One of our goals is to provide neuroscientists at Janelia with the ability to measure electrical properties of active neural circuits in model animal systems. We have fabricated and deployed custom electrodes and recording systems for Drosophila, dragonflys, mice and rats. Most advanced are the systems for high channel count probes in mice and the necessary multiplexing head stages to enable their use on awake, freely moving mice. We fabricate 128 channel probes with up to 64 sites per shank, while keeping the shank width below 70 um and the shank thickness at 15 um. Examples of two of 60+ new probe designs is shown below along with the multiplexing headstage. Data taken from the olfactory bulb of a mouse show the excellent signal and low noise of these probes.
Optogenetics is rapidly becoming a powerful new tool in experimental neuroscience. We are actively involved in developing new technology for Janelia neuroscientists to maximize the potential of optogenetics by integrating micro-fabricated extracellular recording electrodes with optical waveguides, as shown below, for simultaneous optical and electrical interrogation of active neural systems.
Intracellular recordings provide the most fundamental information about electrical activity in neurons. These measurements are extremely delicate and very sensitive to mechanical disturbances, and therefore nearly impossible in active neural systems. The primary goal in this project is to provide the neuroscientists at Janelia with new devices that allow them to reliably measure intracellular electrical properties of active neural circuits with single-cell resolution. Images below show the performance of a novel device as compared to the standard patch electrode when subject to a mechanical impact.
We continually look at opportunities to utilize modern advances in semiconductor fabrication, optoelectronics, and microdevice development (shown below) to advance the field of experimental electrophysiology at Janelia. We believe that in miniaturizing present neural recording systems, by integration of novel micro-devices into complete recording systems, shown below, new opportunities will arise in obtaining fundamental intracellular information from active neural systems with single-cell resolution.
Optics and electrophysiology are becoming more intertwined, as neuroscientists look at novel ways to access information from single cells in active brains. As part of our efforts to provide the tools they need, we are developing two-photon visible patch-clamp electrodes (shown below) that will help Janelia neuroscientists target neural cells of interest with increasing precision and reliability.
The array tomography project is focused on automating and determining the limits of optical reconstructions of embedded, ultrathin sliced brain samples. This technique, pioneered by the Smith lab at Stanford University, has significant potential for combining high-resolution data with molecular information—determining the location of functional proteins within connected neurons. To realize that potential in an accessible experiment, many obstacles of sample preparation, imaging, and analysis must be overcome: Bill Karsh, Kelly Chamberlain, and Jennifer Colonell in APIG are collaborating with many scientists throughout Janelia to tackle these issues.
We have implemented high-throughput, automated imaging tailored to array tomography samples; this is the imaging engine for an ambitious large-volume reconstruction in the mouse barrel cortex led by JC Rah, a visitor in the Svoboda lab. Bill has adapted Lou Scheffer’s EM tools to enable the alignment of large optical data sets on Janelia’s computing cluster. Jennifer is currently working on the automation of slice collection suitable for optical imaging; specifically, optimizing an ATUM tape-based pickup scheme (ATUM provided by Ken Hayworth of the Lichtman Lab at Harvard).
We are collaborating with Tanya Wolff of the Rubin lab to complete an optical reconstruction of selected neurons in the fly lamina, which has been fully reconstructed in EM. These data will allow us to validate measurements made by array tomography.
Volume rendering of a portion of the data shown in the maximum intensity projection. Only the labeled layer 5 pyramidal neurons are shown here.
Zoomed in on apical dendrites. This volume is 30 x 60 x 20 microns.
This is an offshoot of APIG’s Array Tomography Tools project for which it was decided that we should maximize utility for our client/customers by creating the means to acquire high quality serial section data, and deliver the result as a fully aligned stack. Originally we sought a local expert to just take the alignment piece off our plate. Failing that, we next sought an existing large capacity software package, then a willing developer. The best we could get was local source code (from Louis Scheffer), made specifically for a particular EM data set; neither particularly general nor suited to optical data.
Coming from a commercial software background Bill Karsh always designs for mass production, never for once-only usage. As he modified the code to work for our optical data it also became more capable on EM stacks and Bill slowly became a de facto expert resource for aligning both. Alignment had become its own APIG project. Soon Bill was acting as the principal alignment resource for the Bock Lab here at Janelia. Davi’s ambitions to cover a whole mouse barrel with 4X108 EM images pushed aligner development in the direction of ultra high capacity. Moreover, in such large data sets one encounters many challenging pathologies such as burned, distorted or missing tissue. Solving such problems has increased the generality and robustness of our alignment tools.
Ultimately we have a very capable high capacity aligner that Bill has now published along with extensive documentation and a tutorial. It’s freely available here: https://github.com/billkarsh.
We study the photophysics of fluorescent proteins and dye molecules under development by Janelia researchers. Much of our work involves two-photon studies of calcium indicators that may improve the imaging of neural activity in vivo, which are being developed by Jasper Akerboom in Loren Looger’s group, and the GECI project team. We also have a considerable effort exploring photoswitchable and photoactivatable fluorescent proteins used in super-resolution microscopy and for correlative optical/EM microscopy. These proteins are being developed by Gaby Paez Segala and Eric Schreiter in the Looger Lab. All of the molecules we study were designed to undergo a change in fluorescence intensity or emission wavelength in the presence of an effector such as uv light, or the presence of a signaling molecule like calcium or a neurotransmitter. So we quantify how a molecule responds to the absorption of light, how the absorbed energy is distributed and dissipated in the molecule, and what chemical or structural changes result. Importantly, we try to relate the photophysics in solution to observations in cells and other relevant contexts. Understanding a molecule’s photophysics can be crucial to designing better optical probes for one-photon and two-photon fluorescence microscopy, and better optical probes together with advances in imaging techniques should help researchers further understand the molecular processes that direct the organization of cells and complex organisms.
Instruments and techniques
We measure two-photon properties of proteins and dyes using either a scanning 2-photon microscope to study bleaching and spectral properties of fluorophores in cells or tissue, or a non-scanned 2-photon microscope for spectroscopy and FCS measurements on proteins or dyes in buffer solution. In both cases, laser excitation comes from a Ti:sapphire laser or OPO. Both produce an 80 MHz train of ~200 fsec pulses at a wavelength from 700 nm to 1080 nm (Ti:S) or 1000 - 1600 nm (OPO). We use various detectors to obtain emission spectra, 2p excitation spectra, time-resolved fluorescence lifetime, and FCS measurements. The systems and data acquisition are run under computer control.
FCS measures the fluctuations in fluorescent signal which are quantified by taking an autocorrelation of the signal. The fluctuations arise from transient diffusion of individual fluorophores through the laser excitation volume, protonation /deprotonation kinetics of titratable groups of the chromophore, and internal transitions within the chromophores from singlet states to metastable dark states (e.g. triplet and radical states). All of these effects lead to fast or slow blinking of single molecule fluorescence, and therefore fluctuations on the mean signal from an ensemble of molecules, and these can be measured with FCS. We use FCS to count the mean number of fluorophores in the excitation beam and determine concentration, and knowing concentration we can determine extinction coefficient. We can also determine specific brightness of a molecule (mean fluorescence rate per molecule), and for a given wavelength can find the peak brightness, where photobleaching limits further increases in signal as the laser power is raised.
We have investigated the peak brightness spectra of a number of fluorophores. In the figure below, we show the spectra for GECI and dye-based calcium indicators.
Genetically Encoded Calcium indicators – GCAMPs and RCaMPs
Calcium indicators combined with two-photon imaging allows researchers to record the activity of a field of neurons in an awake behaving animal. The GCaMP calcium indicator has been a focus of the looger Lab and the GECI Project, and improvements over the last several years has moved these indicators past the best dye-based indicators, now enabling detection of single action potentials and imaging the activity of a field of individual dendritic spines.
In the figure below, we compare the absorption, emission, and 2-photon-excited spectra for the progression of GCaMP indicators, where the curves in red are obtained with Ca2+ present, and the curves in blue obtained in the absence of Ca2+.
Dragonfly Power Packs
Researchers strive to make measurements on animals in a way that does not inhibit or modify the behavior under study. Anthony Leonardo at Janelia is investigating the neural basis for prey capture by dragonflies in midflight, a behavior requiring untethered and unrestricted motion of the animal. He and colleagues Reid Harrison and Rob Olberg have developed and demonstrated a lightweight telemetry circuit powered by a small battery that when combined with custom electrical probes, can record and transmit extracellular and neuromuscular signals from the dragonfly to a stationary receiver.
In collaboration with Anthony, we are exploring ways to provide a long-life source of lightweight and untethered power for the dragonfly electronics, as an alternative to the rather heavy batteries commercially available. One approach we are pursuing involves using solar cells, which are photodiodes that generate a current and voltage proportional to the amount of light falling on the active area of the device. While ambient light is a convenient source of optical power, in order to keep the size of the solar cells small, a near-IR light source that cannot be seen by the animal can illuminate the body-mounted solar cell while the dragonfly is perched. An additional component, a small lightweight supercapacitor, is added to store electrical energy required for the telemetry circuit during the brief period of flight of the dragonfly during prey capture. This is a rechargeable process; each time the dragonfly returns to the perch it is illuminated with near-IR light, which is converted to electrical power by the solar cell, recharging the supercapacitor for the next flight.
Devices have been constructed and used to successfully record neural activity in perched dragonflies, and experiments are under way to map the complex dynamical behavior and the underlying neural computations involved in midflight prey capture.
Tim Harris Group Leader
Mladen Barbic Senior Scientist
Jennifer Colonell Senior Scientist
James Jun Postdoctoral Associate
Bill Karsh Senior Scientist
Chongxi Lai Visiting Scientist
John Macklin Senior Scientist
Ronak Patel Research Staff
A general method to improve fluorophores for live-cell and single-molecule microscopy.Nature methods 2015
J. B. Grimm, B. P. English, J. Chen, J. P. Slaughter, Z. Zhang, A. Revyakin, R. Patel, J. J. Macklin, D. Normanno, R. H. Singer, T. Lionnet, and L. D. Lavis Nature methods, (2015)
Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.
Fixation-resistant photoactivatable fluorescent proteins for CLEM.Nature methods 2015
M. G. Paez-Segala, M. G. Sun, G. Shtengel, S. Viswanathan, M. A. Baird, J. J. Macklin, R. Patel, J. R. Allen, E. S. Howe, G. Piszczek, H. F. Hess, M. W. Davidson, Y. Wang, and L. L. Looger Nature methods, (2015)
Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5-1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy. Additional material is available at: http://research.janelia.org/loogerlab/.
Large-scale, high-density (up to 512 channels) recording of local circuits in behaving animals.Journal of neurophysiology 2014
A. Berényi, Z. Somogyvári, A. J. Nagy, L. Roux, J. D. Long, S. Fujisawa, E. Stark, A. Leonardo, T. D. Harris, and G. Buzsáki Journal of neurophysiology, 111:1132-49 (2014)
Monitoring representative fractions of neurons from multiple brain circuits in behaving animals is necessary for understanding neuronal computation. Here, we describe a system that allows high-channel-count recordings from a small volume of neuronal tissue using a lightweight signal multiplexing headstage that permits free behavior of small rodents. The system integrates multishank, high-density recording silicon probes, ultraflexible interconnects, and a miniaturized microdrive. These improvements allowed for simultaneous recordings of local field potentials and unit activity from hundreds of sites without confining free movements of the animal. The advantages of large-scale recordings are illustrated by determining the electroanatomic boundaries of layers and regions in the hippocampus and neocortex and constructing a circuit diagram of functional connections among neurons in real anatomic space. These methods will allow the investigation of circuit operations and behavior-dependent interregional interactions for testing hypotheses of neural networks and brain function.
Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics.Frontiers in molecular neuroscience 2013
J. Akerboom, N. Carreras Calderón, L. Tian, S. Wabnig, M. Prigge, J. Tolö, A. Gordus, M. B. Orger, K. E. Severi, J. J. Macklin, R. Patel, S. R. Pulver, T. J. Wardill, E. Fischer, C. Schüler, T. Chen, K. S. Sarkisyan, J. S. Marvin, C. I. Bargmann, D. S. Kim, S. Kügler, L. Lagnado, P. Hegemann, A. Gottschalk, E. R. Schreiter, and L. L. Looger Frontiers in molecular neuroscience, 6:2 (2013)
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, "RCaMPs," engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca(2+)-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca(2+)]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca(2+) affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.
Thalamocortical input onto layer 5 pyramidal neurons measured using quantitative large-scale array tomography.Frontiers in neural circuits 2013
J. Rah, E. Bas, J. Colonell, Y. Mishchenko, B. Karsh, R. D. Fetter, E. W. Myers, D. B. Chklovskii, K. Svoboda, T. D. Harris, and J. T R. Isaac Frontiers in neural circuits, 7:177 (2013)
The subcellular locations of synapses on pyramidal neurons strongly influences dendritic integration and synaptic plasticity. Despite this, there is little quantitative data on spatial distributions of specific types of synaptic input. Here we use array tomography (AT), a high-resolution optical microscopy method, to examine thalamocortical (TC) input onto layer 5 pyramidal neurons. We first verified the ability of AT to identify synapses using parallel electron microscopic analysis of TC synapses in layer 4. We then use large-scale array tomography (LSAT) to measure TC synapse distribution on L5 pyramidal neurons in a 1.00 × 0.83 × 0.21 mm(3) volume of mouse somatosensory cortex. We found that TC synapses primarily target basal dendrites in layer 5, but also make a considerable input to proximal apical dendrites in L4, consistent with previous work. Our analysis further suggests that TC inputs are biased toward certain branches and, within branches, synapses show significant clustering with an excess of TC synapse nearest neighbors within 5-15 μm compared to a random distribution. Thus, we show that AT is a sensitive and quantitative method to map specific types of synaptic input on the dendrites of entire neurons. We anticipate that this technique will be of wide utility for mapping functionally-relevant anatomical connectivity in neural circuits.
Optimization of a GCaMP calcium indicator for neural activity imagingJournal of Neuroscience 2012
J. Akerboom, T. Chen, T. J. Wardill, J. S. Marvin, S. Mutlu, N. Carreras Caldero, F. Esposti, B. G. Borghuis, X. Sun, A. Gordus, M. B. Orger, R. Portugues, F. Engert, J. J. Macklin, A. Filosa, A. Aggarwal, R. Kerr, R. Takagi, S. Kracun, E. Shigetomi, B. S. Khakh, H. Baier, L. Lagnado, S. S-H. Wang, C. Bargmann, B. Kimmel, V. Jayaraman, K. Svoboda, D. S. Kim, E. R. Schreiter, and L. L. Looger Journal of Neuroscience, 32:13819-13840 (2012)
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo . Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3.GCaMP5allows more sensitive detection of neural activity in vivo andmayfind widespread applications for cellular imaging in general.
Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution.Proceedings of the National Academy of Sciences of the United States of America 2012
H. E. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G L. Gustafsson Proceedings of the National Academy of Sciences of the United States of America, 109:E135-43 (2012)
Using ultralow light intensities that are well suited for investigating biological samples, we demonstrate whole-cell superresolution imaging by nonlinear structured-illumination microscopy. Structured-illumination microscopy can increase the spatial resolution of a wide-field light microscope by a factor of two, with greater resolution extension possible if the emission rate of the sample responds nonlinearly to the illumination intensity. Saturating the fluorophore excited state is one such nonlinear response, and a realization of this idea, saturated structured-illumination microscopy, has achieved approximately 50-nm resolution on dye-filled polystyrene beads. Unfortunately, because saturation requires extremely high light intensities that are likely to accelerate photobleaching and damage even fixed tissue, this implementation is of limited use for studying biological samples. Here, reversible photoswitching of a fluorescent protein provides the required nonlinearity at light intensities six orders of magnitude lower than those needed for saturation. We experimentally demonstrate approximately 40-nm resolution on purified microtubules labeled with the fluorescent photoswitchable protein Dronpa, and we visualize cellular structures by imaging the mammalian nuclear pore and actin cytoskeleton. As a result, nonlinear structured-illumination microscopy is now a biologically compatible superresolution imaging method.
Two-photon probe excitation data are commonly presented as absorption cross section or molecular brightness (the detected fluorescence rate per molecule). We report two-photon molecular brightness spectra for a diverse set of organic and genetically encoded probes with an automated spectroscopic system based on fluorescence correlation spectroscopy. The two-photon action cross section can be extracted from molecular brightness measurements at low excitation intensities, while peak molecular brightness (the maximum molecular brightness with increasing excitation intensity) is measured at higher intensities at which probe photophysical effects become significant. The spectral shape of these two parameters was similar across all dye families tested. Peak molecular brightness spectra, which can be obtained rapidly and with reduced experimental complexity, can thus serve as a first-order approximation to cross-section spectra in determining optimal wavelengths for two-photon excitation, while providing additional information pertaining to probe photostability. The data shown should assist in probe choice and experimental design for multiphoton microscopy studies. Further, we show that, by the addition of a passive pulse splitter, nonlinear bleaching can be reduced-resulting in an enhancement of the fluorescence signal in fluorescence correlation spectroscopy by a factor of two. This increase in fluorescence signal, together with the observed resemblance of action cross section and peak brightness spectra, suggests higher-order photobleaching pathways for two-photon excitation.
Prior Publications (6)
A formalism is given in which the optical field generated by a near-field optical aperture is described as an analytic expansion over a complete set of optical modes. This vectoral solution preserves the divergent behavior of the near field and the dipolar nature of the far field. Numerical calculation of the fields requires only evaluation of a well behaved, one-dimensional integral. The formalism is directly applicable to experiments in near-field scanning optical microscopy when relatively flat samples are evaluated.
Luminescent centers with sharp (<0.07 millielectron volt), spectrally distinct emission lines were imaged in a GaAs/AIGaAs quantum well by means of low-temperature near-field scanning optical microscopy. Temperature, magnetic field, and linewidth measurements establish that these centers arise from excitons laterally localized at interface fluctuations. For sufficiently narrow wells, virtually all emission originates from such centers. Near-field microscopy/spectroscopy provides a means to access energies and homogeneous line widths for the individual eigenstates of these centers, and thus opens a rich area of physics involving quantum resolved systems.
Recent advances in probe design have led to enhanced resolution (currently as significant as ~ 12 nm) in optical microscopes based on near-field imaging. We demonstrate that the polarization of emitted and detected light in such microscopes can be manipulated sensitively to generate contrast. We show that the contrast on certain patterns is consistent with a simple interpretation of the requisite boundary conditions, whereas in other cases a more complicated interaction between the probe and the sample is involved. Finally application of the technique to near-filed magneto-optic imaging is demonstrated.
In near-field scanning optical microscopy, a light source or detector with dimensions less than the wavelength (lambda) is placed in close proximity (lambda/50) to a sample to generate images with resolution better than the diffraction limit. A near-field probe has been developed that yields a resolution of approximately 12 nm ( approximately lambda/43) and signals approximately 10(4)- to 10(6)-fold larger than those reported previously. In addition, image contrast is demonstrated to be highly polarization dependent. With these probes, near-field microscopy appears poised to fulfill its promise by combining the power of optical characterization methods with nanometric spatial resolution.