset with dense calcium imaging of around 75,000 neurons in primary visual cortex (VISp) and higher visual areas (VISrl, VISal and VISlm) in an awake mouse that is viewing natural and synthetic stimuli. These data are co-registered with an electron microscopy reconstruction containing more than 200,000 cells and 0.5 billion synapses. Proofreading of a subset of neurons yielded reconstructions that include complete dendritic trees as well the local and inter-areal axonal projections that map up to thousands of cell-to-cell connections per neuron. Released as an open-access resource, this dataset includes the tools for data retrieval and analysis^1,2. Accompanying studies describe its use for comprehensive characterization of cell types^3–6, a synaptic level connectivity diagram of a cortical column⁴, and uncovering cell-type-specific inhibitory connectivity that can be linked to gene expression data^4,7. Functionally, we identify new computational principles of how information is integrated across visual space⁸, characterize novel types of neuronal invariances⁹and bring structure and function together to uncover a general principle for connectivity between excitatory neurons within and across areas^10,11. 

We built a simple and versatile setup to measure tissue ballistic and total transmission with customizable wavelength range, spatial resolution, and sample sizes. We performed ballistic transmission and total transmission measurements of overlying structures from biological samplesex vivo. We obtained spatially resolved transmission maps to reveal transmission heterogeneity from five microscale tissue samples:Danionellaskin, mouse skull bone, mosquito cuticle, wasp cuticle, and rat dura over a wide spectral range from 450 nm to 1624 nm at a spatial resolution of ∼25µm for ballistic transmission measurements and ∼50µm for total transmission measurements. We expect our method can be straightforwardly applied to measuring the transmission of other samples. The measurement results will be valuable for multiphoton microscopy. The total transmission of a sample is important for the collection of multiphoton excited fluorescence and the assessment of laser-induced sample heating. The ballistic transmission determines the excitation power at the focus and hence the fluorescence signal generation. Therefore, knowledge of ballistic transmission, total transmission, and transmission heterogeneity of overlying structures of animals and organs are essential to determine the optimal excitation wavelength and fluorophores for non-invasive multiphoton microscopy. 

Optical coherence microscopy (OCM) uses interferometric detection to capture the complex optical field with high sensitivity, which enables computational wavefront retrieval using back-scattered light from the sample. Compared to a conventional wavefront sensor, aberration sensing with OCM via computational adaptive optics (CAO) leverages coherence and confocal gating to obtain signals from the focus with less cross-talk from other depths or transverse locations within the field-of-view. Here, we present an investigation of the performance of CAO-based aberration sensing in simulation, bead phantoms, andex vivomouse brain tissue. We demonstrate that, due to the influence of the double-pass confocal OCM imaging geometry on the shape of computed pupil functions, computational sensing of high-order aberrations can suffer from signal attenuation in certain spatial-frequency bands and shape similarity with lower order counterparts. However, by sensing and correcting only low-order aberrations (astigmatism, coma, and trefoil), we still successfully corrected tissue-induced aberrations, leading to 3× increase in OCM signal intensity at a depth of ∼0.9 mm in a freshly dissectedex vivomouse brain. 

Abstract Emergent trends in the device development for neural prosthetics have focused on establishing stimulus localization, improving longevity through immune compatibility, reducing energy re-quirements, and embedding active control in the devices. Ultrasound stimulation can single-handedly address several of these challenges. Ultrasonic stimulus of neurons has been studied extensively from 100 kHz to 10 MHz, with high penetration but less localization. In this paper, a chip-scale device consisting of piezoelectric Aluminum Nitride ultrasonic transducers was engineered to deliver gigahertz (GHz) ultrasonic stimulus to the human neural cells. These devices provide a path towards complementary metal oxide semiconductor (CMOS) integration towards fully controllable neural devices. At GHz frequencies, ultrasonic wavelengths in water are a few microns and have an absorption depth of 10–20 µm. This confinement of energy can be used to control stimulation volume within a single neuron. This paper is the first proof-of-concept study to demonstrate that GHz ultrasound can stimulate neuronsin vitro. By utilizing optical calcium imaging, which records calcium ion flux indicating occurrence of an action potential, this paper demonstrates that an application of a nontoxic dosage of GHz ultrasonic waves$$(\ge 0.05\frac{W}{c{m}^{2}})$$ $\left(\ge 0.05\frac{W}{c{m}^2}\right)$caused an average normalized fluorescence intensity recordings >1.40 for the calcium transients. Electrical effects due to chip-scale ultrasound delivery was discounted as the sole mechanism in stimulation, with effects tested atα = 0.01 statistical significance amongst all intensities and con-trol groups. Ionic transients recorded optically were confirmed to be mediated by ion channels and experimental data suggests an insignificant thermal contributions to stimulation, with a predicted increase of 0.03^oCfor$$1.2\frac{W}{c{m}^{2}}\cdot $$ $1.2\frac{W}{c{m}^2}\cdot$This paper paves the experimental framework to further explore chip-scale axon and neuron specific neural stimulation, with future applications in neural prosthetics, chip scale neural engineering, and extensions to different tissue and cell types. 

Abstract The last decade has seen the development of a wide set of tools, such as wavefront shaping, computational or fundamental methods, that allow us to understand and control light propagation in a complex medium, such as biological tissues or multimode fibers. A vibrant and diverse community is now working in this field, which has revolutionized the prospect of diffraction-limited imaging at depth in tissues. This roadmap highlights several key aspects of this fast developing field, and some of the challenges and opportunities ahead. 

We present a platform for parallel production of standalone, untethered electronic sensors that are truly microscopic, i.e., smaller than the resolution of the naked eye. This platform heterogeneously integrates silicon electronics and inorganic microlight emitting diodes (LEDs) into a 100-μm-scale package that is powered by and communicates with light. The devices are fabricated, packaged, and released in parallel using photolithographic techniques, resulting in ∼10,000 individual sensors per square inch. To illustrate their use, we show proof-of-concept measurements recording voltage, temperature, pressure, and conductivity in a variety of environments.