High-speed, miniaturized fluorescence microscopy in freely moving mice

Flusberg, Benjamin A; Nimmerjahn, Axel; Cocker, Eric D.; Mukamel, Eran A.; Barretto, Robert P. J.; Ko, Tony H.; Burns, Laurie D.; Jung, Juergen C.; Schnitzer, Mark J.

doi:10.1038/nmeth.1256

Cited by 372 publications

(314 citation statements)

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“…It is possible to use optical fibers to deliver short-pulse light suitable for two-photon excitation, scan the excitation focus, and collect the emitted fluorescence (14). There have been a number of recent advances in scanning technology (15,16) that have been applied to anesthetized animals by using two-photon excitation (17) or freely moving animals by using one-photon wide-field imaging (18). Here we show that it is possible to use two-photon microscopy to record activity from neuronal populations with cellular resolution in freely moving animals.…”

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confidence: 85%

Visually evoked activity in cortical cells imaged in freely moving animals

Sawiński

Wallace

Greenberg

et al. 2009

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

207

172

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We describe a miniaturized head-mounted multiphoton microscope and its use for recording Ca 2؉ transients from the somata of layer 2/3 neurons in the visual cortex of awake, freely moving rats. Images contained up to 20 neurons and were stable enough to record continuously for >5 min per trial and 20 trials per imaging session, even as the animal was running at velocities of up to 0.6 m/s. Neuronal Ca 2؉ transients were readily detected, and responses to various static visual stimuli were observed during free movement on a running track. Neuronal activity was sparse and increased when the animal swept its gaze across a visual stimulus. Neurons showing preferential activation by specific stimuli were observed in freely moving animals. These results demonstrate that the multiphoton fiberscope is suitable for functional imaging in awake and freely moving animals.calcium imaging ͉ head-mounted microscope ͉ neuronal activity ͉ two-photon ͉ visual cortex T he observation of neural activity has been central to the vast majority of efforts to understand information processing in the mammalian brain. Although some aspects of sensory processing can be studied in animals that are anesthetized or awake but head-fixed, to fully understand awake information processing, animals must be able to interact fully with their environment. Previously, this approach, which includes both allothetic and idiothetic cues, lead to the discovery of place cells (1), head-direction cells (2), and grid cells (3). Multiphoton (MP) imaging (4) of neurons bulk-loaded with calcium indicators (5-7) allows not only the unambiguous identification and precise anatomical localization of active neurons but also the simultaneous recording of activity in multiple neurons even at very low firing rates (8-10). Although one major limitation of conventional MP imaging has been the need to firmly hold the skull in position to prevent the brain from moving relative to the microscope objective, some aspects of free movement can be simulated in head-fixed animals by placing them in a virtual reality situation (11). For measurements in truly free-moving animals, the recording apparatus must be miniaturized and attached to the skull, similar to the approach used for the recording of extracellular (1, 12) and intracellular (13) electrical signals in freely moving rodents. It is possible to use optical fibers to deliver short-pulse light suitable for two-photon excitation, scan the excitation focus, and collect the emitted fluorescence (14). There have been a number of recent advances in scanning technology (15, 16) that have been applied to anesthetized animals by using two-photon excitation (17) or freely moving animals by using one-photon wide-field imaging (18). Here we show that it is possible to use two-photon microscopy to record activity from neuronal populations with cellular resolution in freely moving animals. Results and DiscussionWe developed a fiberscope (Fig. 1 A) that employs a customdesigned water-immersion lens and a leveraged, nonresonant fiber scan...

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confidence: 85%

Visually evoked activity in cortical cells imaged in freely moving animals

Sawiński

Wallace

Greenberg

et al. 2009

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

207

172

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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).…”

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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).…”

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confidence: 99%

Pan-neuronal imaging in roaming Caenorhabditis elegans

Venkatachalam

et al. 2015

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

221

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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“…1). A fiberbased epifluorescence miniaturized microscope with a single lens objective was developed and used for this purpose (6). However, as with the van Leeuwenhoek microscope, this miniature solution does not include the illumination source (and filters) or the detector, the sun and the eye, corresponding to a mercury arc lamp and an EM-CCD camera in the modern implementation.…”

Section: Single Lens Miniature Microscopesmentioning

confidence: 99%