High-speed Volumetric Imaging Research Articles

Robust single-shot 3D fluorescence imaging in scattering media with a simulator-trained neural network.

Imaging through scattering is a pervasive and difficult problem in many biological applications. The high background and the exponentially attenuated target signals due to scattering fundamentally limits the imaging depth of fluorescence microscopy. Light-field systems are favorable for high-speed volumetric imaging, but the 2D-to-3D reconstruction is fundamentally ill-posed, and scattering exacerbates the condition of the inverse problem. Here, we develop a scattering simulator that models low-contrast target signals buried in heterogeneous strong background. We then train a deep neural network solely on synthetic data to descatter and reconstruct a 3D volume from a single-shot light-field measurement with low signal-to-background ratio (SBR). We apply this network to our previously developed computational miniature mesoscope and demonstrate the robustness of our deep learning algorithm on scattering phantoms with different scattering conditions. The network can robustly reconstruct emitters in 3D with a 2D measurement of SBR as low as 1.05 and as deep as a scattering length. We analyze fundamental tradeoffs based on network design factors and out-of-distribution data that affect the deep learning model's generalizability to real experimental data. Broadly, we believe that our simulator-based deep learning approach can be applied to a wide range of imaging through scattering techniques where experimental paired training data is lacking.

The mesencephalic locomotor region recruits V2a reticulospinal neurons to drive forward locomotion in larval zebrafish

The mesencephalic locomotor region (MLR) is a brain stem area whose stimulation triggers graded forward locomotion. How MLR neurons recruit downstream vsx2+ (V2a) reticulospinal neurons (RSNs) is poorly understood. Here, to overcome this challenge, we uncovered the locus of MLR in transparent larval zebrafish and show that the MLR locus is distinct from the nucleus of the medial longitudinal fasciculus. MLR stimulations reliably elicit forward locomotion of controlled duration and frequency. MLR neurons recruit V2a RSNs via projections onto somata in pontine and retropontine areas, and onto dendrites in the medulla. High-speed volumetric imaging of neuronal activity reveals that strongly MLR-coupled RSNs are active for steering or forward swimming, whereas weakly MLR-coupled medullary RSNs encode the duration and frequency of the forward component. Our study demonstrates how MLR neurons recruit specific V2a RSNs to control the kinematics of forward locomotion and suggests conservation of the motor functions of V2a RSNs across vertebrates.

High-resolution multi-z confocal microscopy with a diffractive optical element.

There has been recent interest in the development of fluorescence microscopes that provide high-speed volumetric imaging for life-science applications. For example, multi-z confocal microscopy enables simultaneous optically-sectioned imaging at multiple depths over relatively large fields of view. However, to date, multi-z microscopy has been hampered by limited spatial resolution owing to its initial design. Here we present a variant of multi-z microscopy that recovers the full spatial resolution of a conventional confocal microscope while retaining the simplicity and ease of use of our initial design. By introducing a diffractive optical element in the illumination path of our microscope, we engineer the excitation beam into multiple tightly focused spots that are conjugated to axially distributed confocal pinholes. We discuss the performance of this multi-z microscope in terms of resolution and detectability and demonstrate its versatility by performing in-vivo imaging of beating cardiomyocytes in engineered heart tissues and neuronal activity in c. elegans and zebrafish brains.

Recent advances in oblique plane microscopy

Abstract Oblique plane microscopy (OPM) directly captures object information in a plane tilted from the focal plane of the objective lens without the need for slow z-stack acquisition. This unconventional widefield imaging approach is made possible by using a remote focusing principle that eliminates optical aberrations for object points beyond the focal plane. Together with oblique lightsheet illumination, OPM can make conventional lightsheet imaging fully compatible with standard biological specimens prepared on microscope slides. OPM is not only an excellent high-speed volumetric imaging platform by sweeping oblique lightsheet illumination without mechanically moving either the sample or objective lens in sample space, but also provides a solution for direct oblique plane imaging along any orientation of interest on the sample in a single shot. Since its first demonstration in 2008, OPM has continued to evolve into an advanced microscope platform for biological, medical, and materials science applications. In recent years, many technological advances have been made in OPM with the goal of super-resolution, fast volumetric imaging, and a large imaging field of view, etc. This review gives an overview of OPM’s working principle and imaging performance and introduces recent technical developments in OPM methods and applications. OPM has strong potential in a variety of research fields, including cellular and developmental biology, clinical diagnostics in histology and ophthalmology, flow cytometry, microfluidic devices, and soft materials.

Compact, Hybrid Light-Sheet and Fourier Light-Field Microscopy with a Single Objective for High-Speed Volumetric Imaging In Vivo.

Volumetric imaging of biodynamics at high spatiotemporal resolutions in vivo is vital in biomedical studies, in which Fourier light field microscopy (FLFM) is a promising technique. However, the commonly used wide-field illumination strategy in FLFM introduces intense out of depth-of-field background, which not only degrades the image quality, but also introduces reconstruction artifacts. Employing light sheet illumination is an effective way to alleviate the background and reduce photobleaching in light-field microscopy. Unfortunately, the introduction of light-sheet illumination often requires an extra objective and precise alignment, which increases the system complexity. Here, we propose the compact, hybrid light-sheet and FLFM (CLS-FLFM), which uses only a single objective to achieve both light-sheet illumination and Fourier light-field imaging simultaneously. With a micromirror under the objective, we focus the light sheet, which ensures selective-volume-illumination, on the imaging plane of the FLFM to perform volumetric imaging. We demonstrate the superior performance of CLS-FLFM in inhibiting background in both structural and dynamical imaging of larval zebrafish in vivo. We envision that CLS-FLFM finds wide applications in high-speed, background-inhibited volumetric imaging of biodynamics in vivo.

Weakly supervised learning analysis of Aβ plaque distribution in the whole rat brain.

Alzheimer's disease (AD) is a great challenge for the world and hardly to be cured, partly because of the lack of animal models that fully mimic pathological progress. Recently, a rat model exhibiting the most pathological symptoms of AD has been reported. However, high-resolution imaging and accurate quantification of beta-amyloid (Aβ) plaques in the whole rat brain have not been fulfilled due to substantial technical challenges. In this paper, a high-efficiency data analysis pipeline is proposed to quantify Aβ plaques in whole rat brain through several terabytes of image data acquired by a high-speed volumetric imaging approach we have developed previously. A novel segmentation framework applying a high-performance weakly supervised learning method which can dramatically reduce the human labeling consumption is described in this study. The effectiveness of our segmentation framework is validated with different metrics. The segmented Aβ plaques were mapped to a standard rat brain atlas for quantitative analysis of the Aβ distribution in each brain area. This pipeline may also be applied to the segmentation and accurate quantification of other non-specific morphology objects.

Dual-resonant scanning multiphoton microscope with ultrasound lens and resonant mirror for rapid volumetric imaging

A dual-resonant scanning multiphoton (DRSM) microscope incorporating a tunable acoustic gradient index of refraction lens with a resonant mirror is developed for high-speed volumetric imaging. In the proposed microscope, the pulse train signal of a femtosecond laser is used to trigger an embedded field programmable gate array to sample the multiphoton excited fluorescence signal at the rate of one pixel per laser pulse. It is shown that a frame rate of around 8000 Hz can be obtained in the x–z plane for an image region with a size of 256 × 80 pixels. Moreover, a volumetric imaging rate of over 30 Hz can be obtained for a large image volume of 343 × 343 × 120 μm3 with an image size of 256 × 256 × 80 voxels. Moreover, a volumetric imaging rate of over 30 Hz can be obtained for a large image volume of 256 × 256 × 80 voxels, which represents 343 × 343 × 120 μm3 in field-of-view. The rapid volumetric imaging rate eliminates the aliasing effect for observed temporal frequencies lower than 15 Hz. The practical feasibility of the DRSM microscope is demonstrated by observing the mushroom bodies of a drosophila brain and performing 3D dynamic observations of moving 10-μm fluorescent beads.

400 Hz Volume Rate Swept-Source Optical Coherence Tomography at 1060 nm Using a KTN Deflector

In this Letter, a swept-source optical coherence tomography (SS-OCT) instrument employing an innovative scanning protocol for high-speed volumetric rate imaging is demonstrated. The optical source is a tunable laser based on a supercontinuum source pumped with femtosecond pulses, followed by a time-stretched delay fiber. The instrument is equipped with an ultra-fast lateral scanner, based on a KTN crystal, driven at 100 kHz. The letter proves the utility of combining an ultra-fast lateral scanner with an ultra-fast swept laser to provide A-scans at a repetition rate of 40 MHz and an unprecedented 3D-OCT volume acquisition rate of 400 Hz.

Three-dimensional fluorescence microscopy through virtual refocusing using a recursive light propagation network.

Three-dimensional fluorescence microscopy has an intrinsic performance limit set by the number of photons that can be collected from the sample in a given time interval. Here, we extend our earlier work - a recursive light propagation network (RLP-Net) - which is a computational microscopy technique that overcomes such limitations through virtual refocusing that enables volume reconstruction from two adjacent 2-D wide-field fluorescence images. RLP-Net employs a recursive inference scheme in which the network progressively predicts the subsequent planes along the axial direction. This recursive inference scheme reflects that the law of physics for the light propagation remains spatially invariant and therefore a fixed function (i.e., a neural network) for a short distance light propagation can be recursively applied for a longer distance light propagation. In addition, we employ a self-supervised denoising method to enable accurate virtual light propagation over a long distance. We demonstrate the capability of our method through high-speed volumetric imaging of neuronal activity of a live zebrafish brain. The source code used in the paper is available at https://github.com/NICALab/rlpnet.

Single-shot optical projection tomography for high-speed volumetric imaging of dynamic biological samples.

A single-shot adaptation of Optical Projection Tomography (OPT) for high-speed volumetric snapshot imaging of dynamic mesoscopic biological samples is presented. Conventional OPT has been applied to in vivo imaging of animal models such as D. rerio, but the sequential acquisition of projection images typically requires samples to be immobilized during the acquisition. A proof-of-principle system capable of single-shot tomography of a ~1 mm3 volume is presented, demonstrating camera-limited rates of up to 62.5 volumes/s, which has been applied to 3D imaging of a freely swimming zebrafish embryo. This is achieved by recording eight projection views simultaneously on four low-cost CMOS cameras. With no stage required to rotate the sample, this single-shot OPT system can be implemented with a component cost of under £5000. The system design can be adapted to different sized fields of view and may be applied to a broad range of dynamic samples, including high throughput flow cytometry applied to model organisms and fluid dynamics studies.

Noise-robust phase-space deconvolution for light-field microscopy.

.SignificanceLight-field microscopy has achieved success in various applications of life sciences that require high-speed volumetric imaging. However, existing light-field reconstruction algorithms degrade severely in low-light conditions, and the deconvolution process is time-consuming.AimThis study aims to develop a noise robustness phase-space deconvolution method with low computational costs.ApproachWe reformulate the light-field phase-space deconvolution model into the Fourier domain with random-subset ordering and total-variation (TV) regularization. Additionally, we build a time-division-based multicolor light-field microscopy and conduct the three-dimensional (3D) imaging of the heart beating in zebrafish larva at over 95 Hz with a low light dose.ResultsWe demonstrate that this approach reduces computational resources, brings a tenfold speedup, and achieves a tenfold improvement for the noise robustness in terms of SSIM over the state-of-the-art approach.ConclusionsWe proposed a phase-space deconvolution algorithm for 3D reconstructions in fluorescence imaging. Compared with the state-of-the-art method, we show significant improvement in both computational effectiveness and noise robustness; we further demonstrated practical application on zebrafish larva with low exposure and low light dose.

Improving signal-to-background ratio by orders of magnitude in high-speed volumetric imaging in vivo by robust Fourier light field microscopy

Fourier light field microscopy (FLFM) shows great potential in high-speed volumetric imaging of biodynamics. However, due to the inherent disadvantage of wide-field illumination, it suffers from intense background, arising from out of the depth-of-field signal and tissue scattered noise. The background will not only deteriorate the image contrast, making quantitative measurement difficult, but also introduce artifacts, especially in functional imaging of the neuronal network activity in vivo. Here, we propose the robust Fourier light field microscopy (RFLFM), which suppresses the background in FLFM by introducing structured illumination and computational reconstruction based on HiLo. The superior performance of RFLFM is verified by volumetric imaging of biological dynamics in larval zebrafish and mouse in vivo, at a volumetric imaging rate up to 33.3 Hz. The statistical results show that the fluorescence background can be significantly depressed, with the signal-to-background ratio improved by orders of magnitude and the whole image contrast improved by as much as ∼ 10.4 times. Moreover, we stress that, in functional imaging of neuronal network activity in turbid brain tissues, our system can avoid artifacts resulting from background fluctuations, while conventional light field microscopy fails. As a simple but powerful tool, we anticipate our technique to be widely adopted in robust, high-contrast, high-speed volumetric imaging.

Plug-and-play adaptive optics for two photon high-speed volumetric imaging

To understand brain functions, it is important to study functional connectivity among stereoscopically distributed neurons. Since the brain is composed of 3D neuron networks, volumetric imaging with high spatiotemporal resolution is highly desirable. Two-photon microscopy (2PM) conveniently offers 3D tissue imaging with sub-micrometer resolution based on its intrinsic optical sectioning and deep penetration capabilities. However, the main challenge lies in the volumetric imaging speed and contrast reduction in deep tissue due to aberration. In this study, we integrate a tunable acoustic gradient lens and a plug-and-play adaptive-optics lens into 2PM. The former provides ∼100 kHz axial scan rate, achieving volumetric imaging rate in 1–10 Hz range, while the latter enhances image contrast by nearly two-fold in deep brain regions via correcting both systematic and sample aberrations. The combination offers a practical approach toward high-speed, high-contrast optical volumetric imaging of brain tissues.

Implantable photonic neural probes for light-sheet fluorescence brain imaging.

.Significance: Light-sheet fluorescence microscopy (LSFM) is a powerful technique for high-speed volumetric functional imaging. However, in typical light-sheet microscopes, the illumination and collection optics impose significant constraints upon the imaging of non-transparent brain tissues. We demonstrate that these constraints can be surmounted using a new class of implantable photonic neural probes.Aim: Mass manufacturable, silicon-based light-sheet photonic neural probes can generate planar patterned illumination at arbitrary depths in brain tissues without any additional micro-optic components.Approach: We develop implantable photonic neural probes that generate light sheets in tissue. The probes were fabricated in a photonics foundry on 200-mm-diameter silicon wafers. The light sheets were characterized in fluorescein and in free space. The probe-enabled imaging approach was tested in fixed, in vitro, and in vivo mouse brain tissues. Imaging tests were also performed using fluorescent beads suspended in agarose.Results: The probes had 5 to 10 addressable sheets and average sheet thicknesses for propagation distances up to in free space. Imaging areas were as large as in brain tissue. Image contrast was enhanced relative to epifluorescence microscopy.Conclusions: The neural probes can lead to new variants of LSFM for deep brain imaging and experiments in freely moving animals.

Real-time volumetric reconstruction of biological dynamics with light-field microscopy and deep learning.

Light-field microscopy has emerged as a technique of choice for high-speed volumetric imaging of fast biological processes. However, artefacts, non-uniform resolution, and a slow reconstruction speed have limited its full capabilities for in toto extraction of the dynamic spatiotemporal patterns in samples. Here, we combined a view-channel-depth (VCD) neural network with light-field microscopy to mitigate these limitations, yielding artefact-free three-dimensional image sequences with uniform spatial resolution and high video-rate reconstruction throughput. We imaged neuronal activities across moving C. elegans and blood flow in a beating zebrafish heart at single-cell resolution with volumetric imaging rates up to 200 Hz.

Dual GRIN lens two-photon endoscopy for high-speed volumetric and deep brain imaging.

Studying neural connections and activities in vivo is fundamental to understanding brain functions. Given the cm-size brain and three-dimensional neural circuit dynamics, deep-tissue, high-speed volumetric imaging is highly desirable for brain study. With sub-micrometer spatial resolution, intrinsic optical sectioning, and deep-tissue penetration capability, two-photon microscopy (2PM) has found a niche in neuroscience. However, the current 2PM typically relies on a slow axial scan for volumetric imaging, and the maximal penetration depth is only about 1 mm. Here, we demonstrate that by integrating a gradient-index (GRIN) lens and a tunable acoustic GRIN (TAG) lens into 2PM, both penetration depth and volume-imaging rate can be significantly improved. Specifically, an ∼ 1-cm long GRIN lens allows imaging relay from any target region of a mouse brain, while a TAG lens provides a sub-second volume rate via a 100 kHz ∼ 1 MHz axial scan. This technique enables the study of calcium dynamics in cm-deep brain regions with sub-cellular and sub-second spatiotemporal resolution, paving the way for interrogating deep-brain functional connectome.

Distal convoluted tubule sexual dimorphism revealed by advanced 3D imaging.

The thiazide-sensitive Na+-Cl- cotransporter (NCC) is more abundant in kidneys of female subjects than of male subjects. Because morphological remodeling of the distal convoluted tubule (DCT) is dependent on NCC activity, it has been generally assumed that there is a corresponding sexual dimorphism in the structure of the DCT, leading to a larger female DCT. Until now, this has never been directly examined. Here, optical clearing techniques were combined with antibody labeling of DCT segment markers, state-of-the-art high-speed volumetric imaging, and analysis tools to visualize and quantify DCT morphology in male and female mice and study the DCT remodeling response to furosemide. We found an unexpected sex difference in the structure of the DCT. Compared with the male mice, female mice had a shorter DCT, a higher cellular density of NCC, and a greater capacity to elongate in response to loop diuretics. Our study revealed a sexual dimorphism of the DCT. Female mice expressed a greater density of NCC transporters in a shorter structure to protect Na+ balance in the face of greater basal distal Na+ delivery yet have a larger reserve and structural remodeling capacity to adapt to unique physiological stresses. These observations provide insight into mechanisms that may drive sex differences in the therapeutic responses to diuretics.

Performance tradeoffs for single- and dual-objective open-top light-sheet microscope designs: a simulation-based analysis.

Light-sheet microscopy (LSM) has emerged as a powerful tool for high-speed volumetric imaging of live model organisms and large optically cleared specimens. When designing cleared-tissue LSM systems with certain desired imaging specifications (e.g. resolution, contrast, and working distance), various design parameters must be taken into consideration. In order to elucidate some of the key design tradeoffs for LSM systems, we present a diffraction-based analysis of single- and dual-objective LSM configurations using simulations of LSM point spread functions. We assume Gaussian illumination is utilized. Specifically, we analyze the effects of the illumination and collection numerical aperture (NA), as well as their crossing angle, on spatial resolution and contrast. Assuming an open-top light-sheet (OTLS) architecture, we constrain these parameters based on fundamental geometric considerations as well as those imposed by currently available microscope objectives. In addition to revealing the performance tradeoffs of various single- and dual-objective LSM configurations, our analysis showcases the potential advantages of a novel, non-orthogonal dual-objective (NODO) architecture, especially for moderate-resolution imaging applications (collection NA of 0.5 to 0.8).

High-speed volumetric imaging of formaldehyde in a lifted turbulent jet flame using an acousto-optic deflector

The development of high-speed volumetric laser-induced fluorescence measurements of formaldehyde ($$\hbox {CH}_2\hbox {O}$$-LIF) using a pulse-burst laser operated at a repetition rate of $${100} \hbox { kHz}$$ is presented. A novel laser scanning system employing an acousto-optic deflector (AOD) enables quasi-4D $$\hbox {CH}_2\hbox {O}$$-LIF imaging at a scan frequency of $${10}\hbox { kHz}$$. The diagnostic capability of time-resolved volumetric imaging is demonstrated in a partially premixed DME/air lifted turbulent jet flame near the flame base. Simultaneous imaging of laser beam profiles is performed to account for the laser pulse energy fluctuation and laser sheet inhomogeneity. With the accurate registration of laser sheet positions, the volumetric reconstruction of $$\hbox {CH}_2\hbox {O}$$-LIF signals is performed within a detection volume of $$17.3 \times 11.9 \times {2.3}\, \hbox { mm}^3$$ with an average out-of-plane spatial resolution of $${250}\,\upmu \hbox {m}$$. A surface detection algorithm with adaptive thresholding is used to determine the global maximum intensity gradient by calculating gradient percentiles. The flame topology characteristics are investigated by evaluating the 3D curvatures of $$\hbox {CH}_2\hbox {O}$$ surfaces. Curvatures calculated using 2D data systematically underestimate the full 3D curvature due to the lack of out-of-plane information. The inner surfaces near the turbulent fuel jet exhibit higher probabilities of large mean curvature than the outer surfaces. The saddle and cylindrical structures are dominant on both the inner and outer surfaces and the elliptic structures occur with lower probability. The results suggest that the damping of turbulent fluctuations by the temperature increase through the $$\hbox {CH}_2\hbox {O}$$ region reduces the curvature, but the local structure topology remains self-similar.

Assessing the progression of systemic sclerosis by monitoring the tissue optic axis using PS-OCT

The clinical assessment of fibrosis is critical to the diagnosis and management of patients with systemic sclerosis. Current clinical standards for patient assessment is to use skin fibrosis as an indicator of organ involvement, though this approach is highly subjective and relies on manual palpation. The development of a new method for accurately quantifying collagen content may therefore significantly improve the accuracy of the traditional skin score in patients with systemic sclerosis and may additionally aid in the monitoring of anti-fibrotic therapies in clinical practice. Polarization-sensitive optical coherence tomography (PS-OCT) is a high-speed volumetric imaging modality that can be used to assess birefringent tissues including collagen. In this work we demonstrate a novel computational approach using PS-OCT for the assessment of fibrosis. This approach, based on the measured distribution of optic axis values associated with a given volume of collagen orientation, characterizes fibrotic changes independently from the depth of the region of interest in the tissue. This approach has the potential to accurately quantify collagen content and orientation faster and more robustly compared to traditional PS-OCT metrics. We investigate the viability of this approach for assessing the development of fibrosis in a bleomycin induced skin fibrosis mouse model.