CRASH2p: Closed-loop Two Photon Imaging in Freely Moving Animal
Advances in functional brain imaging now allow sustained rapid 3D visualization of large numbers of neurons inside behaving animals. To decode circuit activity, imaged neurons must be individually segmented and tracked. This is particularly challenging when the brain itself moves and deforms inside a flexible body. The field has lacked general methods for solving this problem effectively. To address this need, we developed a method based on a convolutional neural network (CNN) with specific enhancements which we apply to freely moving Caenorhabditis elegans. For a traditional CNN to track neurons across images of a brain with different postures, the CNN must be trained with ground truth (GT) annotations of similar postures. When these postures are diverse, an adequate number of GT annotations can be prohibitively large to generate manually. We introduce ‘targeted augmentation’, a method to automatically synthesize reliable annotations from a few manual annotations. Our method effectively learns the internal deformations of the brain. The learned deformations are used to synthesize annotations for new postures by deforming the manual annotations of similar postures in GT images. The technique is germane to 3D images, which are generally more difficult to analyze than 2D images. The synthetic annotations, which are added to diversify training datasets, drastically reduce manual annotation and proofreading. Our method is effective both when neurons are represented as individual points or as 3D volumes. We provide a GUI that incorporates targeted augmentation in an end-to-end pipeline, from manual GT annotation of a few images to final proofreading of all images. We apply the method to simultaneously measure activity in the second-layer interneurons in C. elegans: RIA, RIB, and RIM, including the RIA neurite. We find that these neurons show rich behaviors, including switching entrainment on and off dynamically when the animal is exposed to periodic odor pulses.
CRASH2p: Closed-loop Two Photon Imaging in Freely Moving Animal
Advances in functional brain imaging now allow sustained rapid 3D visualization of large numbers of neurons inside behaving animals. To decode circuit activity, imaged neurons must be individually segmented and tracked. This is particularly challenging when the brain itself moves and deforms inside a flexible body. The field has lacked general methods for solving this problem effectively. To address this need, we developed a method based on a convolutional neural network (CNN) with specific enhancements which we apply to freely moving Caenorhabditis elegans. For a traditional CNN to track neurons across images of a brain with different postures, the CNN must be trained with ground truth (GT) annotations of similar postures. When these postures are diverse, an adequate number of GT annotations can be prohibitively large to generate manually. We introduce ‘targeted augmentation’, a method to automatically synthesize reliable annotations from a few manual annotations. Our method effectively learns the internal deformations of the brain. The learned deformations are used to synthesize annotations for new postures by deforming the manual annotations of similar postures in GT images. The technique is germane to 3D images, which are generally more difficult to analyze than 2D images. The synthetic annotations, which are added to diversify training datasets, drastically reduce manual annotation and proofreading. Our method is effective both when neurons are represented as individual points or as 3D volumes. We provide a GUI that incorporates targeted augmentation in an end-to-end pipeline, from manual GT annotation of a few images to final proofreading of all images. We apply the method to simultaneously measure activity in the second-layer interneurons in C. elegans: RIA, RIB, and RIM, including the RIA neurite. We find that these neurons show rich behaviors, including switching entrainment on and off dynamically when the animal is exposed to periodic odor pulses.
Multi-neuronal recording in unrestrained animals with all acousto-optic random-access line-scanning two-photon microscopy
To understand how neural activity encodes and coordinates behavior, it is desirable to record multi-neuronal activity in freely behaving animals. Imaging in unrestrained animals is challenging, especially for those, like larval Drosophila melanogaster, whose brains are deformed by body motion. A previously demonstrated two-photon tracking microscope recorded from individual neurons in freely crawling Drosophila larvae but faced limits in multi-neuronal recording. Here we demonstrate a new tracking microscope using acousto-optic deflectors (AODs) and an acoustic GRIN lens (TAG lens) to achieve axially resonant 2D random access scanning, sampling along arbitrarily located axial lines at a line rate of 70 kHz. With a tracking latency of 0.1 ms, this microscope recorded activities of various neurons in moving larval Drosophila CNS and VNC including premotor neurons, bilateral visual interneurons, and descending command neurons. This technique can be applied to the existing two-photon microscope to allow for fast 3D tracking and scanning.
Multi-neuronal recording in unrestrained animals with all acousto-optic random-access line-scanning two-photon microscopy
To understand how neural activity encodes and coordinates behavior, it is desirable to record multi-neuronal activity in freely behaving animals. Imaging in unrestrained animals is challenging, especially for those, like larval Drosophila melanogaster, whose brains are deformed by body motion. A previously demonstrated two-photon tracking microscope recorded from individual neurons in freely crawling Drosophila larvae but faced limits in multi-neuronal recording. Here we demonstrate a new tracking microscope using acousto-optic deflectors (AODs) and an acoustic GRIN lens (TAG lens) to achieve axially resonant 2D random access scanning, sampling along arbitrarily located axial lines at a line rate of 70 kHz. With a tracking latency of 0.1 ms, this microscope recorded activities of various neurons in moving larval Drosophila CNS and VNC including premotor neurons, bilateral visual interneurons, and descending command neurons. This technique can be applied to the existing two-photon microscope to allow for fast 3D tracking and scanning.
Direction Selectivity in Drosophila Proprioceptors Requires the Mechanosensory Channel Tmc
Drosophila Transmembrane channel-like (Tmc) is a protein that functions in larval proprioception. The closely related TMC1 protein is required for mammalian hearing and is a pore-forming subunit of the hair cell mechanotransduction channel. In hair cells, TMC1 is gated by small deflections of microvilli that produce tension on extracellular tip-links that connect adjacent villi. How Tmc might be gated in larval proprioceptors, which are neurons having a morphology that is completely distinct from hair cells, is unknown. Here, we have used high-speed confocal microscopy both to measure displacements of proprioceptive sensory dendrites during larval movement and to optically measure neural activity of the moving proprioceptors. Unexpectedly, the pattern of dendrite deformation for distinct neurons was unique and differed depending on the direction of locomotion: ddaE neuron dendrites were strongly curved by forward locomotion, while the dendrites of ddaD were more strongly deformed by backward locomotion. Furthermore, GCaMP6f calcium signals recorded in the proprioceptive neurons during locomotion indicated tuning to the direction of movement. ddaE showed strong activation during forward locomotion, while ddaD showed responses that were strongest during backward locomotion. Peripheral proprioceptive neurons in animals mutant for Tmc showed a near-complete loss of movement related calcium signals. As the strength of the responses of wild-type animals was correlated with dendrite curvature, we propose that Tmc channels may be activated by membrane curvature in dendrites that are exposed to strain. Our findings begin to explain how distinct cellular systems rely on a common molecular pathway for mechanosensory responses.
Direction Selectivity in Drosophila Proprioceptors Requires the Mechanosensory Channel Tmc
Drosophila Transmembrane channel-like (Tmc) is a protein that functions in larval proprioception. The closely related TMC1 protein is required for mammalian hearing and is a pore-forming subunit of the hair cell mechanotransduction channel. In hair cells, TMC1 is gated by small deflections of microvilli that produce tension on extracellular tip-links that connect adjacent villi. How Tmc might be gated in larval proprioceptors, which are neurons having a morphology that is completely distinct from hair cells, is unknown. Here, we have used high-speed confocal microscopy both to measure displacements of proprioceptive sensory dendrites during larval movement and to optically measure neural activity of the moving proprioceptors. Unexpectedly, the pattern of dendrite deformation for distinct neurons was unique and differed depending on the direction of locomotion: ddaE neuron dendrites were strongly curved by forward locomotion, while the dendrites of ddaD were more strongly deformed by backward locomotion. Furthermore, GCaMP6f calcium signals recorded in the proprioceptive neurons during locomotion indicated tuning to the direction of movement. ddaE showed strong activation during forward locomotion, while ddaD showed responses that were strongest during backward locomotion. Peripheral proprioceptive neurons in animals mutant for Tmc showed a near-complete loss of movement related calcium signals. As the strength of the responses of wild-type animals was correlated with dendrite curvature, we propose that Tmc channels may be activated by membrane curvature in dendrites that are exposed to strain. Our findings begin to explain how distinct cellular systems rely on a common molecular pathway for mechanosensory responses.
Recording neural activity in unrestrained animals with 3D tracking two photon microscopy
Optical recordings of neural activity in behaving animals can reveal the neural correlates of decision making, but brain motion, which often accompanies behavior, compromises these measurements. Two-photon point-scanning microscopy is especially sensitive to motion artifacts, and two-photon recording of activity has required rigid coupling between the brain and microscope. We developed a two-photon tracking microscope with extremely low-latency (360 μs) feedback implemented in hardware. This microscope can maintain continuous focus on neurons moving with velocities of 3 mm/s and accelerations of 1 m/s2 both in-plane and axially. We recorded calcium dynamics of motor neurons and inter-neurons in unrestrained freely behaving fruit fly larvae, correlating neural activity with stimulus presentations and behavioral outputs, and we measured light-induced depolarization of a visual interneuron in a moving animal using a genetically encoded voltage indicator. Our technique can be extended to stabilize recordings in a variety of moving substrates.
Recording neural activity in unrestrained animals with 3D tracking two photon microscopy
Optical recordings of neural activity in behaving animals can reveal the neural correlates of decision making, but brain motion, which often accompanies behavior, compromises these measurements. Two-photon point-scanning microscopy is especially sensitive to motion artifacts, and two-photon recording of activity has required rigid coupling between the brain and microscope. We developed a two-photon tracking microscope with extremely low-latency (360 μs) feedback implemented in hardware. This microscope can maintain continuous focus on neurons moving with velocities of 3 mm/s and accelerations of 1 m/s2 both in-plane and axially. We recorded calcium dynamics of motor neurons and inter-neurons in unrestrained freely behaving fruit fly larvae, correlating neural activity with stimulus presentations and behavioral outputs, and we measured light-induced depolarization of a visual interneuron in a moving animal using a genetically encoded voltage indicator. Our technique can be extended to stabilize recordings in a variety of moving substrates.
The Nanopore Mass Spectrometer
We report the design of a mass spectrometer featuring an ion source that delivers ions directly into high vacuum from liquid inside a capillary with a sub-micrometer-diameter tip. The surface tension of water and formamide is sufficient to maintain a stable interface with high vacuum at the tip, and the gas load from the interface is negligible, even during electrospray. These conditions lifted the usual requirement of a differentially pumped system. The absence of a background gas also opened up the possibility of designing ion optics to collect and focus ions in order to achieve high overall transmission and detection efficiencies. We describe the operation and performance of the instrument and present mass spectra from solutions of salt ions and DNA bases in formamide and salt ions in water. The spectra show singly charged solute ions clustered with a small number of solvent molecules.
The Nanopore Mass Spectrometer
We report the design of a mass spectrometer featuring an ion source that delivers ions directly into high vacuum from liquid inside a capillary with a sub-micrometer-diameter tip. The surface tension of water and formamide is sufficient to maintain a stable interface with high vacuum at the tip, and the gas load from the interface is negligible, even during electrospray. These conditions lifted the usual requirement of a differentially pumped system. The absence of a background gas also opened up the possibility of designing ion optics to collect and focus ions in order to achieve high overall transmission and detection efficiencies. We describe the operation and performance of the instrument and present mass spectra from solutions of salt ions and DNA bases in formamide and salt ions in water. The spectra show singly charged solute ions clustered with a small number of solvent molecules.
Computations underlying Drosophila photo-taxis odor-taxis and multi-sensory integration
To better understand how organisms make decisions on the basis of temporally varying multi-sensory input, we identified computations made by Drosophila larvae responding to visual and optogenetically induced fictive olfactory stimuli. We modeled the larva's navigational decision to initiate turns as the output of a Linear-Nonlinear-Poisson cascade. We used reverse-correlation to fit parameters to this model; the parameterized model predicted larvae's responses to novel stimulus patterns. For multi-modal inputs, we found that larvae linearly combine olfactory and visual signals upstream of the decision to turn. We verified this prediction by measuring larvae's responses to coordinated changes in odor and light. We studied other navigational decisions and found that larvae integrated odor and light according to the same rule in all cases. These results suggest that photo-taxis and odor-taxis are mediated by a shared computational pathway.
Computations underlying Drosophila photo-taxis odor-taxis and multi-sensory integration
To better understand how organisms make decisions on the basis of temporally varying multi-sensory input, we identified computations made by Drosophila larvae responding to visual and optogenetically induced fictive olfactory stimuli. We modeled the larva's navigational decision to initiate turns as the output of a Linear-Nonlinear-Poisson cascade. We used reverse-correlation to fit parameters to this model; the parameterized model predicted larvae's responses to novel stimulus patterns. For multi-modal inputs, we found that larvae linearly combine olfactory and visual signals upstream of the decision to turn. We verified this prediction by measuring larvae's responses to coordinated changes in odor and light. We studied other navigational decisions and found that larvae integrated odor and light according to the same rule in all cases. These results suggest that photo-taxis and odor-taxis are mediated by a shared computational pathway.
Entropic Cages for Trapping DNA Near a Nanopore
Nanopores can probe the structure of biopolymers in solution; however, diffusion makes it difficult to study the same molecule for extended periods. Here we report devices that entropically trap single DNA molecules in a 6.2-femtolitre cage near a solid-state nanopore. We electrophoretically inject DNA molecules into the cage through the nanopore, pause for preset times and then drive the DNA back out through the nanopore. The saturating recapture time and high recapture probability after long pauses, their agreement with a convection–diffusion model and the observation of trapped DNA under fluorescence microscopy all confirm that the cage stably traps DNA. Meanwhile, the cages have 200 nm openings that make them permeable to small molecules, like the restriction endonuclease we use to sequence-specifically cut trapped DNA into fragments whose number and sizes are analysed upon exiting through the nanopore. Entropic cages thus serve as reactors for chemically modifying single DNA molecules.
Entropic Cages for Trapping DNA Near a Nanopore
Nanopores can probe the structure of biopolymers in solution; however, diffusion makes it difficult to study the same molecule for extended periods. Here we report devices that entropically trap single DNA molecules in a 6.2-femtolitre cage near a solid-state nanopore. We electrophoretically inject DNA molecules into the cage through the nanopore, pause for preset times and then drive the DNA back out through the nanopore. The saturating recapture time and high recapture probability after long pauses, their agreement with a convection–diffusion model and the observation of trapped DNA under fluorescence microscopy all confirm that the cage stably traps DNA. Meanwhile, the cages have 200 nm openings that make them permeable to small molecules, like the restriction endonuclease we use to sequence-specifically cut trapped DNA into fragments whose number and sizes are analysed upon exiting through the nanopore. Entropic cages thus serve as reactors for chemically modifying single DNA molecules.
Statistics of DNA Capture by a Solid-State Nanopore
A solid-state nanopore can electrophoretically capture a DNA molecule and pull it through in a folded configuration. The resulting ionic current signal indicates where along its length the DNA was captured. A statistical study using an 8-nm-wide nanopore reveals a strong bias favoring the capture of molecules near their ends. A theoretical model shows that bias to be a consequence of configurational entropy rather than a search by the polymer for an energetically favorable configuration. We also quantified the fluctuations and length dependence of the speed of simultaneously translocating polymer segments from our study of folded DNA configurations.
Statistics of DNA Capture by a Solid-State Nanopore
A solid-state nanopore can electrophoretically capture a DNA molecule and pull it through in a folded configuration. The resulting ionic current signal indicates where along its length the DNA was captured. A statistical study using an 8-nm-wide nanopore reveals a strong bias favoring the capture of molecules near their ends. A theoretical model shows that bias to be a consequence of configurational entropy rather than a search by the polymer for an energetically favorable configuration. We also quantified the fluctuations and length dependence of the speed of simultaneously translocating polymer segments from our study of folded DNA configurations.
Fabrication of nanopores with embedded annular electrodes and transverse carbon nanotube electrodes
Nanopores with one or two embedded nanoelectrodes can be fabricated by high resolution, milling-based methods. We first demonstrate how a focused ion beam, whose sputtering mechanism is well understood, can create a nanopore containing an annular electrode of an arbitrary metal, and with a regular perimeter. The inner surface of the nanopore can be insulated, and its diameter can be reduced with nanometer precision, by conformally coating a dielectric material by atomic layer deposition. We then investigate the mechanism of pore formation using a transmission electron microscope (TEM) through studies of the milling rate, and its dependence on the flux of electrons and on the atomic number of different target metals. Sputtering from the surface is identified as the dominant mechanism. Accordingly, light element conductors should be chosen to enhance the rate and resolution of TEM milling, which we demonstrate by articulating a nanopore with transverse carbon nanotube electrodes. Finally, we electrochemically verify that TEM milling preserves the quality of an annular gold electrode through cyclic voltammetry measurements performed at various stages of the fabrication.
Fabrication of nanopores with embedded annular electrodes and transverse carbon nanotube electrodes
Nanopores with one or two embedded nanoelectrodes can be fabricated by high resolution, milling-based methods. We first demonstrate how a focused ion beam, whose sputtering mechanism is well understood, can create a nanopore containing an annular electrode of an arbitrary metal, and with a regular perimeter. The inner surface of the nanopore can be insulated, and its diameter can be reduced with nanometer precision, by conformally coating a dielectric material by atomic layer deposition. We then investigate the mechanism of pore formation using a transmission electron microscope (TEM) through studies of the milling rate, and its dependence on the flux of electrons and on the atomic number of different target metals. Sputtering from the surface is identified as the dominant mechanism. Accordingly, light element conductors should be chosen to enhance the rate and resolution of TEM milling, which we demonstrate by articulating a nanopore with transverse carbon nanotube electrodes. Finally, we electrochemically verify that TEM milling preserves the quality of an annular gold electrode through cyclic voltammetry measurements performed at various stages of the fabrication.
Devices and Methods for Containing Molecules
Devices and Methods for Containing Molecules
Passive and Electrically Actuated Solid-State Nanopores for Sensing and Manipulating DNA
Passive and Electrically Actuated Solid-State Nanopores for Sensing and Manipulating DNA