Imaging in vivo: watching the brain in action

Kerr, Jason N. D.; Denk, Winfried

doi:10.1038/nrn2338

Cited by 311 publications

(213 citation statements)

References 158 publications

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“…However, reliable in vivo single-AP detection is not yet feasible with the current generation of techniques. For the moment, projects seeking to image sparsely encoded information will best be served by bulk loading of small molecule dyes, despite the limitations of an acute time course, difficult loading, poor cellular specificity, and neuropil signal contamination (Kerr and Denk, 2008). Future protein engineering efforts will primarily focus on improving GECI SNR.…”

Section: Resultsmentioning

confidence: 99%

“…A broad time course allows more complete calcium binding to GECIs, increasing DF. Photons can be collected over a longer period than the underlying voltage spike, increasing N. It also lowers the sampling rate necessary to accurately capture the transient time course (Kerr and Denk, 2008). This allows simultaneous recording of spatially segregated calcium signals with single point excitation, as in two-photon laser scanning microscopy (2PLSM).…”

Section: Extrinsic Properties That Influence the Performance Of Gecismentioning

confidence: 99%

“…These dyes can reliably quantify the changes in intracellular calcium associated with neural activity . However, Brain Cell Biology Volume 36, 69-86, 200869-86, DOI: 10.1007 these small molecule probes have limitations: they can be difficult to introduce into neurons in the intact brain (Kerr and Denk, 2008), they can accumulate in high- [Ca 2+ ] intracellular compartments (Di Virgilio et al, 1988), and many are extruded from the cytoplasm in several hours (Di Virgilio et al, 1988). Furthermore, chronic repeated measurements from the same cells in vivo are impossible, and the dyes cannot be targeted to specific cell types, populations, or subcellular locations without added transgenes (Tour et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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Reporting neural activity with genetically encoded calcium indicators

2008

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Genetically encoded calcium indicators (GECIs), based on recombinant fluorescent proteins, have been engineered to observe calcium transients in living cells and organisms. Through observation of calcium, these indicators also report neural activity. We review progress in GECI construction and application, particularly toward in vivo monitoring of sparse action potentials (APs). We summarize the extrinsic and intrinsic factors that influence GECI performance. A simple model of GECI response to AP firing demonstrates the relative significance of these factors. We recommend a standardized protocol for evaluating GECIs in a physiologically relevant context. A potential method of simultaneous optical control and recording of neuronal circuits is presented.

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Section: Resultsmentioning

confidence: 99%

Section: Extrinsic Properties That Influence the Performance Of Gecismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Reporting neural activity with genetically encoded calcium indicators

2008

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“…T he ability to visualize biological systems in vivo has been a major attraction of optical microscopy, because studying biological systems as they evolve in their natural, physiological state provides relevant information that in vitro preparations often do not allow (1). However, for conventional optical microscopes to achieve their optimal, diffraction-limited resolution, the specimen needs to have identical optical properties to those of the immersion media for which the microscope objective is designed.…”

mentioning

confidence: 99%

Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex

Satō

Betzig

2011

Proc. Natl. Acad. Sci. U.S.A.

178

158

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The signal and resolution during in vivo imaging of the mouse brain is limited by sample-induced optical aberrations. We find that, although the optical aberrations can vary across the sample and increase in magnitude with depth, they remain stable for hours. As a result, two-photon adaptive optics can recover diffraction-limited performance to depths of 450 μm and improve imaging quality over fields of view of hundreds of microns. Adaptive optical correction yielded fivefold signal enhancement for small neuronal structures and a threefold increase in axial resolution. The corrections allowed us to detect smaller neuronal structures at greater contrast and also improve the signal-to-noise ratio during functional Ca 2 imaging in single neurons.T he ability to visualize biological systems in vivo has been a major attraction of optical microscopy, because studying biological systems as they evolve in their natural, physiological state provides relevant information that in vitro preparations often do not allow (1). However, for conventional optical microscopes to achieve their optimal, diffraction-limited resolution, the specimen needs to have identical optical properties to those of the immersion media for which the microscope objective is designed. For example, one of the most widely applied microscopy techniques for in vivo imaging, two-photon fluorescence microscopy, often uses water-dipping objectives. Because biological samples are comprised of structures (i.e., proteins, nuclear acids, and lipids) with refractive indices different from that of water, they induce optical aberrations to the incoming excitation wave and result in an enlarged focal spot within the sample and a concomitant deterioration of signal and resolution (2, 3). As a result, the resolution and contrast of optical microscopes is compromised in vivo, especially deep in tissue.Many questions related to how the brain processes information on both the neuronal circuit level and the cell biological level can be addressed by observing the morphology and activity of neurons inside a living and, preferably, awake and behaving mouse (1). In a typical experiment, an area of the skull is surgically removed and replaced with a cover glass to provide optical access to the underlying structure of interest (4). For imaging during behavior, the cover glass is often attached to an optically transparent plug embedded in the skull to improve mechanical stability and to prevent the skull from growing back and blocking the optical access (5, 6) (Fig. 1A). Before the excitation light of a two-photon microscope reaches the desired focal plane inside the brain, it has to traverse first the cranial window and then the brain tissue, both with optical properties different from water. Thus, they both impart optical aberrations on the excitation light, which leads to a distorted focus, even at the surface of the brain.These sample-induced aberrations can be corrected with adaptive optics (AO) to recover diffraction-limited resolution. In AO, a wavefront-shaping device m...

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“…Since its establishment, IVM has been used extensively for the study of cell biology 1 , immunology 2 , neurobiology 3 , and tumor pathophysiology 4 . Compared to radiologic imaging modalities, such as positron emission tomography (PET) and magnetic resonance imaging (MRI), the enhanced spatial and temporal resolution of IVM facilitates various mechanistic measurements not possible with the current whole-body imaging techniques.…”

Section: Introductionmentioning

confidence: 99%

Real-time intravital microscopy of individual nanoparticle dynamics in liver and tumors of live mice

Ven¹,

Yun²,

Kim³

et al. 2013

Protocol Exchange

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Intravital microscopy is emerging as an important experimental tool for the research and development of multi-functional therapeutic nanoconstructs. The direct visualization of nanoparticle dynamics within live animals provides invaluable insights into the mechanisms that regulate nanotherapeutics transport and cell-particle interactions. Here we present a protocol to image the dynamics of nanoparticles within the liver and tumors of live mice immediately following systemic injection using a high-speed (30-400 fps) confocal or multi-photon laserscanning fluorescence microscope. Techniques for quantifying the real-time accumulation and cellular association of individual particles with a size ranging from several tens of nanometers to micrometers are described, as well as an experimental strategy for labeling Kupffer cells in the liver in vivo. Experimental design considerations and controls are provided, as well as minimum equipment requirements. The entire protocol takes approximately 4-8 hours and yields quantitative information. These techniques can serve to study a wide range of kinetic parameters that drive nanotherapeutics delivery, uptake, and treatment response.

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Imaging in vivo: watching the brain in action

Cited by 311 publications

References 158 publications

Reporting neural activity with genetically encoded calcium indicators

Reporting neural activity with genetically encoded calcium indicators

Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex

Real-time intravital microscopy of individual nanoparticle dynamics in liver and tumors of live mice

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