Hod Dana

Sensitive red protein calcium indicators for imaging neural activity

Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging. DOI: http://dx.doi.org/10.7554/eLife.12727.001

Labeling of active neural circuits in vivo with designed calcium integrators

Taking a snapshot of active brain circuitry Neuroscientists now have a method to mark active populations of neurons in vivo to study circuit activity in the behaving animal. Fosque et al. designed and thoroughly validated a fluorescent protein–based reagent that allows permanent marking of active cells over short time scales. This indicator, termed CaMPARI, switches from its native green to a red fluorescent state by simultaneous illumination with violet light and exposure to increased levels of intracellular calcium. CaMPARI successfully marked active nerve cells in Drosophila, zebrafish, and mouse brains. Science, this issue p. 755 A fluorescent sensor allows cellular-resolution snapshots of activity across the whole brains of freely moving organisms. The identification of active neurons and circuits in vivo is a fundamental challenge in understanding the neural basis of behavior. Genetically encoded calcium (Ca2+) indicators (GECIs) enable quantitative monitoring of cellular-resolution activity during behavior. However, such indicators require online monitoring within a limited field of view. Alternatively, post hoc staining of immediate early genes (IEGs) indicates highly active cells within the entire brain, albeit with poor temporal resolution. We designed a fluorescent sensor, CaMPARI, that combines the genetic targetability and quantitative link to neural activity of GECIs with the permanent, large-scale labeling of IEGs, allowing a temporally precise “activity snapshot” of a large tissue volume. CaMPARI undergoes efficient and irreversible green-to-red conversion only when elevated intracellular Ca2+ and experimenter-controlled illumination coincide. We demonstrate the utility of CaMPARI in freely moving larvae of zebrafish and flies, and in head-fixed mice and adult flies.

Sparsity-based single-shot subwavelength coherent diffractive imaging

We suggest and demonstrate experimentally a method of performing single-shot sub-wavelength resolution Coherent Diffractive Imaging (CDI), i.e. algorithmic object reconstruction from far-field intensity measurements. The method is applicable to objects that are sparse in a known basis. The prior knowledge of the objects sparsity compensates for the loss of high-spatial frequency information associated with free-space propagation, as well as for the loss of phase information (Since only intensity is measured).

Thy1-GCaMP6 Transgenic Mice for Neuronal Population Imaging In Vivo

Genetically-encoded calcium indicators (GECIs) facilitate imaging activity of genetically defined neuronal populations in vivo. The high intracellular GECI concentrations required for in vivo imaging are usually achieved by viral gene transfer using adeno-associated viruses. Transgenic expression of GECIs promises important advantages, including homogeneous, repeatable, and stable expression without the need for invasive virus injections. Here we present the generation and characterization of transgenic mice expressing the GECIs GCaMP6s or GCaMP6f under the Thy1 promoter. We quantified GCaMP6 expression across brain regions and neurons and compared to other transgenic mice and AAV-mediated expression. We tested three mouse lines for imaging in the visual cortex in vivo and compared their performance to mice injected with AAV expressing GCaMP6. Furthermore, we show that GCaMP6 Thy1 transgenic mice are useful for long-term, high-sensitivity imaging in behaving mice.

A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

Numerical evaluation of temporal focusing characteristics in transparent and scattering media

Temporal focusing is a simple approach for achieving tight, optically sectioned excitation in nonlinear microscopy and multiphoton photo-manipulation. Key applications and advantages of temporal focusing involve propagation through scattering media, but the progressive broadening of the temporal focus has not been characterized. By combining a detailed geometrical optics model with Monte-Carlo scattering simulations we introduce and validate a simulation strategy for predicting temporal focusing characteristics in scattering and non-scattering media. The broadening of the temporal focus width with increasing depth in brain tissue is studied using both simulations and experiments for several key optical geometries, and an analytical approximation is found for the dependence of this broadening on the microscopes parameters in a transparent medium. Our results indicate that a multiphoton temporal focus has radically different broadening characteristics in deep tissue than those of a spatial focus.

Hybrid multiphoton volumetric functional imaging of large-scale bioengineered neuronal networks

Planar neural networks and interfaces serve as versatile in vitro models of central nervous system physiology, but adaptations of related methods to three dimensions (3D) have met with limited success. Here, we demonstrate for the first time volumetric functional imaging in a bio-engineered neural tissue growing in a transparent hydrogel with cortical cellular and synaptic densities, by introducing complementary new developments in nonlinear microscopy and neural tissue engineering. Our system uses a novel hybrid multiphoton microscope design combining a 3D scanning-line temporal-focusing subsystem and a conventional laser-scanning multiphoton microscope to provide functional and structural volumetric imaging capabilities: dense microscopic 3D sampling at tens of volumes/sec of structures with mm-scale dimensions containing a network of over 1000 developing cells with complex spontaneous activity patterns. These developments open new opportunities for large-scale neuronal interfacing and for applications of 3D engineered networks ranging from basic neuroscience to the screening of neuroactive substances.

Remotely scanned multiphoton temporal focusing by axial grism scanning

A simple technique for remote scanning of the focal plane in temporal focusing multiphoton microscopy is demonstrated both theoretically and experimentally. A new on-axis light propagation optical setup design enables this scanning, which was considered not feasible in previous studies. The focal plane is axially displaced by the movement of a remote optical device, consisting of a double prism grating, and optionally a cylindrical lens. The displacement is linear, and its slope is inversely proportional to the square of the optical systems magnification.

Line temporal focusing characteristics in transparent and scattering media

Line illumination geometries have advantageous properties for temporal focusing nonlinear microscopy. The characteristics of line temporal focusing (LITEF) in transparent and scattering media are studied here both experimentally and using numerical model simulations. We introduce an approximate analytical formula for the dependence of axial sectioning on the laser and microscopes parameters. Furthermore, we show that LITEF is more robust to tissue scattering than wide-field temporal focusing, and can penetrate much deeper into scattering tissue while maintaining good sectioning capabilities. Based on these observations, we propose a new design for LITEF-based tissue imaging at depths that could potentially exceed the out-of-focus physical excitation limit.

Neural signatures of dynamic stimulus selection in Drosophila

Many animals orient using visual cues, but how a single cue is selected from among many is poorly understood. Here we show that Drosophila ring neurons—central brain neurons implicated in navigation—display visual stimulus selection. Using in vivo two-color two-photon imaging with genetically encoded calcium indicators, we demonstrate that individual ring neurons inherit simple-cell-like receptive fields from their upstream partners. Stimuli in the contralateral visual field suppressed responses to ipsilateral stimuli in both populations. Suppression strength depended on when and where the contralateral stimulus was presented, an effect stronger in ring neurons than in their upstream inputs. This history-dependent effect on the temporal structure of visual responses, which was well modeled by a simple biphasic filter, may determine how visual references are selected for the flys internal compass. Our approach highlights how two-color calcium imaging can help identify and localize the origins of sensory transformations across synaptically connected neural populations.