Harris Lab - APIG

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. 

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.

On the left is a Golgi stained image of representative cell types in the fly lamina (from Fischbach and Dittrich, Cell Tissue Res., 258:441 (1989)). Top right shows ultrathin slice of embedded fly lamina; two neuron types—L2 and L4—have been labeled with a membrane localized epitope tag. Bottom right shows reconstruction through many of these slices. 3D reconstructions of this resolution reveal the anatomy of and potential connectivity of the neurons.

Maximum intensity projection of 1000 slices in mouse barrel cortex; the full size of this volume is 1.3 mm x 1.4 mm x 0.2 mm; a typical barrel is about 0.3 mm in diameter. The resolution of the original data – which is downsampled for display – is 250x250x400 nm. The green somata are layer 5 pyramidal neurons, while the red labels projections from the thalamus. Note that only about 5% of the cells are labeled—the “empty space” is filled with unlabeled cells.

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⁺.