Highly flexible and compact volumetric endoscope by integrating multiple micro-imaging devices.

A light-field endoscope can simultaneously capture the three-dimensional information of in situ lesions and enables single-shot quantitative depth perception with minimal invasion for improving surgical and diagnostic accuracy. However, due to oversized rigid probes, clinical applications of current techniques are limited by their cumbersome devices. To minimize the size and enhance the flexibility, here we report a highly flexible and compact volumetric endoscope by employing precision-machined multiple micro-imaging devices (MIRDs). To further protect the flexibility, the designed MIRD with a diameter and height of 5 mm is packaged in pliable polyamide, using soft data cables for data transmission. It achieves the optimal lateral resolvability of 31 µm and axial resolvability of 255 µm, with an imaging volume over 2.3 × 2.3 × 10 mm3. Our technique allows easy access to the organism interior through the natural entrance, which has been verified through observational experiments of the stomach and rectum of a rabbit. Together, we expect this device can assist in the removal of tumors and polyps as well as the identification of certain early cancers of the digestive tract.

Hippocampal place cell remapping occurs with memory storage of aversive experiences.

Aversive stimuli can cause hippocampal place cells to remap their firing fields, but it is not known whether remapping plays a role in storing memories of aversive experiences. Here, we addressed this question by performing in vivo calcium imaging of CA1 place cells in freely behaving rats (n = 14). Rats were first trained to prefer a short path over a long path for obtaining food reward, then trained to avoid the short path by delivering a mild footshock. Remapping was assessed by comparing place cell population vector similarity before acquisition versus after extinction of avoidance. Some rats received shock after systemic injections of the amnestic drug scopolamine at a dose (1 mg/kg) that impaired avoidance learning but spared spatial tuning and shock-evoked responses of CA1 neurons. Place cells remapped significantly more following remembered than forgotten shocks (drug-free versus scopolamine conditions); shock-induced remapping did not cause place fields to migrate toward or away from the shocked location and was similarly prevalent in cells that were responsive versus non-responsive to shocks. When rats were exposed to a neutral barrier rather than aversive shock, place cells remapped significantly less in response to the barrier. We conclude that place cell remapping occurs in response to events that are remembered rather than merely perceived and forgotten, suggesting that reorganization of hippocampal population codes may play a role in storing memories for aversive events.

The human brain is able to remember experiences that occurred at specific places and times, such as a birthday party held at a particular restaurant. A part of the brain known as the hippocampus helps to store these episodic memories, but how exactly is not fully understood. Within the hippocampus are specialized neurons known as place cells which 'label' locations with unique patterns of brain activity. When we revisit a place, such as the restaurant, place cells recall the stored pattern of brain activity allowing us to recognize the familiar location. It has been shown that a new negative experience at a familiar place ­ for example, if we went back to the restaurant and had a terrible meal ­ triggers place cells to update the brain activity label associated with the location. However, it remains uncertain whether this re-labelling assists in storing the memory of the unpleasant experience. To investigate, Blair et al. used a technique known as calcium imaging to monitor place cells in the hippocampus of freely moving rats. The rats were given a new experience ­ a mild foot shock ­ at a previously explored location. Tiny cameras attached to their heads were then used to record the activity of hundreds of place cells before and after the shock. Initially, the rats remembered the aversive experience and avoided the location where they had been shocked. Over time, the rats began to return to the location; however, their place cells displayed different patterns of activity compared to their previous visits before the shock. To test whether this change in place cell activity corresponded with new memories, another group of rats were administered a mild amnesia-inducing drug before the shock, causing them to forget the experience. These rats did not avoid the shock site or show any changes in place cell activity when they revisited it. These findings imply that new events cause place cells to alter their 'label' for a location only if the event is remembered, not if it is forgotten. This indicates that alterations in place cell activity patterns may play a role in storing memories of unpleasant experiences. Having a better understanding of how episodic memories are stored could lead to better treatments for diseases that impair memory, such as Alzheimer's disease and age-related dementia.

Quantitative structured illumination microscopy via a physical model-based background filtering algorithm reveals actin dynamics.

Despite the prevalence of superresolution (SR) microscopy, quantitative live-cell SR imaging that maintains the completeness of delicate structures and the linearity of fluorescence signals remains an uncharted territory. Structured illumination microscopy (SIM) is the ideal tool for live-cell SR imaging. However, it suffers from an out-of-focus background that leads to reconstruction artifacts. Previous post hoc background suppression methods are prone to human bias, fail at densely labeled structures, and are nonlinear. Here, we propose a physical model-based Background Filtering method for living cell SR imaging combined with the 2D-SIM reconstruction procedure (BF-SIM). BF-SIM helps preserve intricate and weak structures down to sub-70 nm resolution while maintaining signal linearity, which allows for the discovery of dynamic actin structures that, to the best of our knowledge, have not been previously monitored.

Miniscope-LFOV: A large-field-of-view, single-cell-resolution, miniature microscope for wired and wire-free imaging of neural dynamics in freely behaving animals.

Imaging large-population, single-cell fluorescent dynamics in freely behaving animals larger than mice remains a key endeavor of neuroscience. We present a large-field-of-view open-source miniature microscope (MiniLFOV) designed for large-scale (3.6 mm × 2.7 mm), cellular resolution neural imaging in freely behaving rats. It has an electrically adjustable working distance of up to 3.5 mm ± 100 µm, incorporates an absolute head orientation sensor, and weighs only 13.9 g. The MiniLFOV is capable of both deep brain and cortical imaging and has been validated in freely behaving rats by simultaneously imaging >1000 GCaMP7s-expressing neurons in the hippocampal CA1 layer and in head-fixed mice by simultaneously imaging ~2000 neurons in the dorsal cortex through a cranial window. The MiniLFOV also supports optional wire-free operation using a novel, wire-free data acquisition expansion board. We expect that this new open-source implementation of the UCLA Miniscope platform will enable researchers to address novel hypotheses concerning brain function in freely behaving animals.

A hardware system for real-time decoding of in vivo calcium imaging data.

Epifluorescence miniature microscopes ('miniscopes') are widely used for in vivo calcium imaging of neural population activity. Imaging data are typically collected during a behavioral task and stored for later offline analysis, but emerging techniques for online imaging can support novel closed-loop experiments in which neural population activity is decoded in real time to trigger neurostimulation or sensory feedback. To achieve short feedback latencies, online imaging systems must be optimally designed to maximize computational speed and efficiency while minimizing errors in population decoding. Here we introduce DeCalciOn, an open-source device for real-time imaging and population decoding of in vivo calcium signals that is hardware compatible with all miniscopes that use the UCLA Data Acquisition (DAQ) interface. DeCalciOn performs online motion stabilization, neural enhancement, calcium trace extraction, and decoding of up to 1024 traces per frame at latencies of <50 ms after fluorescence photons arrive at the miniscope image sensor. We show that DeCalciOn can accurately decode the position of rats (n = 12) running on a linear track from calcium fluorescence in the hippocampal CA1 layer, and can categorically classify behaviors performed by rats (n = 2) during an instrumental task from calcium fluorescence in orbitofrontal cortex. DeCalciOn achieves high decoding accuracy at short latencies using innovations such as field-programmable gate array hardware for real-time image processing and contour-free methods to efficiently extract calcium traces from sensor images. In summary, our system offers an affordable plug-and-play solution for real-time calcium imaging experiments in behaving animals.

New imaging instrument in animal models: Two-photon miniature microscope and large field of view miniature microscope for freely behaving animals.

Over the past decade, novel optical imaging tools have been developed for imaging neuronal activities along with the evolution of fluorescence indicators with brighter expression and higher sensitivity. Miniature microscopes, as revolutionary approaches, enable the imaging of large populations of neuron ensembles in freely behaving rodents and mammals, which allows exploring the neural basis of behaviors. Recent progress in two-photon miniature microscopes and mesoscale single-photon miniature microscopes further expand those affordable methods to navigate neural activities during naturalistic behaviors. In this review article, two-photon miniature microscopy techniques are summarized historically from the first documented attempt to the latest ones, and comparisons are made. The driving force behind and their potential for neuroscientific inquiries are also discussed. Current progress in terms of the mesoscale, i.e., the large field-of-view miniature microscopy technique, is addressed as well. Then, pipelines for registering single cells from the data of two-photon and large field-of-view miniature microscopes are discussed. Finally, we present the potential evolution of the techniques.

BehaviorDEPOT is a simple, flexible tool for automated behavioral detection based on markerless pose tracking.

Quantitative descriptions of animal behavior are essential to study the neural substrates of cognitive and emotional processes. Analyses of naturalistic behaviors are often performed by hand or with expensive, inflexible commercial software. Recently, machine learning methods for markerless pose estimation enabled automated tracking of freely moving animals, including in labs with limited coding expertise. However, classifying specific behaviors based on pose data requires additional computational analyses and remains a significant challenge for many groups. We developed BehaviorDEPOT (DEcoding behavior based on POsitional Tracking), a simple, flexible software program that can detect behavior from video timeseries and can analyze the results of experimental assays. BehaviorDEPOT calculates kinematic and postural statistics from keypoint tracking data and creates heuristics that reliably detect behaviors. It requires no programming experience and is applicable to a wide range of behaviors and experimental designs. We provide several hard-coded heuristics. Our freezing detection heuristic achieves above 90% accuracy in videos of mice and rats, including those wearing tethered head-mounts. BehaviorDEPOT also helps researchers develop their own heuristics and incorporate them into the software's graphical interface. Behavioral data is stored framewise for easy alignment with neural data. We demonstrate the immediate utility and flexibility of BehaviorDEPOT using popular assays including fear conditioning, decision-making in a T-maze, open field, elevated plus maze, and novel object exploration.

Enhancing the heterojunction component-interaction by in-situ hydrothermal growth toward photocatalytic hydrogen evolution.

The in-situ synthesis method to construct a heterostructure with a tight binding interface can promote the separation and transfer of charges, which is particularly crucial for improving photocatalytic efficiency. Herein, we have successfully synthesized a high-efficiency photoreduction catalyst by in situ growing a layer of flaky nickel chromium layered double hydroxides nanosheets (LDH) on carbon nitride hexagonal tube (CN) in hydrothermal. The tube-flakes like CN-LDH heterostructures have enhanced hydrogen evolution efficiency (14.5 mmol h-1 g-1), which is about 4.7 times that of pure CN (2.7 mmol h-1 g-1) and much higher than that of LDH (0.06 mmol h-1 g-1). We attribute this performance improvement mainly to the close-knit heterostructure formed between LDH and CN. This tight combination strengthens the diffusion of self-charge between the two semiconductors to form a strong built-in electric field and band bending. Under the action of the built-in electric field (BIEF), the photogenerated charge can be efficiently separated and oriented fast transfer, thereby greatly improving the photocatalytic efficiency. This work constructs a tightly connected heterostructure photocatalyst through hydrothermal method, and uses the catalyst to convert high-efficiency solar energy into renewable energy.

Efficient Suzuki-Miyaura cross-coupling reaction by loading trace Pd nanoparticles onto copper-complex-derived Cu/C-700 solid support.

The development of efficient carbon-carbon cross-coupling catalysts with low noble metal amounts attracts much attention recently. Herein, a Cu/C-700/Pd nanocomposite is obtained by loading trace Pd2+ onto carbon support derived from a novel mononuclear copper complex, {[Cu(POP)2(Phen)2]BF4}. The as-prepared nanomaterial features the facial structure of highly dispersed copper phosphide nanoparticles as well as Pd nanoparticles via neighboring Cu-Pd sites. The Cu/C-700/Pd nanocomposite shows excellent catalytic activity (99.73%) and selectivity in Suzuki-Miyaura cross-coupling reaction, at trace Pd loading (0.43 mol%). Compared with the reported palladium nano catalysts, its advantages are proved. The appealing gateway to this stable, innovative and recyclability, Cu/C-700/Pd nanostructure recommends its beneficial utilization in carbon-carbon coupling and other environmentally friendly processes.

Microengineered poly(HEMA) hydrogels for wearable contact lens biosensing.

Microchannels in hydrogels play an essential role in enabling a smart contact lens. However, microchannels have rarely been created in commercial hydrogel contact lenses due to their sensitivity to conventional microfabrication techniques. Here, we report the fabrication of microchannels in poly(2-hydroxyethyl methacrylate) (poly(HEMA)) hydrogels that are used in commercial contact lenses with a three-dimensional (3D) printed mold. We investigated the corresponding capillary flow behaviors in these microchannels. We observed different capillary flow regimes in these microchannels, depending on their hydration level. In particular, we found that a peristaltic pressure could reinstate flow in a dehydrated channel, indicating that the motion of eye-blinking may help tears flow in a microchannel-containing contact lens. Colorimetric pH and electrochemical Na+ sensing capabilities were demonstrated in these microchannels. This work paves the way for the development of microengineered poly(HEMA) hydrogels for various biomedical applications such as eye-care and wearable biosensing.

Airy-beam tomographic microscopy.

We introduce Airy-beam tomographic microscopy (ATM) for high-resolution, volumetric, inertia-free imaging of biological specimens. The work exploits the highly adjustable Airy trajectories in the three-dimensional (3D) space, transforming the conventional telecentric wide-field imaging scheme that requires sample or focal-plane scanning to acquire 3D information. The results present a consistent near-diffraction-limited 3D resolution across a tenfold extended imaging depth compared to wide-field microscopy. We anticipate the strategy to not only offer a promising paradigm for 3D optical microscopy, but also be translated to other non-optical waveforms.

Fast and accurate sCMOS noise correction for fluorescence microscopy.

The rapid development of scientific CMOS (sCMOS) technology has greatly advanced optical microscopy for biomedical research with superior sensitivity, resolution, field-of-view, and frame rates. However, for sCMOS sensors, the parallel charge-voltage conversion and different responsivity at each pixel induces extra readout and pattern noise compared to charge-coupled devices (CCD) and electron-multiplying CCD (EM-CCD) sensors. This can produce artifacts, deteriorate imaging capability, and hinder quantification of fluorescent signals, thereby compromising strategies to reduce photo-damage to live samples. Here, we propose a content-adaptive algorithm for the automatic correction of sCMOS-related noise (ACsN) for fluorescence microscopy. ACsN combines camera physics and layered sparse filtering to significantly reduce the most relevant noise sources in a sCMOS sensor while preserving the fine details of the signal. The method improves the camera performance, enabling fast, low-light and quantitative optical microscopy with video-rate denoising for a broad range of imaging conditions and modalities.

Robust autofocusing method for multi-wavelength lensless imaging.

Lensless imaging based on multi-wavelength phase retrieval becomes a promising technology widely used as it has simple acquisition, miniaturized size and low-cost setup. However, measuring the sample-to-sensor distance with high accuracy, which is the key for high-resolution reconstruction, is still a challenge. In this work, we propose a multi-wavelength criterion to realize autofocusing modulation, i.e., achieving much higher accuracy in determining the sample-to-sensor distance, compared to the conventional methods. Three beams in different spectrums are adopted to illuminate the sample, and the resulting holograms are recorded by a CCD camera. The patterns calculated by performing back propagation of the recorded holograms, with exhaustively searched sample-to-sensor distance value, are adopted to access the criterion. Image sharpness can be accessed and the optimal sample-to-sensor distance can be finely determined by targeting the valley of the curve given by the criterion. Through our novel multi-wavelength based autofocusing strategy and executing further phase retrieval process, high-resolution images can be finally retrieved. The applicability and robustness of our method is validated both in simulations and experiments. Our technique provides a useful tool for multi-wavelength lensless imaging under limited experimental conditions.

Fourier light-field microscopy.

Observing the various anatomical and functional information that spans many spatiotemporal scales with high resolution provides deep understandings of the fundamentals of biological systems. Light-field microscopy (LFM) has recently emerged as a scanning-free, scalable method that allows for high-speed, volumetric imaging ranging from single-cell specimens to the mammalian brain. However, the prohibitive reconstruction artifacts and severe computational cost have thus far limited broader applications of LFM. To address the challenge, in this work, we report Fourier LFM (FLFM), a system that processes the light-field information through the Fourier domain. We established a complete theoretical and algorithmic framework that describes light propagation, image formation and system characterization of FLFM. Compared with conventional LFM, FLFM fundamentally mitigates the artifacts, allowing high-resolution imaging across a two- to three-fold extended depth. In addition, the system substantially reduces the reconstruction time by roughly two orders of magnitude. FLFM was validated by high-resolution, artifact-free imaging of various caliber and biological samples. Furthermore, we proposed a generic design principle for FLFM, as a highly scalable method to meet broader imaging needs across various spatial levels. We anticipate FLFM to be a particularly powerful tool for imaging diverse phenotypic and functional information, spanning broad molecular, cellular and tissue systems.

Fast, volumetric live-cell imaging using high-resolution light-field microscopy.

Visualizing diverse anatomical and functional traits that span many spatial scales with high spatio-temporal resolution provides insights into the fundamentals of living organisms. Light-field microscopy (LFM) has recently emerged as a scanning-free, scalable method that allows for high-speed, volumetric functional brain imaging. Given those promising applications at the tissue level, at its other extreme, this highly-scalable approach holds great potential for observing structures and dynamics in single-cell specimens. However, the challenge remains for current LFM to achieve a subcellular level, near-diffraction-limited 3D spatial resolution. Here, we report high-resolution LFM (HR-LFM) for live-cell imaging with a resolution of 300-700 nm in all three dimensions, an imaging depth of several micrometers, and a volume acquisition time of milliseconds. We demonstrate the technique by imaging various cellular dynamics and structures and tracking single particles. The method may advance LFM as a particularly useful tool for understanding biological systems at multiple spatio-temporal levels.

Depth-extended, high-resolution fluorescence microscopy: whole-cell imaging with double-ring phase (DRiP) modulation.

We report a depth-extended, high-resolution fluorescence microscopy system based on interfering Bessel beams generated with double-ring phase (DRiP) modulation. The DRiP method effectively suppresses the Bessel side lobes, exhibiting a high resolution of the main lobe throughout a four- to five-fold improved depth of focus (DOF), compared to conventional wide-field microscopy. We showed both theoretically and experimentally the generation and propagation of a DRiP point-spread function (DRiP-PSF) of the imaging system. We further developed an approach for creating an axially-uniform DRiP-PSF and successfully demonstrated diffraction-limited, depth-extended imaging of cellular structures. We expect the DRiP method to contribute to the fast-developing field of non-diffracting-beam-enabled optical microscopy and be useful for various types of imaging modalities.

Measuring refractive index of glass by using speckle.

A method to measure the refractive index of an optically flat, regularly shaped slab of glass using speckle correlation-based techniques is reported. The intensity of the diffraction field of the diffuser is captured by a CCD both with and without the glass present. As the position of the peak correlation coefficient is quantitatively related to the change in optical path length arising due to the presence of the glass, the refractive index of the glass can be evaluated by cross-correlating the two captured images. The theoretical correlation function that describes the effects of such an optical path length change is discussed, and the resulting speckle decorrelation function derived. Two glass samples are measured to demonstrate the accuracy and robustness of the proposed technique.

Holographic characterization of diffraction grating modulation in photopolymers.

In general, the holographic grating refractive index profiles in photopolymer materials are not identical to the exposing pattern. During exposure, high harmonics of the fundamental refractive index period are generated within the layer volume. A set of equations to calculate the amplitudes of the higher harmonics of refractive index induced in the grating is introduced. Then, an algorithm involving the use of the 3D nonlocal photopolymerization-driven diffusion model is presented and applied to calculate the resulting grating diffraction efficiencies. The experimental observation that the grating diffraction efficiency cannot reach the theoretical maximum value (ηmax=100%) and that, in the case of over-modulation, the minimum value (ηmin=0%) is also never achieved, are explained theoretically. The predictions of the simulations are also fit to experimental data for an acrylamide/polyvinyl alcohol photopolymer material with good agreement being achieved.

Low photon count based digital holography for quadratic phase cryptography.

Recently, the vulnerability of the linear canonical transform-based double random phase encryption system to attack has been demonstrated. To alleviate this, we present for the first time, to the best of our knowledge, a method for securing a two-dimensional scene using a quadratic phase encoding system operating in the photon-counted imaging (PCI) regime. Position-phase-shifting digital holography is applied to record the photon-limited encrypted complex samples. The reconstruction of the complex wavefront involves four sparse (undersampled) dataset intensity measurements (interferograms) at two different positions. Computer simulations validate that the photon-limited sparse-encrypted data has adequate information to authenticate the original data set. Finally, security analysis, employing iterative phase retrieval attacks, has been performed.

Volumetric Light-field Encryption at the Microscopic Scale.

We report a light-field based method that allows the optical encryption of three-dimensional (3D) volumetric information at the microscopic scale in a single 2D light-field image. The system consists of a microlens array and an array of random phase/amplitude masks. The method utilizes a wave optics model to account for the dominant diffraction effect at this new scale, and the system point-spread function (PSF) serves as the key for encryption and decryption. We successfully developed and demonstrated a deconvolution algorithm to retrieve both spatially multiplexed discrete data and continuous volumetric data from 2D light-field images. Showing that the method is practical for data transmission and storage, we obtained a faithful reconstruction of the 3D volumetric information from a digital copy of the encrypted light-field image. The method represents a new level of optical encryption, paving the way for broad industrial and biomedical applications in processing and securing 3D data at the microscopic scale.