ABSTRACT
Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits its speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and computationally recovers high-resolution images, attaining voxel rates of over 1 billion Hz in structured samples. Using a static image as a prior for recording neural activity, we imaged visually evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 µm and frame rates over 1 kHz. Dendritic glutamate transients in anesthetized mice are synchronized within spatially contiguous domains spanning tens of micrometers at frequencies ranging from 1-100 Hz. We demonstrate millisecond-resolved recordings of acetylcholine and voltage indicators, three-dimensional single-particle tracking and imaging in densely labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.
Subject(s)
Cerebral Cortex/physiology , Glutamic Acid/metabolism , Neurons/physiology , Tomography/methods , Animals , Calcium/metabolism , Cerebral Cortex/cytology , Female , Mice , Mice, Inbred C57BL , Neurons/cytology , Photons , RatsABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
The version of this paper originally published cited a preprint version of ref. 12 instead of the published version (Proc. Natl. Acad. Sci. USA 115, 5594-5599; 2018), which was available before this Nature Methods paper went to press. The reference information has been updated in the PDF and HTML versions of the article.
ABSTRACT
In the version of this paper originally published, important figure labels in Fig. 3d were not visible. An image layer present in the authors' original figure that included two small dashed outlines and text labels indicating ROI 1 and ROI 2, as well as a scale bar and the name of the cell label, was erroneously altered during image processing. The figure has been corrected in the HTML and PDF versions of the paper.
ABSTRACT
Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.
Subject(s)
Glutamic Acid/metabolism , Green Fluorescent Proteins/metabolism , Microscopy, Fluorescence/methods , Neurons/cytology , Retina/cytology , Visual Cortex/cytology , Animals , Color , Female , Ferrets , Fluorescent Dyes , Glutamic Acid/analysis , Male , Mice, Inbred C57BL , Neurons/metabolism , Retina/metabolism , Visual Cortex/metabolismABSTRACT
OBJECTIVE: Common biological measurements are in the form of noisy convolutions of signals of interest with possibly unknown and transient blurring kernels. Examples include EEG and calcium imaging data. Thus, signal deconvolution of these measurements is crucial in understanding the underlying biological processes. The objective of this paper is to develop fast and stable solutions for signal deconvolution from noisy, blurred, and undersampled data, where the signals are in the form of discrete events distributed in time and space. METHODS: We introduce compressible state-space models as a framework to model and estimate such discrete events. These state-space models admit abrupt changes in the states and have a convergent transition matrix, and are coupled with compressive linear measurements. We consider a dynamic compressive sensing optimization problem and develop a fast solution, using two nested expectation maximization algorithms, to jointly estimate the states as well as their transition matrices. Under suitable sparsity assumptions on the dynamics, we prove optimal stability guarantees for the recovery of the states and present a method for the identification of the underlying discrete events with precise confidence bounds. RESULTS: We present simulation studies as well as application to calcium deconvolution and sleep spindle detection, which verify our theoretical results and show significant improvement over existing techniques. CONCLUSION: Our results show that by explicitly modeling the dynamics of the underlying signals, it is possible to construct signal deconvolution solutions that are scalable, statistically robust, and achieve high temporal resolution. SIGNIFICANCE: Our proposed methodology provides a framework for modeling and deconvolution of noisy, blurred, and undersampled measurements in a fast and stable fashion, with potential application to a wide range of biological data.
