Dual-Color 3D Superresolution Microscopy by Combined Spectral-Demixing and Biplane Imaging

Winterflood, Christian M.; Platonova, Evgenia; Albrecht, David; Ewers, Helge

doi:10.1016/j.bpj.2015.05.026

Cited by 38 publications

(37 citation statements)

References 22 publications

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“…To this end, we imaged Bsn and Ca v 2.1 using dual-color direct stochastic optical reconstruction microscopy (dSTORM) (Winterflood et al., 2015) (Figures S1A–S1C).…”

Section: Resultsmentioning

confidence: 99%

“…For correlative live-fixed imaging of presynaptic structure and function, live images of SypHy responses were aligned with the fixed ratiometric images using a MATLAB routine (Figure S6). For STORM imaging, samples were fixed, permeabilized, stained for the proteins of interest, and imaged using either a commercially available N-STORM Nikon system or a custom-built setup as described before (Winterflood et al., 2015). Imaging was performed in objective-type near-total internal reflection fluorescence (TIRF) mode.…”

Section: Methodsmentioning

confidence: 99%

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Nanoscale Structural Plasticity of the Active Zone Matrix Modulates Presynaptic Function

et al. 2017

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SummaryThe active zone (AZ) matrix of presynaptic terminals coordinates the recruitment of voltage-gated calcium channels (VGCCs) and synaptic vesicles to orchestrate neurotransmitter release. However, the spatial organization of the AZ and how it controls vesicle fusion remain poorly understood. Here, we employ super-resolution microscopy and ratiometric imaging to visualize the AZ structure on the nanoscale, revealing segregation between the AZ matrix, VGCCs, and putative release sites. Long-term blockade of neuronal activity leads to reversible AZ matrix unclustering and presynaptic actin depolymerization, allowing for enrichment of AZ machinery. Conversely, patterned optogenetic stimulation of postsynaptic neurons retrogradely enhanced AZ clustering. In individual synapses, AZ clustering was inversely correlated with local VGCC recruitment and vesicle cycling. Acute actin depolymerization led to rapid (5 min) nanoscale AZ matrix unclustering. We propose a model whereby neuronal activity modulates presynaptic function in a homeostatic manner by altering the clustering state of the AZ matrix.

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“…To this end, we imaged Bsn and Ca v 2.1 using dual-color direct stochastic optical reconstruction microscopy (dSTORM) (Winterflood et al., 2015) (Figures S1A–S1C).…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Nanoscale Structural Plasticity of the Active Zone Matrix Modulates Presynaptic Function

et al. 2017

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“…The imaging of microtubules as parallel lines around 40 nm apart we achieved here is a benchmark for high quality SMLM super-resolution imaging [15][16][17]19,26 . Similarly, the hollow nature of spectrin rings in neuronal axons has so far only been reported for 3D-SMLM 20,27,28 . ExSTED thus provides at least similar resolution to the most powerful superresolution approaches available at the time, both in 2D and in 3D.…”

Section: Discussionmentioning

confidence: 98%

Expansion stimulated emission depletion microscopy (ExSTED)

Gao

Maraspini

Beutel

et al. 2018

Preprint

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Stimulated emission depletion (STED) microscopy is routinely used to resolve the ultrastructure of cells with a ~10-fold higher resolution compared to diffraction limited imaging. While STED microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy (ExM) provides sub-diffraction resolution by physically enlarging the sample before microscopy. Expansion of fixed cells by crosslinking and swelling of hydrogels easily enlarges the sample ~4-fold and hence increases the effective optical resolution by this factor. To overcome the current limits of these complimentary approaches, we here combined ExM with STED (ExSTED) and demonstrate an increase in resolution of up to 30-fold compared to conventional microscopy (<10 nm lateral and ~50 nm isotropic). While the increase in resolution is straight forward, we found that high fidelity labelling via multi-epitopes is required to obtain emitter densities that allow to resolve ultra-structural details with ExSTED. Our work provides a robust template for super resolution microscopy of entire cells in the ten nanometer range.

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“…The machine-learning algorithm we developed could correct for color cross-talk with high accuracy (>95% correctly classified fluorophores). This algorithm should be applicable to correct for color cross-talk that arises in emission-based superresolution microscopy when fluorophores with overlapping emission spectra are used (31,32), as long as the training dataset is generated using the specific experimental configuration, making it a versatile approach for correcting color cross-talk in superresolution imaging.…”

Section: Discussionmentioning

confidence: 99%

Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT

Gómez-García

Garbacik

Otterstrom

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Recent advancements in single-molecule-based superresolution microscopy have made it possible to visualize biological structures with unprecedented spatial resolution. Determining the spatial coorganization of these structures within cells under physiological and pathological conditions is an important biological goal. This goal has been stymied by the current limitations of carrying out superresolution microscopy in multiple colors. Here, we develop an approach for simultaneous multicolor superresolution imaging which relies solely on fluorophore excitation, rather than fluorescence emission properties. By modulating the intensity of the excitation lasers at different frequencies, we show that the color channel can be determined based on the fluorophore’s response to the modulated excitation. We use this frequency multiplexing to reduce the image acquisition time of multicolor superresolution DNA-PAINT while maintaining all its advantages: minimal color cross-talk, minimal photobleaching, maximal signal throughput, ability to maintain the fluorophore density per imaged color, and ability to use the full camera field of view. We refer to this imaging modality as “frequency multiplexed DNA-PAINT,” or fm-DNA-PAINT for short. We also show that frequency multiplexing is fully compatible with STORM superresolution imaging, which we term fm-STORM. Unlike fm-DNA-PAINT, fm-STORM is prone to color cross-talk. To overcome this caveat, we further develop a machine-learning algorithm to correct for color cross-talk with more than 95% accuracy, without the need for prior information about the imaged structure.

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Dual-Color 3D Superresolution Microscopy by Combined Spectral-Demixing and Biplane Imaging

Cited by 38 publications

References 22 publications

Nanoscale Structural Plasticity of the Active Zone Matrix Modulates Presynaptic Function

Nanoscale Structural Plasticity of the Active Zone Matrix Modulates Presynaptic Function

Expansion stimulated emission depletion microscopy (ExSTED)

Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT

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