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Here we report SUPPORT (statistically unbiased prediction utilizing spatiotemporal information in imaging data), a self-supervised learning method for removing Poisson-Gaussian noise in voltage imaging data. SUPPORT is based on the insight that a pixel value in voltage imaging data is highly dependent on its spatiotemporal neighboring pixels, even when its temporally adjacent frames alone do not provide useful information for statistical prediction. Such dependency is captured and used by a convolutional neural network with a spatiotemporal blind spot to accurately denoise voltage imaging data in which the existence of the action potential in a time frame cannot be inferred by the information in other frames. Through simulations and experiments, we show that SUPPORT enables precise denoising of voltage imaging data and other types of microscopy image while preserving the underlying dynamics within the scene.
Assuntos
Microscopia , Redes Neurais de Computação , Razão Sinal-Ruído , Distribuição Normal , Processamento de Imagem Assistida por Computador/métodosRESUMO
We report the development of deep decomposition and deconvolution microscopy (3DM), a computational microscopy method for the volumetric imaging of neural activity. 3DM overcomes the major challenge of deconvolution microscopy, the ill-posed inverse problem. We take advantage of the temporal sparsity of neural activity to reformulate and solve the inverse problem using two neural networks which perform sparse decomposition and deconvolution. We demonstrate the capability of 3DM via in vivo imaging of the neural activity of a whole larval zebrafish brain with a field of view of 1040 µm × 400 µm × 235 µm and with estimated lateral and axial resolutions of 1.7 µm and 5.4 µm, respectively, at imaging rates of up to 4.2 volumes per second.
Assuntos
Encéfalo/diagnóstico por imagem , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Peixe-Zebra/fisiologia , Animais , Encéfalo/fisiologia , Microscopia Intravital/métodos , Larva , Microscopia Confocal , Redes Neurais de Computação , Neurônios/fisiologia , Medula Espinal/diagnóstico por imagem , Medula Espinal/fisiologiaRESUMO
We recently developed a method called expansion microscopy, in which preserved biological specimens are physically magnified by embedding them in a densely crosslinked polyelectrolyte gel, anchoring key labels or biomolecules to the gel, mechanically homogenizing the specimen, and then swelling the gel-specimen composite by â¼4.5× in linear dimension. Here we describe iterative expansion microscopy (iExM), in which a sample is expanded â¼20×. After preliminary expansion a second swellable polymer mesh is formed in the space newly opened up by the first expansion, and the sample is expanded again. iExM expands biological specimens â¼4.5 × 4.5, or â¼20×, and enables â¼25-nm-resolution imaging of cells and tissues on conventional microscopes. We used iExM to visualize synaptic proteins, as well as the detailed architecture of dendritic spines, in mouse brain circuitry.
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Aumento da Imagem/métodos , Micromanipulação/métodos , Microscopia/métodos , Polímeros/química , Manejo de Espécimes/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
In the version of this article originally published, the bottom of Figure 4f,g was partially truncated in the PDF. The error has been corrected in the PDF version of this article.
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We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.
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Evolução Molecular Direcionada/métodos , Proteínas Luminescentes/química , Engenharia de Proteínas/métodos , Robótica , Peixe-Zebra/embriologia , Animais , Encéfalo/diagnóstico por imagem , Caenorhabditis elegans , Separação Celular , Feminino , Citometria de Fluxo , Fluorescência , Biblioteca Gênica , Genes Reporter , Células HEK293 , Hipocampo/citologia , Humanos , Masculino , Camundongos , Microscopia de Fluorescência , Neurônios/citologia , OptogenéticaRESUMO
High-speed, large-scale three-dimensional (3D) imaging of neuronal activity poses a major challenge in neuroscience. Here we demonstrate simultaneous functional imaging of neuronal activity at single-neuron resolution in an entire Caenorhabditis elegans and in larval zebrafish brain. Our technique captures the dynamics of spiking neurons in volumes of â¼700 µm × 700 µm × 200 µm at 20 Hz. Its simplicity makes it an attractive tool for high-speed volumetric calcium imaging.
Assuntos
Cálcio/metabolismo , Imageamento Tridimensional/métodos , Microscopia/métodos , Neurônios/fisiologia , Animais , Caenorhabditis elegans , Sinalização do Cálcio , Larva/ultraestrutura , Microscopia de Fluorescência/métodos , Peixe-ZebraRESUMO
As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the cellular resolution. Here, we provide an optimized protocol for performing whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy, including sample preparation and immobilization, sample embedding, image acquisition, and visualization after imaging. The current protocol enables in vivo imaging of the structure and neuronal activity of a larval zebrafish brain at a cellular resolution for over 1 h using confocal microscopy and custom-designed fluorescence microscopy. The critical steps in the protocol are also discussed, including sample mounting and positioning, preventing bubble formation and dust in the agarose gel, and avoiding motion in images caused by incomplete solidification of the agarose gel and paralyzation of the fish. The protocol has been validated and confirmed in multiple settings. This protocol can be easily adapted for imaging other organs of a larval zebrafish.
Assuntos
Encéfalo , Imageamento Tridimensional , Microscopia Intravital , Microscopia de Fluorescência , Neuroimagem , Peixe-Zebra , Animais , Encéfalo/diagnóstico por imagem , Imageamento Tridimensional/instrumentação , Imageamento Tridimensional/métodos , Neuroimagem/instrumentação , Neuroimagem/métodos , Sefarose , Microscopia Intravital/instrumentação , Microscopia Intravital/métodosRESUMO
Three-dimensional fluorescence microscopy has an intrinsic performance limit set by the number of photons that can be collected from the sample in a given time interval. Here, we extend our earlier work - a recursive light propagation network (RLP-Net) - which is a computational microscopy technique that overcomes such limitations through virtual refocusing that enables volume reconstruction from two adjacent 2-D wide-field fluorescence images. RLP-Net employs a recursive inference scheme in which the network progressively predicts the subsequent planes along the axial direction. This recursive inference scheme reflects that the law of physics for the light propagation remains spatially invariant and therefore a fixed function (i.e., a neural network) for a short distance light propagation can be recursively applied for a longer distance light propagation. In addition, we employ a self-supervised denoising method to enable accurate virtual light propagation over a long distance. We demonstrate the capability of our method through high-speed volumetric imaging of neuronal activity of a live zebrafish brain. The source code used in the paper is available at https://github.com/NICALab/rlpnet.
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Software , Peixe-Zebra , Animais , Microscopia de Fluorescência/métodos , Redes Neurais de Computação , NeurôniosRESUMO
Ultra-multiplexed fluorescence imaging requires the use of spectrally overlapping fluorophores to label proteins and then to unmix the images of the fluorophores. However, doing this remains a challenge, especially in highly heterogeneous specimens, such as the brain, owing to the high degree of variation in the emission spectra of fluorophores in such specimens. Here, we propose PICASSO, which enables more than 15-color imaging of spatially overlapping proteins in a single imaging round without using any reference emission spectra. PICASSO requires an equal number of images and fluorophores, which enables such advanced multiplexed imaging, even with bandpass filter-based microscopy. We show that PICASSO can be used to achieve strong multiplexing capability in diverse applications. By combining PICASSO with cyclic immunofluorescence staining, we achieve 45-color imaging of the mouse brain in three cycles. PICASSO provides a tool for multiplexed imaging with high accessibility and accuracy for a broad range of researchers.
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Corantes Fluorescentes , Imagem Óptica , Animais , Camundongos , Microscopia de Fluorescência/métodos , Proteínas , Coloração e RotulagemRESUMO
Methods for one-photon fluorescent imaging of calcium dynamics can capture the activity of hundreds of neurons across large fields of view at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk from neuropil, resulting in a decreased signal-to-noise ratio and artifactual correlations of neural activity. We address this problem by engineering cell-body-targeted variants of the fluorescent calcium indicators GCaMP6f and GCaMP7f. We screened fusions of GCaMP to natural, as well as artificial, peptides and identified fusions that localized GCaMP to within 50 µm of the cell body of neurons in mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP in dense neural circuits reported fewer artifactual spikes from neuropil, an increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators facilitates usage of simple, powerful, one-photon methods for imaging neural calcium dynamics.
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Encéfalo/diagnóstico por imagem , Cálcio/metabolismo , Corpo Celular/patologia , Neurônios/patologia , Imagem Óptica/métodos , Animais , Artefatos , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Ligação ao Cálcio , Corpo Celular/metabolismo , Proteínas de Fluorescência Verde , Camundongos , Neurônios/metabolismo , Neurópilo , Peixe-ZebraRESUMO
We here introduce and study the properties, via computer simulation, of a candidate automated approach to algorithmic reconstruction of dense neural morphology, based on simulated data of the kind that would be obtained via two emerging molecular technologies-expansion microscopy (ExM) and in-situ molecular barcoding. We utilize a convolutional neural network to detect neuronal boundaries from protein-tagged plasma membrane images obtained via ExM, as well as a subsequent supervoxel-merging pipeline guided by optical readout of information-rich, cell-specific nucleic acid barcodes. We attempt to use conservative imaging and labeling parameters, with the goal of establishing a baseline case that points to the potential feasibility of optical circuit reconstruction, leaving open the possibility of higher-performance labeling technologies and algorithms. We find that, even with these conservative assumptions, an all-optical approach to dense neural morphology reconstruction may be possible via the proposed algorithmic framework. Future work should explore both the design-space of chemical labels and barcodes, as well as algorithms, to ultimately enable routine, high-performance optical circuit reconstruction.
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In this paper, a cepstral analysis based approach to measuring the depth of anesthesia (DoA) is presented. Cepstral analysis is a signal processing technique widely used especially for speech recognition in order to extract speech information regardless of vocal cord characteristics. The resulting index for the DoA is called index based on cepstral analysis (ICep). The Fisher criterion is engaged to evaluate the performance of indices. All analyses are based on a single-channel electroencephalogram (EEG) of 10 human subjects. To validate the proposed technique, ICep is compared with bispectral index (BIS), which is the most commonly used method to estimate the level of consciousness via EEG during general anesthesia. The results show that ICep has high correlation with BIS, and is outstanding in terms of the Fisher criterion and offers faster tracking than BIS in the transition from consciousness to unconsciousness.
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Anestesia Geral/métodos , Eletroencefalografia , Adolescente , Adulto , Algoritmos , Estado de Consciência , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Processamento de Sinais Assistido por Computador , Software , Processos Estocásticos , Inconsciência , Adulto JovemRESUMO
In this paper, an entropy based method for quantifying the depth of anesthesia from rat EEG is presented. The proposed index for the depth of anesthesia called modified Shannon entropy (MShEn) is based on Shannon entropy (ShEn) and spectral entropy (SpEn) which are widely used for analyzing non-stationary signals. Discrimination power (DP), as a performance indicator for indexes, is defined and used to derive the final index for the depth of anesthesia. For experiment, EEG from anesthetized rats are measured and analyzed by using MShEn. MShEn shows both high stability and high correlation with other indexes for depth of anesthesia.