Our projects span the range of target biology, from C. elegans to mice and rats. About one-third of our current projects are directed to electrophysiology, intracellular and extracellular, a second third are advanced optical imaging for neural reconstruction, and the remainder focus on specialty projects.
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.
Researchers strive to make measurements on animals in a way that does not inhibit or modify the behavior under study. Anthony Leonardo here 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.
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+.
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.