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3920 Publications
Showing 3651-3660 of 3920 resultsCalcium imaging is commonly used to measure the neural activity of large groups of neurons in mice. Genetically encoded calcium indicators (GECIs) can be delivered for this purpose using non-invasive genetic methods. Compared to viral gene transfer, transgenic targeting of GECIs provides stable long-term expression and obviates the need for invasive viral injections. Transgenic mice expressing the green GECI GCaMP6 are already widely used. Here we present the generation and characterization of transgenic mice expressing the sensitive red GECI jRGECO1a, driven by the Thy1 promoter. Four transgenic lines with different expression patterns showed sufficiently high expression for cellular in vivo imaging. We used two-photon microscopy to characterize visual responses of individual neurons in the visual cortex in vivo. The signal-to-noise ratio in transgenic mice was comparable to, or better than, mice transduced with adeno-associated virus. In addition, we show that Thy1-jRGECO1a transgenic mice are useful for transcranial population imaging and functional mapping using widefield fluorescence microscopy. We also demonstrate imaging of visual responses in retinal ganglion cells in vitro. Thy1-jRGECO1a transgenic mice are therefore a useful addition to the toolbox for imaging activity in intact neural networks.
Structured illumination microscopy (SIM) is widely used for fast, long-term, live-cell super-resolution imaging. However, SIM images can contain substantial artifacts if the sample does not conform to the underlying assumptions of the reconstruction algorithm. Here we describe a simple, easy to implement, process that can be combined with any reconstruction algorithm to alleviate many common SIM reconstruction artifacts and briefly discuss possible extensions.
A plexus of multidendritic sensory neurons, the dendritic arborization (da) neurons, innervates the epidermis of soft-bodied insects. Previous studies have indicated that the plexus may comprise distinct subtypes of da neurons, which utilize diverse cyclic 3’,5’-guanosine monophosphate signaling pathways and could serve several functions. Here, we identify three distinct classes of da neurons in Manduca, which we term the alpha, beta, and gamma classes. These three classes differ in their sensory responses, branch complexity, peripheral dendritic fields, and axonal projections. The two identified alpha neurons branch over defined regions of the body wall, which in some cases correspond to specific natural folds of the cuticle. These cells project to an intermediate region of the neuropil and appear to function as proprioceptors. Three beta neurons are characterized by long, sinuous dendritic branches and axons that terminate in the ventral neuropil. The function of this group of neurons is unknown. Four neurons belonging to the gamma class have the most complex peripheral dendrites. A representative gamma neuron responds to forceful touch of the cuticle. Although the dendrites of da neurons of different classes may overlap extensively, cells belonging to the same class show minimal dendritic overlap. As a result, the body wall is independently tiled by the beta and gamma da neurons and partially innervated by the alpha neurons. These properties of the da system likely allow insects to discriminate the quality and location of several types of stimuli acting on the cuticle.
The comparison of a pair of electron microscope images recorded at different specimen tilt angles provides a powerful approach for evaluating the quality of images, image-processing procedures, or three-dimensional structures. Here, we analyze tilt-pair images recorded from a range of specimens with different symmetries and molecular masses and show how the analysis can produce valuable information not easily obtained otherwise. We show that the accuracy of orientation determination of individual single particles depends on molecular mass, as expected theoretically since the information in each particle image increases with molecular mass. The angular uncertainty is less than 1° for particles of high molecular mass ( 50 MDa), several degrees for particles in the range 1-5 MDa, and tens of degrees for particles below 1 MDa. Orientational uncertainty may be the major contributor to the effective temperature factor (B-factor) describing contrast loss and therefore the maximum resolution of a structure determination. We also made two unexpected observations. Single particles that are known to be flexible showed a wider spread in orientation accuracy, and the orientations of the largest particles examined changed by several degrees during typical low-dose exposures. Smaller particles presumably also reorient during the exposure; hence, specimen movement is a second major factor that limits resolution. Tilt pairs thus enable assessment of orientation accuracy, map quality, specimen motion, and conformational heterogeneity. A convincing tilt-pair parameter plot, where 60% of the particles show a single cluster around the expected tilt axis and tilt angle, provides confidence in a structure determined using electron cryomicroscopy.
Kernel regression or classification (also referred to as weighted ε-NN methods in Machine Learning) are appealing for their simplicity and therefore ubiquitous in data analysis. How- ever, practical implementations of kernel regression or classification consist of quantizing or sub-sampling data for improving time efficiency, often at the cost of prediction quality. While such tradeoffs are necessary in practice, their statistical implications are generally not well understood, hence practical implementations come with few performance guaran- tees. In particular, it is unclear whether it is possible to maintain the statistical accuracy of kernel prediction—crucial in some applications—while improving prediction time. The present work provides guiding principles for combining kernel prediction with data- quantization so as to guarantee good tradeoffs between prediction time and accuracy, and in particular so as to approximately maintain the good accuracy of vanilla kernel prediction. Furthermore, our tradeoff guarantees are worked out explicitly in terms of a tuning parameter which acts as a knob that favors either time or accuracy depending on practical needs. On one end of the knob, prediction time is of the same order as that of single-nearest- neighbor prediction (which is statistically inconsistent) while maintaining consistency; on the other end of the knob, the prediction risk is nearly minimax-optimal (in terms of the original data size) while still reducing time complexity. The analysis thus reveals the interaction between the data-quantization approach and the kernel prediction method, and most importantly gives explicit control of the tradeoff to the practitioner rather than fixing the tradeoff in advance or leaving it opaque. The theoretical results are validated on data from a range of real-world application domains; in particular we demonstrate that the theoretical knob performs as expected.
Time-integrated fluorescence cumulant analysis (TIFCA) is a data analysis technique for fluorescence fluctuation spectroscopy (FFS) that extracts information from the cumulants of the integrated fluorescence intensity. It is the first exact theory that describes the effect of sampling time on FFS experiment. Rebinning of data to longer sampling times helps to increase the signal/noise ratio of the experimental cumulants of the photon counts. The sampling time dependence of the cumulants encodes both brightness and diffusion information of the sample. TIFCA analysis extracts this information by fitting the cumulants to model functions. Generalization of TIFCA to multicolor FFS experiment is straightforward. Here, we present an overview of the theory, its implementation, as well as the benefits and requirements of TIFCA. The questions of why, when, and how to use TIFCA will be discussed. We give several examples of practical applications of TIFCA, particularly focused on measuring molecular interaction in living cells.
Previous implementations of structured-illumination microscopy (SIM) were slow or designed for one-color excitation, sacrificing two unique and extremely beneficial aspects of light microscopy: live-cell imaging in multiple colors. This is especially unfortunate because, among the resolution-extending techniques, SIM is an attractive choice for live-cell imaging; it requires no special fluorophores or high light intensities to achieve twice diffraction-limited resolution in three dimensions. Furthermore, its wide-field nature makes it light-efficient and decouples the acquisition speed from the size of the lateral field of view, meaning that high frame rates over large volumes are possible. Here, we report a previously undescribed SIM setup that is fast enough to record 3D two-color datasets of living whole cells. Using rapidly programmable liquid crystal devices and a flexible 2D grid pattern algorithm to switch between excitation wavelengths quickly, we show volume rates as high as 4 s in one color and 8.5 s in two colors over tens of time points. To demonstrate the capabilities of our microscope, we image a variety of biological structures, including mitochondria, clathrin-coated vesicles, and the actin cytoskeleton, in either HeLa cells or cultured neurons.
Much of systems neuroscience posits the functional importance of brain activity patterns that lack natural scales of sizes, durations, or frequencies. The field has developed prominent, and sometimes competing, explanations for the nature of this scale-free activity. Here, we reconcile these explanations across species and modalities. First, we link estimates of excitation-inhibition (E-I) balance with time-resolved correlation of distributed brain activity. Second, we develop an unbiased method for sampling time series constrained by this time-resolved correlation. Third, we use this method to show that estimates of E-I balance account for diverse scale-free phenomena without need to attribute additional function or importance to these phenomena. Collectively, our results simplify existing explanations of scale-free brain activity and provide stringent tests on future theories that seek to transcend these explanations.
Recording transcriptional histories of a cell would enable deeper understanding of cellular developmental trajectories and responses to external perturbations. Here we describe an engineered protein fiber that incorporates diverse fluorescent marks during its growth to store a ticker tape-like history. An embedded HaloTag reporter incorporates user-supplied dyes, leading to colored stripes that map the growth of each individual fiber to wall clock time. A co-expressed eGFP tag driven by a promoter of interest records a history of transcriptional activation. High-resolution multi-spectral imaging on fixed samples reads the cellular histories, and interpolation of eGFP marks relative to HaloTag timestamps provides accurate absolute timing. We demonstrate recordings of doxycycline-induced transcription in HEK cells and cFos promoter activation in cultured neurons, with a single-cell absolute accuracy of 30-40 minutes over a 12-hour recording. The protein-based ticker tape design we present here could be generalized to achieve massively parallel single-cell recordings of diverse physiological modalities.
In the current model of endothelial barrier regulation, the tyrosine kinase SRC is purported to induce disassembly of endothelial adherens junctions (AJs) via phosphorylation of VE cadherin, and thereby increase junctional permeability. Here, using a chemical biology approach to temporally control SRC activation, we show that SRC exerts distinct time-variant effects on the endothelial barrier. We discovered that the immediate effect of SRC activation was to transiently enhance endothelial barrier function as the result of accumulation of VE cadherin at AJs and formation of morphologically distinct reticular AJs. Endothelial barrier enhancement via SRC required phosphorylation of VE cadherin at Y731. In contrast, prolonged SRC activation induced VE cadherin phosphorylation at Y685, resulting in increased endothelial permeability. Thus, time-variant SRC activation differentially phosphorylates VE cadherin and shapes AJs to fine-tune endothelial barrier function. Our work demonstrates important advantages of synthetic biology tools in dissecting complex signaling systems.