High-speed, random-access fluorescence microscopy: I. High-resolution optical recording with voltage-sensitive dyes and ion indicators.

The design and implementation of a high-speed, random-access, laser-scanning fluorescence microscope configured to record fast physiological signals from small neuronal structures with high spatiotemporal resolution is presented. The laser-scanning capability of this nonimaging microscope is provided by two orthogonal acousto-optic deflectors under computer control. Each scanning point can be randomly accessed and has a positioning time of 3-5 microseconds. Sampling time is also computer-controlled and can be varied to maximize the signal-to-noise ratio. Acquisition rates up to 200k samples/s at 16-bit digitizing resolution are possible. The spatial resolution of this instrument is determined by the minimal spot size at the level of the preparation (i.e., 2-7 microns). Scanning points are selected interactively from a reference image collected with differential interference contrast optics and a video camera. Frame rates up to 5 kHz are easily attainable. Intrinsic variations in laser light intensity and scanning spot brightness are overcome by an on-line signal-processing scheme. Representative records obtained with this instrument by using voltage-sensitive dyes and calcium indicators demonstrate the ability to make fast, high-fidelity measurements of membrane potential and intracellular calcium at high spatial resolution (2 microns) without any temporal averaging.     

Efficient and cost‐effective 3D cellular imaging by sub‐voxel‐resolving light‐sheet add‐on microscopy

Light‐sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high‐speed and with low photo‐toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub‐voxel‐resolving (SVR) light‐sheet add‐on microscopy (SLAM), is presented to enable fast, resolution‐enhanced light‐sheet fluorescence imaging from a conventional wide‐field microscope. This method contains two components: a miniature add‐on device to regular wide‐field microscopes, which contains a horizontal laser light‐sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off‐axis scanning strategy and a SVR algorithm that utilizes sub‐voxel spatial shifts to reconstruct the image volume that results in a twofold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost‐effective solution to convert the vast number of in‐service microscopes for fast 3D live imaging with voxel‐super‐resolved capability.     

一种低成本、高性价比的穿戴式扩散光学断层人脑成像系统

现有扩散光学断层成像(diffuse optical tomography,DOT)系统常采用高档光探测器和放大器,令系统成本居高不下、性价比较低.本文旨在使用全新技术降低系统成本、增加成像深度,研发一套可实用于人脑成像的新型扩散光学断层成像系统.为此采用了成本较低的新型光探测器和一款自主设计研制的光电二极管前置放大器.相较于实验室原有DOT系统,新系统整体成本节约了40%以上.其次,设计了一款具有弹性并可适应多种不规则待测物(subject)外形的无光纤穿戴式脑-机接口(brain computer interface,BCI)装置.本文首次将手持式3D激光扫描仪应用于待测物外形结构和光源-探测器位置信息的精确获取.最后综合应用上述技术装备于人脑形状仿体成像实验中,新系统的目标成像深度提升至35 mm.验证结果说明本系统已具备了实际人脑功能成像能力.     

Computer aided fluorescence imaging of photosynthetic systems

A fluorescence video imaging system utilizing relatively inexpensive commercial components is described. The instrument utilizes a black and white CCD video camera detector, a commercial video imaging board and a IBM-AT compatible computer. The color output of the imaging board greatly aids in the users ability to visually discriminate areas of interest in the video field. Software development that enables the user to capture kinetic traces in real time from the video images is also described. The system is used to monitor fluorescence from photosynthetic systems. The usefulness of the system in screening for photosynthetic mutants is also demonstrated. The cost of the system can be kept below $12,000.Abbreviations CCD charge-coupled device - DCMU diuron, 3-[3,4-Dichlorophenyl]1,1-dimethylurea - AGC automatic gain control - LUT look-up table - AOI area of interest - CPU central processing unit - RAM random access memory - ADC analog-to-digital converter - FVIPS fluorescence video image processing software - I/O input/output - F₀ dark-level fluorescence - OIDPSMT characteristic transient components, where O is dark level, I is intermediary peak, D is dip, P is peak of fast transient, S is quasi-steady state level, M is second maximum, T is terminal level     

Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy

Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.     

TrakEM2 software for neural circuit reconstruction

A key challenge in neuroscience is the expeditious reconstruction of neuronal circuits. For model systems such as Drosophila and C. elegans, the limiting step is no longer the acquisition of imagery but the extraction of the circuit from images. For this purpose, we designed a software application, TrakEM2, that addresses the systematic reconstruction of neuronal circuits from large electron microscopical and optical image volumes. We address the challenges of image volume composition from individual, deformed images; of the reconstruction of neuronal arbors and annotation of synapses with fast manual and semi-automatic methods; and the management of large collections of both images and annotations. The output is a neural circuit of 3d arbors and synapses, encoded in NeuroML and other formats, ready for analysis.     

A new multiparameter flow cytometer: optical and electrical cell analysis in combination with video microscopy in flow

BACKGROUND: Flow cytometers, which are commercially available, do not necessarily meet all demands of actual biomedical research. This is the case for the investigation of mechanisms involved in cell volume regulation, which requires electrical volume measurement and ratiometric multichannel fluorescence analysis for the simultaneous assessment of different physiologic parameters (intracellular pH and the intracellular concentration of calcium ions, etc). METHODS AND RESULTS: We describe the construction of a new nonsorting flow cytometer designed for the simultaneous acquisition of seven parameters including fluorescence signals, forward and perpendicular light scatter, cell volume according to the electrical Coulter principle, and flow cytometric imaging. The instrument is equipped with three different light sources. A tunable argon-ion laser generates efficient excitation of the most standard fluorescent probes in the visible spectral range, and an arc lamp provides the means for ultraviolet excitation at low cost. Because of the spatial filtering by the excitation and detection optics, two independent sets of dual fluorescence measurements can be performed, a prerequisite for flexible ratiometric fluorescence analysis. A flow video microscope integrated into the optical system optionally generates either brightfield or phase images of selected flowing particles. Only particles whose individual datasets meet predefined gating conditions are imaged in real time. To avoid smear effects, the motion of the object to be imaged (speed approximately 8 m/s) is frozen on the target of a CCD camera by flash illumination. For this purpose, a high radiance gas discharge lamp with 25-mJ electric pulse energy provides an illumination time of 18 ns (full width half maximum). Test results obtained from latex spheres and cells are shown. CONCLUSIONS: Test results indicate that our instrument can perform Coulter measurements in combination with flexible optical analysis. Moreover, integration of an adapted video microscope into a flow cytometer is an approach to overcome the gap between flow and image cytometry.     

Volume electron microscopy for neuronal circuit reconstruction

The last decade has seen a rapid increase in the number of tools to acquire volume electron microscopy (EM) data. Several new scanning EM (SEM) imaging methods have emerged, and classical transmission EM (TEM) methods are being scaled up and automated. Here we summarize the new methods for acquiring large EM volumes, and discuss the tradeoffs in terms of resolution, acquisition speed, and reliability. We then assess each method's applicability to the problem of reconstructing anatomical connectivity between neurons, considering both the current capabilities and future prospects of the method. Finally, we argue that neuronal 'wiring diagrams' are likely necessary, but not sufficient, to understand the operation of most neuronal circuits: volume EM imaging will likely find its best application in combination with other methods in neuroscience, such as molecular biology, optogenetics, and physiology.     

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g. obesity) increase with age.     

In situ optical mapping of voltage and calcium in the heart

Electroanatomic mapping the interrelation of intracardiac electrical activation with anatomic locations has become an important tool for clinical assessment of complex arrhythmias. Optical mapping of cardiac electrophysiology combines high spatiotemporal resolution of anatomy and physiological function with fast and simultaneous data acquisition. If applied to the clinical setting, this could improve both diagnostic potential and therapeutic efficacy of clinical arrhythmia interventions. The aim of this study was to explore this utility in vivo using a rat model. To this aim, we present a single-camera imaging and multiple light-emitting-diode illumination system that reduces economic and technical implementation hurdles to cardiac optical mapping. Combined with a red-shifted calcium dye and a new near-infrared voltage-sensitive dye, both suitable for use in blood-perfused tissue, we demonstrate the feasibility of in vivo multi-parametric imaging of the mammalian heart. Our approach combines recording of electrophysiologically-relevant parameters with observation of structural substrates and is adaptable, in principle, to trans-catheter percutaneous approaches.     

Construction of a two-photon microscope for video-rate Ca imaging

We describe the construction of a video-rate two-photon laser scanning microscope, compare its performance to a similar confocal microscope, and illustrate its use for imaging local Ca(2+) transients from cortical neurons in brain slices. Key features include the use of a Ti-sapphire femtosecond laser allowing continuous tuning over a wide (700-1000 nm) wavelength range, a resonant scanning mirror to permit frame acquisition at 30 Hz, and efficient wide-field fluorescence detection. Two-photon imaging provides compelling advantages over confocal microscopy in terms of improved imaging depth and reduced phototoxicity and photobleaching, but the high cost of commercial instruments has limited their widespread adoption. By constructing one's own system the expense is greatly reduced without sacrifice of performance, and the microscope can be more readily tailored to specific applications.     

A novel environmental chamber for neuronal network multisite recordings

Environmental stability is a critical issue for neuronal networks in vitro. Hence, the ability to control the physical and chemical environment of cell cultures during electrophysiological measurements is an important requirement in the experimental design. In this work, we describe the development and the experimental verification of a closed chamber for multisite electrophysiology and optical monitoring. The chamber provides stable temperature, pH and humidity and guarantees cell viability comparable to standard incubators. Besides, it integrates the electronics for long‐term neuronal activity recording. The system is portable and adaptable for multiple network housings, which allows performing parallel experiments in the same environment. Our results show that this device can be a solution for long‐term electrophysiology, for dual network experiments and for coupled optical and electrical measurements. Biotechnol. Bioeng. 2012; 109: 2553–2566. © 2012 Wiley Periodicals, Inc.     

In vitro functional imaging in brain slices using fast voltage-sensitive dye imaging combined with whole-cell patch recording

In many brain areas, circuit connectivity is segregated into specific lamina or glomerula. Functional imaging in these anatomically discrete areas is particularly useful in characterizing circuit properties. Voltage-sensitive dye (VSD) imaging directly assays the spatiotemporal dynamics of neuronal activity, including the functional connectivity of the neurons involved. In spatially segregated structures, VSD imaging can define how physiology and connectivity interact, and can identify functional abnormalities in models of neurological and psychiatric disorders. In the following protocol, we describe the in vitro slice preparation, epifluorescence setup and analyses necessary for fast charge-coupled device (CCD)-based VSD imaging combined with simultaneous whole-cell patch recording. The addition of single-cell recordings validates imaging results, and can reveal the relationship between single-cell activity and the VSD-imaged population response; in synchronously activated neurons, this change in whole-cell recorded V(m) can accurately represent population V(m) changes driving the VSD responses. Thus, the combined VSD imaging and whole-cell patch approach provides experimental resolution spanning single-cell electrophysiology to complex local circuit responses.     

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.     

In vivo imaging of subcutaneous structures using functional photoacoustic microscopy

Functional photoacoustic microscopy (fPAM) is a hybrid technology that permits noninvasive imaging of the optical absorption contrast in subcutaneous biological tissues. fPAM uses a focused ultrasonic transducer to detect high-frequency photoacoustic (PA) signals. Volumetric images of biological tissues can be formed by two-dimensional raster scanning, and functional parameters can be further extracted from spectral measurements. fPAM is safe and applicable to animals as well as humans. This protocol provides guidelines for parameter selection, system alignment, imaging operation, laser safety and data processing for in vivo fPAM. It currently takes approximately 100 min to carry out this protocol, including approximately 50 min for data acquisition using a 10-Hz pulse-repetition-rate laser system. The data acquisition time, however, can be significantly reduced by using a laser system with a higher pulse repetition rate.     

Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy

Unraveling the functions of the diverse neural types in any local circuit ultimately requires methods to record from most or all of its cells simultaneously. One promising approach to this goal is fluorescence imaging, but existing methods using laser-scanning microscopy (LSM) are severely limited in their ability to resolve rapid phenomena, like neuronal action potentials, over wide fields. Here we present a microscope that rapidly sections a three-dimensional volume using a thin illumination sheet whose position is rigidly coupled to the objective and aligned with its focal plane. We demonstrate that this approach allows exceptionally low-noise imaging of large neuronal populations at pixel rates at least 100-fold higher than with LSM. Using this microscope, we studied the pheromone-sensing neurons of the mouse vomeronasal organ and found that responses to dilute urine are largely or exclusively restricted to cells in the apical layer, the location of V1r-family-expressing neurons.     

Precise spatiotemporal control of optogenetic activation using an acousto-optic device

Light activation and inactivation of neurons by optogenetic techniques has emerged as an important tool for studying neural circuit function. To achieve a high resolution, new methods are being developed to selectively manipulate the activity of individual neurons. Here, we report that the combination of an acousto-optic device (AOD) and single-photon laser was used to achieve rapid and precise spatiotemporal control of light stimulation at multiple points in a neural circuit with millisecond time resolution. The performance of this system in activating ChIEF expressed on HEK 293 cells as well as cultured neurons was first evaluated, and the laser stimulation patterns were optimized. Next, the spatiotemporally selective manipulation of multiple neurons was achieved in a precise manner. Finally, we demonstrated the versatility of this high-resolution method in dissecting neural circuits both in the mouse cortical slice and the Drosophila brain in vivo. Taken together, our results show that the combination of AOD-assisted laser stimulation and optogenetic tools provides a flexible solution for manipulating neuronal activity at high efficiency and with high temporal precision.     

Determinants of electrical properties in developing neurons

The regulatory mechanisms that orchestrate the developmental acquisition of electrical properties in embryonic neurons are poorly understood. Progress in this important area is dependent on the availability of preparations that allow electrophysiology to be married with genetics. The powerful genetics of the fruitfly Drosophila melanogaster has long been exploited to describe fundamental mechanisms associated with structural neuronal development (i.e. axon guidance). It has not, however, been fully employed to study the final stages of embryonic neural development and in particular the acquisition of electrical activity. This review focuses on the recent development of a Drosophila preparation that allows central neurons to be accessed by patch electrodes at both embryonic and larval stages. This preparation, which allows electrophysiology to be coupled with genetics, offers the prospect of making significant advances in our understanding of functional neuron development.     

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals^1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo^3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex^5-9.This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.