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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.

 

Comprehensive understanding of interconnected networks within the brain requires access to high resolution information within large field of views and over time. Currently, methods that enable mapping structural changes of the entire brain in vivo are extremely limited. Third harmonic generation (THG) can resolve myelinated structures, blood vessels, and cell bodies throughout the brain without the need for any exogenous labeling. Together with deep penetration of long wavelengths, this enables in vivo brain‐mapping of large fractions of the brain in small animals and over time. Here, we demonstrate that THG microscopy allows non‐invasive label‐free mapping of the entire brain of an adult vertebrate,Danionella dracula, which is a miniature species of cyprinid fish. We show this capability in multiple brain regions and in particular the identification of major commissural fiber bundles in the midbrain and the hindbrain. These features provide readily discernable landmarks for navigation and identification of regional‐specific neuronal groups and even single neurons during in vivo experiments. We further show how this label‐free technique can easily be coupled with fluorescence microscopy and used as a comparative tool for studies of other species with similar body features toDanionella, such as zebrafish (Danio rerio)and tetras (Trochilocharax ornatus). This new evidence, building on previous studies, demonstrates how small size and relative transparency, combined with the unique capabilities of THG microscopy, can enable label‐free access to the entire adult vertebrate brain.

 

Much of fluorescence-based microscopy involves detection of if an object is present or absent (i.e., binary detection). The imaging depth of three-dimensionally resolved imaging, such as multiphoton imaging, is fundamentally limited by out-of-focus background fluorescence, which when compared to the in-focus fluorescence makes detecting objects in the presence of noise difficult. Here, we use detection theory to present a statistical framework and metric to quantify the quality of an image when binary detection is of interest. Our treatment does not require acquired or reference images, and thus allows for a theoretical comparison of different imaging modalities and systems.

 

Multiphoton fluorescence microscopy enables deepin vivoimaging by using long excitation wavelengths to increase the penetration depth of ballistic photons and nonlinear excitation to suppress the out-of-focus fluorescence. However, the imaging depth of multiphoton microscopy is limited by tissue scattering and absorption. This fundamental depth limit for two-photon microscopy has been studied theoretically and experimentally. Long wavelength three-photon fluorescence microscopy was developed to image beyond the depth limit of two-photon microscopy and has achieved unprecedentedin vivoimaging depth. Here we extend the theoretical framework for characterizing the depth limit of two-photon microscopy to three-photon microscopy. We further verify the theoretical predictions with experimental results from tissue phantoms. We demonstrate experimentally that high spatial resolution diffraction-limited imaging at a depth of 10 scattering mean free paths, which is nearly twice the transport mean free path, is possible with multiphoton microscopy. Our results indicate that the depth limit of three-photon microscopy is significantly beyond what has been achieved in biological tissues so far, and further technological development is required to reach the full potential of three-photon microscopy.

 

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.

 

The mouse subventricular zone (SVZ) produces neurons throughout life. It is useful for mechanism discovery and is relevant for regeneration. However, the SVZ is deep, significantly restricting live imaging since current methods do not extend beyond a few hundred microns. We developed and adapted three-photon microscopy (3PM) for non-invasive deep brain imaging in live mice, but its utility in imaging the SVZ niche was unknown. Here, with fluorescent dyes and genetic labeling, we show successful 3PM imaging in the whole SVZ, extending to a maximum depth of 1.5 mm ventral to the dura mater. 3PM imaging distinguished multiple SVZ cell types in postnatal and juvenile mice. We also detected fine processes on neural stem cells interacting with the vasculature. Previous live imaging removed overlying cortical tissue or lowered lenses into the brain, which could cause inflammation and alter neurogenesis. We found that neither astrocytes nor microglia become activated in the SVZ, suggesting 3PM does not induce major damage in the niche. Thus, we show for the first time 3PM imaging of the SVZ in live mice. This strategy could be useful for intravital visualization of cell dynamics, molecular, and pathological perturbation and regenerative events.

 

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.

 

Three-photon microscopy has been increasingly adopted for probing neural activities beyond the typical two-photon imaging depth. In this review, we outline the unique properties that differentiate three-photon microscopy from two-photon microscopy forin vivoimaging in biological samples, especially in the mouse brain. We present a systematic summary of the optimization of three-photon imaging parameters for neural imaging, based on their effects on calcium imaging quality and perturbation to brain tissues. Furthermore, we review the existing techniques for volumetric imaging and discuss their prospects in mesoscale three-photon imaging in deep tissue.

 

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. 

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.