Large-scale deep tissue voltage imaging with targeted-illumination confocal microscopy.

Voltage imaging with cellular specificity has been made possible by advances in genetically encoded voltage indicators. However, the kilohertz rates required for voltage imaging lead to weak signals. Moreover, out-of-focus fluorescence and tissue scattering produce background that both undermines the signal-to-noise ratio and induces crosstalk between cells, making reliable in vivo imaging in densely labeled tissue highly challenging. We describe a microscope that combines the distinct advantages of targeted illumination and confocal gating while also maximizing signal detection efficiency. The resulting benefits in signal-to-noise ratio and crosstalk reduction are quantified experimentally and theoretically. Our microscope provides a versatile solution for enabling high-fidelity in vivo voltage imaging at large scales and penetration depths, which we demonstrate across a wide range of imaging conditions and different genetically encoded voltage indicator classes.

Multiplane HiLo microscopy with speckle illumination and non-local means denoising.

Significance: HiLo microscopy synthesizes an optically sectioned image from two images, one obtained with uniform and another with patterned illumination, such as laser speckle. Speckle-based HiLo has the advantage of being robust to aberrations but is susceptible to residual speckle noise that is difficult to control. We present a computational method to reduce this residual noise without undermining resolution. In addition, we improve the versatility of HiLo microscopy by enabling simultaneous multiplane imaging (here nine planes). Aim: Our goal is to perform fast, high-contrast, multiplane imaging with a conventional camera-based fluorescence microscope. Approach: Multiplane HiLo imaging is achieved with the use of a single camera and z-splitter prism. Speckle noise reduction is based on the application of a non-local means (NLM) denoising method to perform ensemble averaging of speckle grains. Results: We demonstrate the capabilities of multiplane HiLo with NLM denoising both with synthesized data and by imaging cardiac and brain activity in zebrafish larvae at 40 Hz frame rates. Conclusions: Multiplane HiLo microscopy aided by NLM denoising provides a simple tool for fast optically sectioned volumetric imaging that can be of general utility for fluorescence imaging applications.

Assessing depth sensitivity in laser interferometry speckle visibility spectroscopy (iSVS) through source-to-detector distance variation and cerebral blood flow monitoring in humans and rabbits.

Recently, speckle visibility spectroscopy (SVS) was non-invasively applied on the head to monitor cerebral blood flow. The technique, using a multi-pixel detecting device (e.g., camera), allows the detection of a larger number of speckles, increasing the proportion of light that is detected. Due to this increase, it is possible to collect light that has propagated deeper through the brain. As a direct consequence, cerebral blood flow can be monitored. However, isolating the cerebral blood flow from the other layers, such as the scalp or skull components, remains challenging. In this paper, we report our investigations on the depth-sensitivity of laser interferometry speckle visibility spectroscopy (iSVS). Specifically, we varied the depth of penetration of the laser light into the head by tuning the source-to-detector distance, and identified the transition point at which cerebral blood flow in humans and rabbits starts to be detected.

Interferometric speckle visibility spectroscopy (iSVS) for measuring decorrelation time and dynamics of moving samples with enhanced signal-to-noise ratio and relaxed reference requirements.

Diffusing wave spectroscopy (DWS) is a group of techniques used to measure the dynamics of a scattering medium in a non-invasive manner. DWS methods rely on detecting the speckle light field from the moving scattering medium and measuring the speckle decorrelation time to quantify the scattering medium's dynamics. For DWS, the signal-to-noise (SNR) is determined by the ratio between measured decorrelation time to the standard error of the measurement. This SNR is often low in certain applications because of high noise variances and low signal intensity, especially in biological applications with restricted exposure and emission levels. To address this photon-limited signal-to-noise ratio problem, we investigated, theoretically and experimentally, the SNR of an interferometric speckle visibility spectroscopy (iSVS) compared to more traditional DWS methods. We found that iSVS can provide excellent SNR performance through its ability to overcome camera noise. We also proved an iSVS system has more relaxed constraints on the reference beam properties. For an iSVS system to function properly, we only require the reference beam to exhibit local temporal stability, while incident angle, reference phase and intensity uniformity do not need to be constrained. This flexibility can potentially enable more unconventional iSVS implementation schemes.

High-speed multiplane confocal microscopy for voltage imaging in densely labeled neuronal populations.

Genetically encoded voltage indicators (GEVIs) hold immense potential for monitoring neuronal population activity. To date, best-in-class GEVIs rely on one-photon excitation. However, GEVI imaging of dense neuronal populations remains difficult because out-of-focus background fluorescence produces low contrast and excess noise when paired with conventional one-photon widefield imaging methods. To address this challenge, we developed an imaging system capable of efficient, high-contrast GEVI imaging at near-kHz rates and demonstrate it for in vivo and ex vivo imaging applications in the mouse neocortex. Our approach uses simultaneous multiplane imaging to monitor activity within contiguous tissue volumes with no penalty in speed or requirement for high excitation power. This approach, multi-Z imaging with confocal detection (MuZIC), permits high signal-to-noise ratio voltage imaging in densely labeled neuronal populations and is compatible with imaging through micro-optics. Moreover, it minimizes artifacts associated with concurrent imaging and optogenetic photostimulation for all-optical electrophysiology.

Theta and gamma rhythmic coding through two spike output modes in the hippocampus during spatial navigation.

Hippocampal CA1 neurons generate single spikes and stereotyped bursts of spikes. However, it is unclear how individual neurons dynamically switch between these output modes and whether these two spiking outputs relay distinct information. We performed extracellular recordings in spatially navigating rats and cellular voltage imaging and optogenetics in awake mice. We found that spike bursts are preferentially linked to cellular and network theta rhythms (3-12 Hz) and encode an animal's position via theta phase precession, particularly as animals are entering a place field. In contrast, single spikes exhibit additional coupling to gamma rhythms (30-100 Hz), particularly as animals leave a place field. Biophysical modeling suggests that intracellular properties alone are sufficient to explain the observed input frequency-dependent spike coding. Thus, hippocampal neurons regulate the generation of bursts and single spikes according to frequency-specific network and intracellular dynamics, suggesting that these spiking modes perform distinct computations to support spatial behavior.

Resolution enhancement with deblurring by pixel reassignment (DPR).

Improving the spatial resolution of a fluorescence microscope has been an ongoing challenge in the imaging community. To address this challenge, a variety of approaches have been taken, ranging from instrumentation development to image post-processing. An example of the latter is deconvolution, where images are numerically deblurred based on a knowledge of the microscope point spread function. However, deconvolution can easily lead to noise-amplification artifacts. Deblurring by post-processing can also lead to negativities or fail to conserve local linearity between sample and image. We describe here a simple image deblurring algorithm based on pixel reassignment that inherently avoids such artifacts and can be applied to general microscope modalities and fluorophore types. Our algorithm helps distinguish nearby fluorophores even when these are separated by distances smaller than the conventional resolution limit, helping facilitate, for example, the application of single-molecule localization microscopy in dense samples. We demonstrate the versatility and performance of our algorithm under a variety of imaging conditions.

Large-scale deep tissue voltage imaging with targeted illumination confocal microscopy.

Voltage imaging with cellular specificity has been made possible by the tremendous advances in genetically encoded voltage indicators (GEVIs). However, the kilohertz rates required for voltage imaging lead to weak signals. Moreover, out-of-focus fluorescence and tissue scattering produce background that both undermines signal-to-noise ratio (SNR) and induces crosstalk between cells, making reliable in vivo imaging in densely labeled tissue highly challenging. We describe a microscope that combines the distinct advantages of targeted illumination and confocal gating, while also maximizing signal detection efficiency. The resulting benefits in SNR and crosstalk reduction are quantified experimentally and theoretically. Our microscope provides a versatile solution for enabling high-fidelity in vivo voltage imaging at large scales and penetration depths, which we demonstrate across a wide range of imaging conditions and different GEVI classes.

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.

Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator.

The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

Correcting sampling bias in speckle contrast imaging.

When performing spatial or temporal laser speckle contrast imaging (LSCI), contrast is generally estimated from localized windows containing limited numbers of independent speckle grains NS. This leads to a systematic bias in the estimated speckle contrast. We describe an approach to determine NS and largely correct for this bias, enabling a more accurate estimation of the speckle decorrelation time without recourse to numerical fitting of data. Validation experiments are presented where measurements are ergodic or non-ergodic, including in vivo imaging of mouse brain.

Direct characterization of tissue dynamics with laser speckle contrast imaging.

Laser speckle contrast imaging (LSCI) has gained broad appeal as a technique to monitor tissue dynamics (broadly defined to include blood flow dynamics), in part because of its remarkable simplicity. When laser light is backscattered from a tissue, it produces speckle patterns that vary in time. A measure of the speckle field decorrelation time provides information about the tissue dynamics. In conventional LSCI, this measure requires numerical fitting to a specific theoretical model for the field decorrelation. However, this model may not be known a priori, or it may vary over the image field of view. We describe a method to reconstruct the speckle field decorrelation time that is completely model free, provided that the measured speckle dynamics are ergodic. We also extend our approach to allow for the possibility of non-ergodic measurements caused by the presence of a background static speckle field. In both ergodic and non-ergodic cases, our approach accurately retrieves the correlation time without any recourse to numerical fitting and is largely independent of camera exposure time. We apply our method to tissue phantom and in-vivo mouse brain imaging. Our aim is to facilitate and add robustness to LSCI processing methods for potential clinical or pre-clinical applications.

Flatfield ultrafast imaging with single-shot non-synchronous array photography.

We present a method for acquiring a sequence of time-resolved images in a single shot, called single-shot non-synchronous array photography (SNAP). In SNAP, a pulsed laser beam is split by a diffractive optical element into an array of angled beamlets whose illumination fronts remain perpendicular to the optical axis. Different time delays are imparted to each beamlet by an echelon, enabling them to probe ultrafast dynamics in rapid succession. The beamlets are imaged onto different regions of a camera by a lenslet array. Because the illumination fronts remain flat (head-on) independently of beamlet angle, the exposure time in SNAP is fundamentally limited only by the laser pulse duration, akin to a "global shutter" in conventional imaging. We demonstrate SNAP by capturing the evolution of a laser induced plasma filament over 20 frames at an average rate of 4.2 trillion frames per second (Tfps) and a peak rate of 5.7 Tfps.

Large-scale voltage imaging in behaving mice using targeted illumination.

Recent improvements in genetically encoded voltage indicators enabled optical imaging of action potentials and subthreshold transmembrane voltage in vivo. To perform high-speed voltage imaging of many neurons simultaneously over a large anatomical area, widefield microscopy remains an essential tool. However, the lack of optical sectioning makes widefield microscopy prone to background cross-contamination. We implemented a digital-micromirror-device-based targeted illumination strategy to restrict illumination to the cells of interest and quantified the resulting improvement both theoretically and experimentally with SomArchon expressing neurons. We found that targeted illumination increased SomArchon signal contrast, decreased photobleaching, and reduced background cross-contamination. With the use of a high-speed, large-area sCMOS camera, we routinely imaged tens of spiking neurons simultaneously over minutes in behaving mice. Thus, the targeted illumination strategy described here offers a simple solution for widefield voltage imaging of many neurons over a large field of view in behaving animals.

High-speed multifocus phase imaging in thick tissue.

Phase microscopy is widely used to image unstained biological samples. However, most phase imaging techniques require transmission geometries, making them unsuited for thick sample applications. Moreover, when applied to volumetric imaging, phase imaging generally requires large numbers of measurements, often making it too slow to capture live biological processes with fast 3D index-of-refraction variations. By combining oblique back-illumination microscopy and a z-splitter prism, we perform phase imaging that is both epi-mode and multifocus, enabling high-speed 3D phase imaging in thick, scattering tissues with a single camera. We demonstrate here 3D qualitative phase imaging of blood flow in chick embryos over a field of view of 546 × 546 × 137 µm3 at speeds up to 47 Hz.

Depth resolution in multifocus laser speckle contrast imaging.

Laser speckle contrast imaging (LSCI) can be used to evaluate blood flow based on spatial or temporal speckle statistics, but its accuracy is undermined by out-of-focus image blur. In this Letter, we show how the fraction of dynamic versus static light scattering is dependent on focus, and describe a deconvolution strategy to correct for out-of-focus blur. With the aid of a z-splitter, which enables instantaneous multifocus imaging, we demonstrate depth-resolved LSCI that can robustly extract multi-plane structural and flow-speed information simultaneously. This method is applied to in vivo imaging of blood vessels in a mouse cortex and provides improved estimates of blood flow speed throughout a depth range of 300µm.

Fast, multiplane line-scan confocal microscopy using axially distributed slits.

The inherent constraints on resolution, speed and field of view have hindered the development of high-speed, three-dimensional microscopy techniques over large scales. Here, we present a multiplane line-scan imaging strategy, which uses a series of axially distributed reflecting slits to probe different depths within a sample volume. Our technique enables the simultaneous imaging of an optically sectioned image stack with a single camera at frame rates of hundreds of hertz, without the need for axial scanning. We demonstrate the applicability of our system to monitor fast dynamics in biological samples by performing calcium imaging of neuronal activity in mouse brains and voltage imaging of cardiomyocytes in cardiac samples.

Ultrasound differential phase contrast using backscattering and the memory effect.

We describe a simple and fast technique to perform ultrasound differential phase contrast (DPC) imaging in arbitrarily thick scattering media. Although configured in a reflection geometry, DPC is based on transmission imaging and is a direct analog of optical differential interference contrast. DPC exploits the memory effect and works in combination with standard pulse-echo imaging, with no additional hardware or data requirements, enabling complementary phase contrast (in the transverse direction) without any need for intensive numerical computation. We experimentally demonstrate the principle of DPC using tissue phantoms with calibrated speed-of-sound inclusions.

Scan multiplier unit for ultrafast laser scanning beyond the inertia limit.

A passive add-on greatly multiplies the sweep rate of any mechanical scanner while also enhancing throughput, enabling a single linear scanner to produce ultrafast 1D or 2D laser scans for general applications.

In vivo corneal and lenticular microscopy with asymmetric fundus retroillumination.

We describe a new technique for non-contact in vivo corneal and lenticular microscopy. It is based on fundus retro-reflection and back-illumination of the crystalline lens and cornea. To enhance phase-gradient contrast, we apply asymmetric illumination by illuminating one side of the fundus. The technique produces micron-scale lateral resolution images across a 1 mm diagonal field of view in the central cornea. We show representative images of the epithelium, the subbasal nerve plexus, large stromal nerves, dendritic immune cells, endothelial nuclei, and the anterior crystalline lens, demonstrating the potential of this instrument for clinical applications.