Improved deep two-photon calcium imaging in vivo

Two-photon laser scanning calcium imaging has emerged as a useful method for the exploration of neural function and structure at the cellular and subcellular level in vivo. The applications range from imaging of subcellular compartments such as dendrites, spines and axonal boutons up to the functional analysis of large neuronal or glial populations. However, the depth penetration is often limited to a few hundred micrometers, corresponding, for example, to the upper cortical layers of the mouse brain. Light scattering and aberrations originating from refractive index inhomogeneties of the tissue are the reasons for these limitations. The depth penetration of two-photon imaging can be enhanced through various approaches, such as the implementation of adaptive optics, the use of three-photon excitation and/or labeling cells with red-shifted genetically encoded fluorescent sensors. However, most of the approaches used so far require the implementation of new instrumentation and/or time consuming staining protocols. Here we present a simple approach that can be readily implemented in combination with standard two-photon microscopes. The method involves an optimized protocol for depth-restricted labeling with the red-shifted fluorescent calcium indicator Cal-590 and benefits from the use of ultra-short laser pulses. The approach allows in vivo functional imaging of neuronal populations with single cell resolution in all six layers of the mouse cortex. We demonstrate that stable recordings in deep cortical layers are not restricted to anesthetized animals but are well feasible in awake, behaving mice. We anticipate that the improved depth penetration will be beneficial for two-photon functional imaging in larger species, such as non-human primates.     

Ultra-Fast Biosensors and Multi-Photon Microscopy in the Future of Brain Studies

The direct, highly selective and sensitive real-time imaging of neuro- and biochemical mediators is the only way to clarify precisely the chemistry of the brain and to discover the key molecular targets involved in regulation of brain homeostasis. To realize that, we need: high-speed deep-tissue imaging techniques with high spatial and temporal resolution; and ultra-fast and highly selective molecular sensors, giving a possibility to monitor target molecules directly in their physiological environment; in addition, these molecular sensors have to be comparatively small and permeable for blood-brain barrier, to be applicable in brain studies. The present view accents on the perspectives for development of direct approach for investigation of function/flow coupling phenomenon in the brain, based on the current progress in development of ultra-fast molecular sensors for direct visualization of biochemical mediators (e.g., nitric oxide, Ca ions), and high-speed two-photon/multi-photon deep-tissue imaging.     

Current and emerging methods for probing neuropeptide transmission

Neuropeptides comprise the most diverse category of neurochemicals in the brain, playing critical roles in a wide range of physiological and pathophysiological processes. Monitoring neuropeptides with high spatial and temporal resolution is essential for understanding how peptidergic transmission is regulated throughout the central nervous system. In this review, we provide an overview of current non-optical and optical approaches used to detect neuropeptides, including their design principles, intrinsic properties, and potential limitations. We also highlight the advantages of using G protein‒coupled receptor (GPCR) activation‒based (GRAB) sensors to monitor neuropeptides in vivo with high sensitivity, good specificity, and high spatiotemporal resolution. Finally, we present a promising outlook regarding the development and optimization of new GRAB neuropeptide sensors, as well as their potential applications.     

Cranial MRI of small rodents using a clinical MR scanner

Increasing numbers of small animal models are in use in the field of neuroscience research. Magnetic resonance imaging (MRI) provides an excellent method for non-invasive imaging of the brain. Using three-dimensional (3D) MR sequences allows lesion volumetry, e.g. for the quantification of tumor size. Specialized small-bore animal MRI scanners are available for high-resolution MRI of small rodents' brain, but major drawbacks of this dedicated equipment are its high costs and thus its limited availability. Therefore, more and more research groups use clinical MR scanners for imaging small animal models. But to achieve a reasonable spatial resolution at an acceptable signal-to-noise ratio with these scanners, some requirements concerning sequence parameters have to be matched. Thus, the aim of this paper was to present in detail a method how to perform MRI of small rodents brain using a standard clinical 1.5 T scanner and clinically available radio frequency coils to keep material costs low and to circumvent the development of custom-made coils.     

A Neuron-Based Screening Platform for Optimizing Genetically-Encoded Calcium Indicators

Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc.) are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs). Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs) and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude.     

Optical calcium imaging in the nervous system of Drosophila melanogaster

BACKGROUND: Drosophila melanogaster is one of the best-studied model organisms in biology, mainly because of the versatility of methods by which heredity and specific expression of genes can be traced and manipulated. Sophisticated genetic tools have been developed to express transgenes in selected cell types, and these techniques can be utilized to target DNA-encoded fluorescence probes to genetically defined subsets of neurons. Neuroscientists make use of this approach to monitor the activity of restricted types or subsets of neurons in the brain and the peripheral nervous system. Since membrane depolarization is typically accompanied by an increase in intracellular calcium ions, calcium-sensitive fluorescence proteins provide favorable tools to monitor the spatio-temporal activity across groups of neurons. SCOPE OF REVIEW: Here we describe approaches to perform optical calcium imaging in Drosophila in consideration of various calcium sensors and expression systems. In addition, we outline by way of examples for which particular neuronal systems in Drosophila optical calcium imaging have been used. Finally, we exemplify briefly how optical calcium imaging in the brain of Drosophila can be carried out in practice. MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE: Drosophila provides an excellent model organism to combine genetic expression systems with optical calcium imaging in order to investigate principles of sensory coding, neuronal plasticity, and processing of neuronal information underlying behavior. This article is part of a Special Issue entitled Biochemical, Biophysical and Genetic Approaches to Intracellular Calcium Signaling.     

Methods for in vivo molecular imaging

Visualization of single molecules and specific subsets of cells is widely used for studies of biological processes and particularly in immunological research. Recent technological advances have provided a qualitative change in biological visualization from studying of ??snapshot?? pictures to real-time continuous observation of cellular dynamics in vivo. Contemporary methods of in vivo imaging make it possible to localize specific cells within organs and tissues, to study their differentiation, migration, and cell-to-cell interactions, and to follow some intracellular events. Fluorescence intravital microscopy plays an especially important role in high resolution molecular imaging. The methods of intravital microscopy are quickly advancing thanks to improvements in molecular sensors, labeling strategies, and detection approaches. Novel techniques allow simultaneous detection of various probes with better resolution and depth of imaging. In this review, we describe current methods for in vivo imaging, with special accent on fluorescence approaches, and discuss their applications for medical and biological studies.     

Imaging neuronal networks in behaving animals

Neuronal activity has recently been imaged with single-cell resolution in behaving vertebrates. This was accomplished by using fluorescent calcium indicators in conjunction with confocal or two-photon microscopy. These optical techniques, along with other new approaches for imaging synaptic activity, second messengers, and neurotransmitters and their receptors offer great promise for the study of neuronal networks at high resolution in vivo.     

Synthesis,Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.     

Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes

The understanding of brain computations requires methods that read out neural activity on different spatial and temporal scales. Following signal propagation and integration across a neuron and recording the concerted activity of hundreds of neurons pose distinct challenges, and the design of imaging systems has been mostly focused on tackling one of the two operations. We developed a high-resolution, acousto-optic two-photon microscope with continuous three-dimensional (3D) trajectory and random-access scanning modes that reaches near-cubic-millimeter scan range and can be adapted to imaging different spatial scales. We performed 3D calcium imaging of action potential backpropagation and dendritic spike forward propagation at sub-millisecond temporal resolution in mouse brain slices. We also performed volumetric random-access scanning calcium imaging of spontaneous and visual stimulation-evoked activity in hundreds of neurons of the mouse visual cortex in vivo. These experiments demonstrate the subcellular and network-scale imaging capabilities of our system.     

Denoising two-photon calcium imaging data

Two-photon calcium imaging is now an important tool for in vivo imaging of biological systems. By enabling neuronal population imaging with subcellular resolution, this modality offers an approach for gaining a fundamental understanding of brain anatomy and physiology. Proper analysis of calcium imaging data requires denoising, that is separating the signal from complex physiological noise. To analyze two-photon brain imaging data, we present a signal plus colored noise model in which the signal is represented as harmonic regression and the correlated noise is represented as an order autoregressive process. We provide an efficient cyclic descent algorithm to compute approximate maximum likelihood parameter estimates by combing a weighted least-squares procedure with the Burg algorithm. We use Akaike information criterion to guide selection of the harmonic regression and the autoregressive model orders. Our flexible yet parsimonious modeling approach reliably separates stimulus-evoked fluorescence response from background activity and noise, assesses goodness of fit, and estimates confidence intervals and signal-to-noise ratio. This refined separation leads to appreciably enhanced image contrast for individual cells including clear delineation of subcellular details and network activity. The application of our approach to in vivo imaging data recorded in the ferret primary visual cortex demonstrates that our method yields substantially denoised signal estimates. We also provide a general Volterra series framework for deriving this and other signal plus correlated noise models for imaging. This approach to analyzing two-photon calcium imaging data may be readily adapted to other computational biology problems which apply correlated noise models.     

Hemodynamic responses in cortex investigated with optical imaging methods. Implications for functional brain mapping.

During the last 20 years, optical imaging methods - either alone or in combination with other recording techniques - has proven a fruitful approach to explore both the physiological and the functional aspects of activity-evoked hemodynamic responses in cortex. One of the main advantages of optical imaging consists in its high spatio-temporal resolution (in the order of few microns and milliseconds), allowing not only to unambiguously distinguish between activity patterns relating to the underlying functional architecture and those originating from the activation of medium/large blood vessels, but also to investigate the various activity-evoked hemodynamic processes at very fine detail. Here, we briefly review the principal findings obtained by optical imaging about the spatio-temporal properties of the various hemodynamic responses in cortex, i.e., changes in blood-oxygenation, blood-volume, and, to some extent, blood-flow. We also discuss the implications of those findings for non-invasive high-resolution functional brain imaging, in particular for fMRI. Finally, we underscore the importance of novel approaches for high-resolution blood-flow imaging, in the context of the need to gather information at fine spatial detail about the blood-flow response, necessary to constrain the multiple free parameters of hemodynamic response models.     

Spatiotemporal dynamics in large-scale cortical networks

Investigating links between nervous system function and behavior requires monitoring neuronal activity at a range of spatial and temporal scales. Here, we summarize recent progress in applying two distinct but complementary approaches to the study of network dynamics in the neocortex. Mesoscopic calcium imaging allows simultaneous monitoring of activity across most of the cortex at moderate spatiotemporal resolution. Electrophysiological recordings provide extremely high temporal resolution of neural signals at multiple targeted locations. A number of recent studies have used these tools to reveal novel patterns of activity across distributed cortical subnetworks. This growing body of work strongly supports the hypothesis that the dynamic coordination of spatially distinct regions is a fundamental aspect of cortical function that supports cognition and behavior.     

A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals

Two-photon microscopy has enabled anatomical and functional fluorescence imaging in the intact brain of rats. Here, we extend two-photon imaging from anesthetized, head-stabilized to awake, freely moving animals by using a miniaturized head-mounted microscope. Excitation light is conducted to the microscope in a single-mode optical fiber, and images are scanned using vibrations of the fiber tip. Microscope performance was first characterized in the neocortex of anesthetized rats. We readily obtained images of vasculature filled with fluorescently labeled blood and of layer 2/3 pyramidal neurons filled with a calcium indicator. Capillary blood flow and dendritic calcium transients were measured with high time resolution using line scans. In awake, freely moving rats, stable imaging was possible except during sudden head movements.     

多方式认知功能成像研究进展

对大脑结构和功能的深入研究要求认知功能成像技术同时具有高时间分辨率和高空间分辨率.多方式认知功能成像通过不同成像技术fMRI/PET和EEG/MEG的结合,能够同时在空间定位和时间过程上研究大脑认知活动的动态过程.多方式认知功能成像已经被成功地应用于选择性注意、视觉通路、随意运动和语义加工等的研究,并揭示了相关大脑活动的空间和时间特征.今后的研究将进一步提高多方式认知功能成像的时空分辨率和准确性,以更深入地探索认知功能的神经机制.     

基于扩散张量成像的脑组织各向异性电导率计算模型的研究综述

脑组织电导率不仅对脑电源分析起着至关重要的作用,而且也是及时发现脑组织发生功能性病变的重要依据之一.扩散张量成像是一种无损伤功能性成像新技术,具有很高的空间分辨率.基于扩散张量成像的脑组织电导率计算是近年来的一项重要研究课题.本文综述了已有脑组织各向异性电导率的计算模型,主要包括张量特征值线性模型、电场力-粘力平衡模型...     

Quantitative imaging using genetically encoded sensors for small molecules in plants

Quantitative imaging in live cells is a powerful method for monitoring the dynamics of biomolecules at an excellent spatio-temporal resolution. Such an approach, initially limited to a small number of substrates for which specific dyes were available, has become possible for a large number of biomolecules due to the development of genetically encoded, protein-based sensors. These sensors, which can be introduced into live cells through a transgenic approach, offer the benefits of quantitative imaging, with an extra advantage of non-invasiveness. In the past decade there has been a drastic expansion in the number of biomolecules for which genetically encoded sensors are available, and the functional properties of existing sensors are being improved at a dramatic pace. A number of technical improvements have now made the application of genetically encoded sensors in plants rather straightforward, and some of the sensors such as calcium indicator proteins have become standard analytical tools in many plant laboratories. The use of a handful of probes has already revealed an amazing specificity of cellular biomolecule dynamics in plants, which leads us to believe that there are many more discoveries to be made using genetically encoded sensors. In this short review, we will summarize the progress made in the past 15?years in the development in genetically encoded sensors, and highlight significant discoveries made in plant biology.     

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.     

Digitizing life at the level of the cell: high-performance laser-scanning microscopy and image analysis for in toto imaging of development

The field of biological imaging is progressing at an amazing rate. Advances in both laser-scanning microscopy and green fluorescent protein (GFP) technology are combining to make possible imaging-based approaches for studying developmental mechanisms that were previously impossible. Modern confocal and multi-photon microscopes are pushing the envelope of speed, sensitivity, spectral resolution, and depth resolution to allow in vivo imaging of whole, live embryos at cellular resolution over extended periods of time. In toto imaging, in which nearly every cell in an embryo or tissue can be tracked through space and time during development, may become a standard technique for small transparent embryos such as zebrafish and early stage chick and mouse embryos. GFP and its spectral variants can be used to mark a wide range of in vivo biological information for in toto imaging including gene expression patterns, mutant phenotypes, and protein subcellular localization patterns. Combining in toto imaging and GFP transgenic approaches on a large scale may usher in an explosion of in vivo, developmental data as has happened in the past several years with genomic data. There are significant challenges that must be met to reach these goals. This paper will discuss the current state-of-the-art, the challenges, and the prospects of in toto imaging in the areas of imaging, image analysis, and informatics.     

Two-Photon Excitation STED Microscopy in Two Colors in Acute Brain Slices

Many cellular structures and organelles are too small to be properly resolved by conventional light microscopy. This is particularly true for dendritic spines and glial processes, which are very small, dynamic, and embedded in dense tissue, making it difficult to image them under realistic experimental conditions. Two-photon microscopy is currently the method of choice for imaging in thick living tissue preparations, both in acute brain slices and in vivo. However, the spatial resolution of a two-photon microscope, which is limited to ∼350 nm by the diffraction of light, is not sufficient for resolving many important details of neural morphology, such as the width of spine necks or thin glial processes. Recently developed superresolution approaches, such as stimulated emission depletion microscopy, have set new standards of optical resolution in imaging living tissue. However, the important goal of superresolution imaging with significant subdiffraction resolution has not yet been accomplished in acute brain slices. To overcome this limitation, we have developed a new microscope based on two-photon excitation and pulsed stimulated emission depletion microscopy, which provides unprecedented spatial resolution and excellent experimental access in acute brain slices using a long-working distance objective. The new microscope improves on the spatial resolution of a regular two-photon microscope by a factor of four to six, and it is compatible with time-lapse and simultaneous two-color superresolution imaging in living cells. We demonstrate the potential of this nanoscopy approach for brain slice physiology by imaging the morphology of dendritic spines and microglial cells well below the surface of acute brain slices.