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1.
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.  相似文献   

2.
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.  相似文献   

3.
双光子荧光显微镜是神经科学研究中的重要观测仪器,但是现有的商品化仪器受限于较低的成像速度,难以满足脑功能研究中毫秒量级神经信号检测的需要.基于声光偏转器的快速随机扫描双光子显微成像技术,有望在保持信噪比的同时提高观测速度.本文综述了这一研究的最新进展,从飞秒激光经过角色散器件后的时空演化理论、声光偏转器的色散补偿方法、随机扫描成像仪器及仪器应用到神经成像时钙信号的识别方法四个方面分别进行介绍,最后分析了随机扫描双光子显微成像技术的发展趋势.这项技术的系统深入研究将为神经活动观测提供一种全新的方法,推动脑科学研究的发展.  相似文献   

4.
Multiphoton imaging has evolved as an indispensable tool in cell biology and holds prospects for clinical applications. When addressing endogenous signals such as coherent anti-Stokes Raman scattering (CARS) or second harmonic generation, it requires intense laser irradiation that may cause photodamage. We report that increasing endogenous fluorescence signal upon multiphoton imaging constitutes a marker of photodamage. The effect was studied on mouse brain in vivo and ex vivo, on ex vivo human brain tissue samples, as well as on glioblastoma cells in vitro, demonstrating that this phenomenon is common to a variety of different systems, both ex vivo and in vivo. CARS microscopy and vibrational spectroscopy were used to analyze the photodamage. The development of a standard easy-to-use model that employs rehydrated cryosections allowed the characterization of the irradiation-induced fluorescence and related it to nonlinear photodamage. In conclusion, the monitoring of endogenous two-photon excited fluorescence during label-free multiphoton microscopy enables to estimate damage thresholds ex vivo as well as detect photodamage during in vivo experiments.  相似文献   

5.
Hidden Markov modeling (HMM) can be applied to extract single channel kinetics at signal-to-noise ratios that are too low for conventional analysis. There are two general HMM approaches: traditional Baum's reestimation and direct optimization. The optimization approach has the advantage that it optimizes the rate constants directly. This allows setting constraints on the rate constants, fitting multiple data sets across different experimental conditions, and handling nonstationary channels where the starting probability of the channel depends on the unknown kinetics. We present here an extension of this approach that addresses the additional issues of low-pass filtering and correlated noise. The filtering is modeled using a finite impulse response (FIR) filter applied to the underlying signal, and the noise correlation is accounted for using an autoregressive (AR) process. In addition to correlated background noise, the algorithm allows for excess open channel noise that can be white or correlated. To maximize the efficiency of the algorithm, we derive the analytical derivatives of the likelihood function with respect to all unknown model parameters. The search of the likelihood space is performed using a variable metric method. Extension of the algorithm to data containing multiple channels is described. Examples are presented that demonstrate the applicability and effectiveness of the algorithm. Practical issues such as the selection of appropriate noise AR orders are also discussed through examples.  相似文献   

6.
In vivo two-photon calcium imaging would benefit from the use of multiple excitation beams to increase scanning speed, signal-to-noise ratio and field of view or to image different axial planes simultaneously. Using spatiotemporal multiplexing we circumvented light-scattering ambiguity inherent to deep-tissue multifocal two-photon microscopy. We demonstrate calcium imaging at multiple axial planes in the intact mouse brain to monitor network activity of ensembles of cortical neurons in three spatial dimensions.  相似文献   

7.
One of the challenges for modern neuroscience is to understand the rules of concerted neuronal function in vivo. This question can be addressed using noninvasive high-resolution imaging techniques like two-photon microscopy. This protocol describes a versatile approach for in vivo two-photon calcium imaging of neural networks, stained with membrane-permeant fluorescent-indicator dyes. It is based on a targeted pressure ejection of the dye into the tissue of interest and can be used for a large spectrum of indicator dyes, including Oregon Green 488 BAPTA-1 acetoxymethyl ester and Fura-2 acetoxymethyl ester. Through the use of dye mixtures and multicolor imaging, this technique allows the visualization of distinct neurons and glial cells up to 500 microm below the brain surface. It is suitable for staining the brain tissue of various different species (e.g., mouse, rat, cat and zebrafish) at all developmental stages. When combined with brain microendoscopy, it allows the monitoring of intracellular calcium signals in awake, behaving animals. The total time required to carry out the protocol, including dissection and cell staining, is approximately 2 h. Thereafter, imaging experiments might be performed for at least 6 h.  相似文献   

8.
We introduce an optical method to stimulate individual neurons in brain slices in any arbitrary spatiotemporal pattern, using two-photon uncaging of MNI-glutamate with beam multiplexing. This method has single-cell and three-dimensional precision. By sequentially stimulating up to a thousand potential presynaptic neurons, we generated detailed functional maps of inputs to a cell. We combined this approach with two-photon calcium imaging in an all-optical method to image and manipulate circuit activity.  相似文献   

9.
Glial cells have been identified as key signaling components in the brain; however, methods to investigate their structure and function in vivo have been lacking. Here, we describe a new, highly selective approach for labeling astrocytes in intact rodent neocortex that allows in vivo imaging using two-photon microscopy. The red fluorescent dye sulforhodamine 101 (SR101) was specifically taken up by protoplasmic astrocytes after brief exposure to the brain surface. Specificity was confirmed by immunohistochemistry. In addition, SR101 labeled enhanced green fluorescent protein (EGFP)-expressing astrocytes but not microglial cells in transgenic mice. We used SR101 labeling to quantify morphological characteristics of astrocytes and to visualize their close association with the cortical microvasculature. Furthermore, by combining this method with calcium indicator loading of cell populations, we demonstrated distinct calcium dynamics in astroglial and neuronal networks. We expect SR101 staining to become a principal tool for investigating astroglia in vivo.  相似文献   

10.
We report a technique for two-photon fluorescence imaging with cellular resolution in awake, behaving mice with minimal motion artifact. The apparatus combines an upright, table-mounted two-photon microscope with a spherical treadmill consisting of a large, air-supported Styrofoam ball. Mice, with implanted cranial windows, are head restrained under the objective while their limbs rest on the ball's upper surface. Following adaptation to head restraint, mice maneuver on the spherical treadmill as their heads remain motionless. Image sequences demonstrate that running-associated brain motion is limited to approximately 2-5 microm. In addition, motion is predominantly in the focal plane, with little out-of-plane motion, making the application of a custom-designed Hidden-Markov-Model-based motion correction algorithm useful for postprocessing. Behaviorally correlated calcium transients from large neuronal and astrocytic populations were routinely measured, with an estimated motion-induced false positive error rate of <5%.  相似文献   

11.
Ji N  Magee JC  Betzig E 《Nature methods》2008,5(2):197-202
Pulsed lasers are key elements in nonlinear bioimaging techniques such as two-photon fluorescence excitation (TPE) microscopy. Typically, however, only a percent or less of the laser power available can be delivered to the sample before photoinduced damage becomes excessive. Here we describe a passive pulse splitter that converts each laser pulse into a fixed number of sub-pulses of equal energy. We applied the splitter to TPE imaging of fixed mouse brain slices labeled with GFP and show that, in different power regimes, the splitter can be used either to increase the signal rate more than 100-fold or to reduce the rate of photobleaching by over fourfold. In living specimens, the gains were even greater: a ninefold reduction in photobleaching during in vivo imaging of Caenorhabditis elegans larvae, and a six- to 20-fold decrease in the rate of photodamage during calcium imaging of rat hippocampal brain slices.  相似文献   

12.
Neurons in the mammalian brain receive thousands of synaptic inputs on their dendrites. In many types of neurons, such as cortical pyramidal neurons, excitatory synapses are formed on fine dendritic protrusions called spines. Usually, an individual spine forms a single synaptic contact with an afferent axon. In this protocol, we describe a recently established experimental procedure for measuring intracellular calcium signals from dendritic spines in cortical neurons in vivo by using a combination of two-photon microscopy and whole-cell patch-clamp recordings. We have used mice as an experimental model system, but the protocol may be readily adapted to other species. This method involves data acquisition at high frame rates and low-excitation laser power, and is termed low-power temporal oversampling (LOTOS). Because of its high sensitivity of fluorescence detection and reduced phototoxicity, LOTOS allows for prolonged and stable calcium imaging in vivo. Key aspects of the protocol, which can be completed in 5-6 h, include the use of a variant of high-speed two-photon imaging, refined surgery procedures and optimized tissue stabilization.  相似文献   

13.
Dendritic spines are the postsynaptic sites of most excitatory synapses in the mammalian brain. With the advent of two-photon microscopy and transgenic mice expressing fluorescent proteins, dendritic spines can now be imaged in the living cerebral cortex over time scales ranging from seconds to years. Recent studies with this in vivo imaging approach have begun to provide important insights into the development and plasticity of dendritic spines in the intact brain. Here, we review these studies and discuss technical requirements for image acquisition. We envision that intravital two-photon imaging at the level of individual synapses will greatly expand our current understandings of how neuronal networks are assembled and modified throughout life.  相似文献   

14.
Calcium imaging has revolutionized the approaches for functional analyses in the living brain of animal experimental models. Changes in intracellular calcium concentration are strictly linked to the electrical activity in neurons and produce signals that are effectively detected by optical methods. Distinctive features of fluorescence-based calcium imaging are its high temporal resolution in the millisecond range and its high spatial resolution in the micrometer range. Recent progress includes the development of fluorometric calcium sensors, new approaches for targeted labeling with these sensors and the implementation of powerful imaging techniques, especially two-photon microscopy. An important and rapidly evolving field of current research is the use of calcium imaging for the analysis of in vivo mouse models for various brain diseases, such as Alzheimer's disease, stroke and epilepsy.  相似文献   

15.
Targeted whole-cell recordings in the mammalian brain in vivo   总被引:13,自引:0,他引:13  
While electrophysiological recordings from visually identified cell bodies or dendrites are routinely performed in cell culture and acute brain slice preparations, targeted recordings from the mammalian nervous system are currently not possible in vivo. The "blind" approach that is used instead is somewhat random and largely limited to common neuronal cell types. This approach prohibits recordings from, for example, molecularly defined and/or disrupted populations of neurons. Here we describe a method, which we call TPTP (two-photon targeted patching), that uses two-photon imaging to guide in vivo whole-cell recordings to individual, genetically labeled cortical neurons. We apply this technique to obtain recordings from genetically manipulated, parvalbumin-EGFP-positive interneurons in the somatosensory cortex. We find that both spontaneous and sensory-evoked activity patterns involve the synchronized discharge of electrically coupled interneurons. TPTP applied in vivo will therefore provide new insights into the molecular control of neuronal function at the systems level.  相似文献   

16.
H J Koester  D Baur  R Uhl    S W Hell 《Biophysical journal》1999,77(4):2226-2236
The signal and limitations of calcium florescence imaging using nonresonant multiphoton absorption of near-infrared femto- and picosecond laser pulses were examined. The fluorescence changes of various Ca(2+)-indicators induced by transient increases of the intradendritic calcium concentration were evaluated by evoking physiological activity in neocortical neurons in rat brain slices. Photodamage was noticeable as irreversible changes in the parameters describing the calcium fluorescence transients. At higher two-photon excitation rates, a great variety of irregular functional and structural alterations occurred. Thus, signal and observation time were limited by phototoxic effects. At lower excitation rates, photodamage accumulated linearly with exposure time. Femtosecond and picosecond laser pulses were directly compared with respect to this cumulative photodamage. The variation of the pulse length at a constant two-photon excitation rate indicated that a two-photon excitation mechanism is mainly responsible for the cumulative photodamage within the investigated window of 75 fs to 3.2 ps. As a direct consequence, at low excitation rates, the same image quality is achieved irrespective of whether two-photon Ca(2+)-imaging is carried out with femto- or picosecond laser pulses.  相似文献   

17.
Grienberger C  Konnerth A 《Neuron》2012,73(5):862-885
Calcium ions generate versatile intracellular signals that control key functions in all types of neurons. Imaging calcium in neurons is particularly important because calcium signals exert their highly specific functions in well-defined cellular subcompartments. In this Primer, we briefly review the general mechanisms of neuronal calcium signaling. We then introduce the calcium imaging devices, including confocal and two-photon microscopy as well as miniaturized devices that are used in freely moving animals. We provide an overview of the classical chemical fluorescent calcium indicators and of the protein-based genetically encoded calcium indicators. Using application examples, we introduce new developments in the field, such as calcium imaging in awake, behaving animals and the use of calcium imaging for mapping single spine sensory inputs in cortical neurons in vivo. We conclude by providing an outlook on the prospects of calcium imaging for the analysis of neuronal signaling and plasticity in various animal models.  相似文献   

18.
Second harmonic generating (SHG) nanoprobes have recently emerged as versatile and durable labels suitable for in vivo imaging, circumventing many of the inherent drawbacks encountered with classical fluorescent probes. Since their nanocrystalline structure lacks a central point of symmetry, they are capable of generating second harmonic signal under intense illumination - converting two photons into one photon of half the incident wavelength - and can be detected by conventional two-photon microscopy. Because the optical signal of SHG nanoprobes is based on scattering, rather than absorption as in the case of fluorescent probes, they neither bleach nor blink, and the signal does not saturate with increasing illumination intensity. When SHG nanoprobes are used to image live tissue, the SHG signal can be detected with little background signal, and they are physiologically inert, showing excellent long-term photostability. Because of their photophysical properties, SHG nanoprobes provide unique advantages for molecular imaging of living cells and tissues with unmatched sensitivity and temporal resolution.  相似文献   

19.

Background

Living tissues contain a range of intrinsic fluorophores and sources of second harmonic generation which provide contrast that can be exploited for fresh tissue imaging. Microscopic imaging of fresh tissue samples can circumvent the cost and time associated with conventional histology. Further, intrinsic contrast can provide rich information about a tissue''s composition, structure and function, and opens the potential for in-vivo imaging without the need for contrast agents.

Methodology/Principal Findings

In this study, we used hyperspectral two-photon microscopy to explore the characteristics of both normal and diseased gastrointestinal (GI) tissues, relying only on their endogenous fluorescence and second harmonic generation to provide contrast. We obtained hyperspectral data at subcellular resolution by acquiring images over a range of two-photon excitation wavelengths, and found excitation spectral signatures of specific tissue types based on our ability to clearly visualize morphology. We present the two-photon excitation spectral properties of four major tissue types that are present throughout the GI tract: epithelium, lamina propria, collagen, and lymphatic tissue. Using these four excitation signatures as basis spectra, linear unmixing strategies were applied to hyperspectral data sets of both normal and neoplastic tissue acquired in the colon and small intestine. Our results show that hyperspectral unmixing with excitation spectra allows segmentation, showing promise for blind identification of tissue types within a field of view, analogous to specific staining in conventional histology. The intrinsic spectral signatures of these tissue types provide information relating to their biochemical composition.

Conclusions/Significance

These results suggest hyperspectral two-photon microscopy could provide an alternative to conventional histology either for in-situ imaging, or intraoperative ‘instant histology’ of fresh tissue biopsies.  相似文献   

20.
Neurons in cortical sensory regions receive modality-specific information through synapses that are located on their dendrites. Recently, the use of two-photon microscopy combined with whole-cell recordings has helped to identify visually evoked dendritic calcium signals in mouse visual cortical neurons in vivo. The calcium signals are restricted to small dendritic domains ('hotspots') and they represent visual synaptic inputs that are highly tuned for orientation and direction. This protocol describes the experimental procedures for the recording and the analysis of these visually evoked dendritic calcium signals. The key points of this method include delivery of fluorescent calcium indicators through the recording patch pipette, selection of an appropriate optical plane with many dendrites, hyperpolarization of the membrane potential and two-photon imaging. The whole protocol can be completed in 5-6 h, including 1-2 h of two-photon calcium imaging in combination with stable whole-cell recordings.  相似文献   

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