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128 Janelia Publications
Showing 1-10 of 128 resultsWe address the problem of inferring the number of independently blinking fluorescent light emitters, when only their combined intensity contributions can be observed at each timepoint. This problem occurs regularly in light microscopy of objects that are smaller than the diffraction limit, where one wishes to count the number of fluorescently labelled subunits. Our proposed solution directly models the photo-physics of the system, as well as the blinking kinetics of the fluorescent emitters as a fully differentiable hidden Markov model. Given a trace of intensity over time, our model jointly estimates the parameters of the intensity distribution per emitter, their blinking rates, as well as a posterior distribution of the total number of fluorescent emitters. We show that our model is consistently more accurate and increases the range of countable subunits by a factor of two compared to current state-of-the-art methods, which count based on autocorrelation and blinking frequency, Further-more, we demonstrate that our model can be used to investigate the effect of blinking kinetics on counting ability, and therefore can inform experimental conditions that will maximize counting accuracy.
Significance: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy. Aim: We describe the development and applications of T-GECO1-a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1. Approach: We use protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon Ca2+ imaging in hippocampal slices. Results: The Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300M−1cm−1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold, and the apparent Kd for Ca2+ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled the detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant. Conclusions: T-GECO1 is a high-performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants. Keywords: blue-shifted fluorescence; genetically encoded calcium ion indicator; neuronal activity imaging; protein engineering; two-photon excitation.
Anchoring goals to spatial representations enables flexible navigation but is challenging in novel environments when both representations must be acquired simultaneously. We propose a framework for how Drosophila uses internal representations of head direction (HD) to build goal representations upon selective thermal reinforcement. We show that flies use stochastically generated fixations and directed saccades to express heading preferences in an operant visual learning paradigm and that HD neurons are required to modify these preferences based on reinforcement. We used a symmetric visual setting to expose how flies' HD and goal representations co-evolve and how the reliability of these interacting representations impacts behavior. Finally, we describe how rapid learning of new goal headings may rest on a behavioral policy whose parameters are flexible but whose form is genetically encoded in circuit architecture. Such evolutionarily structured architectures, which enable rapidly adaptive behavior driven by internal representations, may be relevant across species.
The endoplasmic reticulum (ER) is an important regulator of Ca2+ in cells and dysregulation of ER calcium homeostasis can lead to numerous pathologies. Understanding how various pharmacological and genetic perturbations of ER Ca2+ homeostasis impacts cellular physiology would likely be facilitated by more quantitative measurements of ER Ca2+ levels that allow easier comparisons across conditions. Here, we developed a ratiometric version of our original ER-GCaMP probe that allows for more quantitative comparisons of the concentration of Ca2+ in the ER across cell types and sub-cellular compartments. Using this approach we show that the resting concentration of ER Ca2+ in primary dissociated neurons is substantially lower than that in measured in embryonic fibroblasts.
Spontaneously blinking fluorophores permit the detection and localization of individual molecules without reducing buffers or caging groups, thus simplifying single-molecule localization microscopy (SMLM). The intrinsic blinking properties of such dyes are dictated by molecular structure and modulated by environment, which can limit utility. We report a series of tuned spontaneously blinking dyes with duty cycles that span two orders of magnitude, allowing facile SMLM in cells and dense biomolecular structures.
Techniques that enable precise manipulations of subsets of neurons in the fly central nervous system have greatly facilitated our understanding of the neural basis of behavior. Split-GAL4 driver lines allow specific targeting of cell types in Drosophila melanogaster and other species. We describe here a collection of 3060 lines targeting a range of cell types in the adult Drosophila central nervous system and 1373 lines characterized in third-instar larvae. These tools enable functional, transcriptomic, and proteomic studies based on precise anatomical targeting. NeuronBridge and other search tools relate light microscopy images of these split-GAL4 lines to connectomes reconstructed from electron microscopy images. The collections are the result of screening over 77,000 split hemidriver combinations. In addition to images and fly stocks for these well-characterized lines, we make available 300,000 new 3D images of other split-GAL4 lines.
Large axon-diameter descending neurons are metabolically costly but transmit information rapidly from sensory neurons in the brain to motor neurons in the nerve cord. They have thus endured as a common feature of escape circuits in many animal species where speed is paramount. Though often considered isolated command neurons triggering fast-reaction-time, all-or-none escape responses, giant neurons are just one of multiple parallel pathways enabling selection between behavioral alternatives. Such degeneracy among escape circuits makes it unclear if and how giant neurons benefit prey fitness. Here we competed Drosophila melanogaster flies with genetically-silenced Giant Fibers (GFs) against flies with functional GFs in an arena with wild-caught damselfly predators and find that GF silencing decreases prey survival. Kinematic analysis of damselfly attack trajectories shows that decreased prey survival fitness results from GF-silenced flies failing to escape during predator attack speeds and approach distances that would normally elicit successful escapes. When challenged with a virtual looming predator, fly GFs promote survival by enforcing selection of a short-duration takeoff sequence as opposed to reducing reaction time. Our findings support a role for the GFs in promoting prey survival by influencing action selection as a means to enhance escape performance during realistically complex predation scenarios.
Near-infrared (NIR) fluorescent reporters provide additional colors for highly multiplexed imaging of cells and organisms, and enable imaging with less toxic light and higher contrast and depth. Here, we present the engineering of nirFAST, a small tunable chemogenetic NIR fluorescent reporter that is brighter than top-performing NIR fluorescent proteins in cultured mammalian cells. nirFAST is a small genetically encoded protein of 14 kDa that binds and stabilizes the fluorescent state of synthetic, highly cell-permeant, fluorogenic chromophores (so-called fluorogens) that are otherwise dark when free. Engineered to emit NIR light, nirFAST can also emit far-red or red lights through change of chromophore. nirFAST allows the imaging of proteins in live cultured mammalian cells, chicken embryo tissues and zebrafish larvae. Its near infrared fluorescence provides an additional color for high spectral multiplexing. We showed that nirFAST is well-suited for stimulated emission depletion (STED) nanoscopy, allowing the efficient imaging of proteins with subdiffraction resolution in live cells. nirFAST enabled the design of a chemogenetic green-NIR fluorescent ubiquitination-based cell cycle indicator (FUCCI) for the monitoring of the different phases of the cell cycle. Finally, bisection of nirFAST allowed the design of a fluorogenic chemically induced dimerization technology with NIR fluorescence readout, enabling the control and visualization of protein proximity.
Third-harmonic generation microscopy is a powerful label-free nonlinear imaging technique, providing essential information about structural characteristics of cells and tissues without requiring external labelling agents. In this work, we integrated a recently developed compact adaptive optics module into a third-harmonic generation microscope, to measure and correct for optical aberrations in complex tissues. Taking advantage of the high sensitivity of the third-harmonic generation process to material interfaces and thin membranes, along with the 1,300-nm excitation wavelength used here, our adaptive optical third-harmonic generation microscope enabled high-resolution in vivo imaging within highly scattering biological model systems. Examples include imaging of myelinated axons and vascular structures within the mouse spinal cord and deep cortical layers of the mouse brain, along with imaging of key anatomical features in the roots of the model plant Brachypodium distachyon. In all instances, aberration correction led to significant enhancements in image quality.
Organismal aging involves functional declines in both somatic and reproductive tissues. Multiple strategies have been discovered to extend lifespan across species. However, how age-related molecular changes differ among various tissues and how those lifespan-extending strategies slow tissue aging in distinct manners remain unclear. Here we generated the transcriptomic Cell Atlas of Worm Aging (CAWA, http://mengwanglab.org/atlas ) of wild-type and long-lived strains. We discovered cell-specific, age-related molecular and functional signatures across all somatic and germ cell types. We developed transcriptomic aging clocks for different tissues and quantitatively determined how three different pro-longevity strategies slow tissue aging distinctively. Furthermore, through genome-wide profiling of alternative polyadenylation (APA) events in different tissues, we discovered cell-type-specific APA changes during aging and revealed how these changes are differentially affected by the pro-longevity strategies. Together, this study offers fundamental molecular insights into both somatic and reproductive aging and provides a valuable resource for in-depth understanding of the diversity of pro-longevity mechanisms.