High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision

Grewe, Benjamin F.; Langer, Dominik; Kasper, Hansjörg; Kampa, Björn M.; Helmchen, Fritjof

doi:10.1038/nmeth.1453

Cited by 465 publications

(486 citation statements)

References 44 publications

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“…This sampling rate can be achieved with low read noise using fast EMCCD or scientific grade CMOS cameras. Alternatively, in two-photon microscopy random-access laser-scanning techniques based on acoustooptic deflectors permit discontinuous scanning between selected regions with low access times (t a ~ 10 μs) 38 compatible with ~2 kHz sampling at each neuron ( Figure 4C,D).…”

Section: Comparisons Of Fluorescence Microscopy Modalities For Voltagmentioning

confidence: 99%

Optical Strategies for Sensing Neuronal Voltage Using Quantum Dots and Other Semiconductor Nanocrystals

Marshall

Schnitzer

2013

ACS Nano

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Biophysicists have long sought optical methods capable of reporting the electrophysiological dynamics of large-scale neural networks with millisecond-scale temporal resolution. Existing fluorescent sensors of cell membrane voltage can report action potentials in individual cultured neurons, but limitations in brightness and dynamic range of both synthetic organic and genetically encoded voltage sensors have prevented concurrent monitoring of spiking activity across large populations of individual neurons. Here we propose a novel, inorganic class of fluorescent voltage sensors: semiconductor nanoparticles, such as ultrabright quantum dots (qdots). Our calculations revealed that transmembrane electric fields characteristic of neuronal spiking (~10 mV/nm) modulate a qdot's electronic structure and can induce ~5% changes in its fluorescence intensity and ~1 nm shifts in its emission wavelength, depending on the qdot's size, composition, and dielectric environment. Moreover, tailored qdot sensors composed of two different materials can exhibit substantial (~30%) changes in fluorescence intensity during neuronal spiking. Using signal detection theory, we show that conventional qdots should be capable of reporting voltage dynamics with millisecond precision across several tens or more individual neurons over a range of optical and neurophysiological conditions. These results unveil promising avenues for imaging spiking dynamics in neural networks and merit in-depth experimental investigation. Toward addressing these challenges, we studied whether tools from nanotechnology, specifically, bright, multifunctional semiconductor quantum dots (qdots), 22,23 might be useful for imaging neuronal membrane potentials. Qdots are known to possess voltagesensitive optical properties, including a red-shifting of the qdot's fluorescence emission peak, spectral broadening, and a decrease in the intensity of fluorescence emission ( Figure 1C,D). [24][25][26][27] Qdots also are highly resistant to photobleaching and exhibit large quantum yields and absorption cross sections for one-(σ 1 ) and two-photon (σ 2 ) excitation. For example, CdSe qdots with radius R ~ 3 nm exhibit cross sections of σ 1 > 1 nm 2 and σ 2 > 3 × 10 4 GM, as compared to σ 1 ~ 10 −2 nm 2 and σ 2 ~ 3 × 10 2 GM for green fluorescent protein. 28,29 Marshall and Schnitzer Page 2 ACS Nano. Author manuscript; available in PMC 2017 December 15. Graphical Abstract HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptTo evaluate the spike detection capabilities of qdot indicators quantitatively, we applied a theoretical approach that incorporates the physical, biophysical, and statistical components of the problem. We calculated the effect of applied electric fields on the qdot's electronic structure and examined how this modulates a qdot's fluorescence intensity and emission spectra. We also studied nonspherical, nonhomogenous qdots, which we refer to more generally as semiconductor nanocrystals, and found that these nanoparticles can demonstrate muc...

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Section: Comparisons Of Fluorescence Microscopy Modalities For Voltagmentioning

confidence: 99%

Optical Strategies for Sensing Neuronal Voltage Using Quantum Dots and Other Semiconductor Nanocrystals

Marshall

Schnitzer

2013

ACS Nano

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“…The nematode Caenorhabditis elegans is particularly ideal for optical neurophysiology owing to its small size, optical transparency, compact nervous system, and ease of genetic manipulation. Imaging systems for tracking the activity of small numbers of neurons have been effective in determining their role during nematode locomotion and navigational behaviors like chemotaxis, thermotaxis, and the escape response (1-6).Recordings from large numbers of interconnected neurons are required to understand how neuronal ensembles carry out the systematic transformations of sensory input into motor patterns that build behavioral decisions.Several methods for fast 3D imaging of neural activity in a fixed imaging volume have been developed for different model organisms (7)(8)(9)(10)(11)(12)(13)(14). High-speed light sheet microscopy, light field microscopy, multifocus microscopy, and two-photon structured illumination microscopy have proved effective for rapidly recording large numbers of neurons in immobilized, intact, transparent animals like larval zebrafish and nematodes (15)(16)(17)(18)(19).…”

mentioning

confidence: 99%

“…Several methods for fast 3D imaging of neural activity in a fixed imaging volume have been developed for different model organisms (7)(8)(9)(10)(11)(12)(13)(14). High-speed light sheet microscopy, light field microscopy, multifocus microscopy, and two-photon structured illumination microscopy have proved effective for rapidly recording large numbers of neurons in immobilized, intact, transparent animals like larval zebrafish and nematodes (15)(16)(17)(18)(19).…”

mentioning

confidence: 99%

Pan-neuronal imaging in roaming Caenorhabditis elegans

Venkatachalam

et al. 2015

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

215

216

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We present an imaging system for pan-neuronal recording in crawling Caenorhabditis elegans. A spinning disk confocal microscope, modified for automated tracking of the C. elegans head ganglia, simultaneously records the activity and position of ∼80 neurons that coexpress cytoplasmic calcium indicator GCaMP6s and nuclear localized red fluorescent protein at 10 volumes per second. We developed a behavioral analysis algorithm that maps the movements of the head ganglia to the animal's posture and locomotion. Image registration and analysis software automatically assigns an index to each nucleus and calculates the corresponding calcium signal. Neurons with highly stereotyped positions can be associated with unique indexes and subsequently identified using an atlas of the worm nervous system. To test our system, we analyzed the brainwide activity patterns of moving worms subjected to thermosensory inputs. We demonstrate that our setup is able to uncover representations of sensory input and motor output of individual neurons from brainwide dynamics. Our imaging setup and analysis pipeline should facilitate mapping circuits for sensory to motor transformation in transparent behaving animals such as C. elegans and Drosophila larva.C. elegans | Drosophila | calcium imaging | thermotaxis U nderstanding how brain dynamics creates behaviors requires quantifying the flow and transformation of sensory information to motor output in behaving animals. Optical imaging using genetically encoded calcium or voltage fluorescent probes offers a minimally invasive method to record neural activity in intact animals. The nematode Caenorhabditis elegans is particularly ideal for optical neurophysiology owing to its small size, optical transparency, compact nervous system, and ease of genetic manipulation. Imaging systems for tracking the activity of small numbers of neurons have been effective in determining their role during nematode locomotion and navigational behaviors like chemotaxis, thermotaxis, and the escape response (1-6).Recordings from large numbers of interconnected neurons are required to understand how neuronal ensembles carry out the systematic transformations of sensory input into motor patterns that build behavioral decisions.Several methods for fast 3D imaging of neural activity in a fixed imaging volume have been developed for different model organisms (7)(8)(9)(10)(11)(12)(13)(14). High-speed light sheet microscopy, light field microscopy, multifocus microscopy, and two-photon structured illumination microscopy have proved effective for rapidly recording large numbers of neurons in immobilized, intact, transparent animals like larval zebrafish and nematodes (15)(16)(17)(18)(19). However, these methods are problematic when attempting to track many neurons within the bending and moving body of a behaving animal. Panneuronal recording in moving animals poses higher demands on spatial and temporal resolution. Furthermore, extracting neuronal signals from recordings in a behaving animal requires an effective analysis pipel...

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“…galvanometer or AOD scanned laser beam and photomultiplier tube) one can record from multiple sites and planes, but only serially. Sequential scanning of targets is still required, and despite sophisticated scanning systems, these systems cannot monitor multiple points with true simultaneity [8][9][10][11]. One technique that helps alleviate this limitation is to rely upon task-based approaches to imaging and leverage joint optical-digital design strategies that can be used to selectively enhance defocus-related performance through engineering of the optical Point Spread Function (PSF) [2,[12][13][14][15][16].…”

Section: Extended Depth-of-field Imaging Using Engineered Point Spreamentioning

confidence: 99%

Instantaneous three-dimensional sensing using spatial light modulator illumination with extended depth of field imaging

2013

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Imaging three-dimensional structures represents a major challenge for conventional microscopies. Here we describe a Spatial Light Modulator (SLM) microscope that can simultaneously address and image multiple targets in three dimensions. A wavefront coding element and computational image processing enables extended depth-of-field imaging. High-resolution, multi-site three-dimensional targeting and sensing is demonstrated in both transparent and scattering media over a depth range of 300-1,000 microns.

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High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision

Cited by 465 publications

References 44 publications

Optical Strategies for Sensing Neuronal Voltage Using Quantum Dots and Other Semiconductor Nanocrystals

Optical Strategies for Sensing Neuronal Voltage Using Quantum Dots and Other Semiconductor Nanocrystals

Pan-neuronal imaging in roaming Caenorhabditis elegans

Instantaneous three-dimensional sensing using spatial light modulator illumination with extended depth of field imaging

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