scholarly journals Multiplexed confocal and super-resolution fluorescence imaging of cytoskeletal and neuronal synapse proteins

2017 ◽  
Author(s):  
Syuan-Ming Guo ◽  
Remi Veneziano ◽  
Simon Gordonov ◽  
Li Li ◽  
Demian Park ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Super Resolution ◽  
Synaptic Proteins ◽  
Locked Nucleic Acid ◽  
Target Number ◽  
Expression Levels ◽  
High Affinity ◽  
Neuronal Synapses ◽  
Resolution Imaging

ABSTRACTNeuronal synapses contain dozens of protein species whose expression levels and localizations are key determinants of synaptic transmission and plasticity. The spectral properties of fluorophores used in conventional microscopy limit the number of measured proteins to four species within a given sample. The ability to perform high-throughput confocal or super-resolution imaging of many proteins simultaneously without limitation in target number imposed by this spectral limit would enable large-scale characterization of synaptic protein networks in situ. Here, we introduce PRISM: Probe-based Imaging for Sequential Multiplexing, a method that sequentially utilizes either high affinity Locked Nucleic Acid (LNA) or low affinity DNA probes to enable diffraction-limited confocal and PAINT-based super-resolution imaging. High-affinity LNA probes offer high-throughput, confocal-based imaging compared with PAINT, which uses low affinity probes to realize localization-based super-resolution imaging. Simultaneous immunostaining of all targets is performed prior to imaging, followed by sequential LNA/DNA probe exchange that requires only minutes under mild wash conditions. We apply PRISM to quantify the co-expression levels and nanometer-scale organization of one dozen cytoskeletal and synaptic proteins within individual neuronal synapses. Our approach is scalable to dozens of target proteins and is compatible with high-content screening platforms commonly used to interrogate phenotypic changes associated with genetic and drug perturbations in a variety of cell types.

scholarly journals Multiplexed and high-throughput neuronal fluorescence imaging with diffusible probes

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Syuan-Ming Guo ◽  
Remi Veneziano ◽  
Simon Gordonov ◽  
Li Li ◽  
Eric Danielson ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Signal Transmission ◽  
Super Resolution ◽  
Confocal Imaging ◽  
Synaptic Proteins ◽  
Locked Nucleic Acid ◽  
Neuronal Synapses ◽  
Imaging Probes ◽  
Synaptic Markers ◽  
Heterogeneous Expression

Abstract Synapses contain hundreds of distinct proteins whose heterogeneous expression levels are determinants of synaptic plasticity and signal transmission relevant to a range of diseases. Here, we use diffusible nucleic acid imaging probes to profile neuronal synapses using multiplexed confocal and super-resolution microscopy. Confocal imaging is performed using high-affinity locked nucleic acid imaging probes that stably yet reversibly bind to oligonucleotides conjugated to antibodies and peptides. Super-resolution PAINT imaging of the same targets is performed using low-affinity DNA imaging probes to resolve nanometer-scale synaptic protein organization across nine distinct protein targets. Our approach enables the quantitative analysis of thousands of synapses in neuronal culture to identify putative synaptic sub-types and co-localization patterns from one dozen proteins. Application to characterize synaptic reorganization following neuronal activity blockade reveals coordinated upregulation of the post-synaptic proteins PSD-95, SHANK3 and Homer-1b/c, as well as increased correlation between synaptic markers in the active and synaptic vesicle zones.

scholarly journals Large-Scale Fabrication of Photonic Nanojet Array via Template-Assisted Self-Assembly

Micromachines ◽  
2020 ◽  
Vol 11 (5) ◽  
pp. 473 ◽  
Author(s):  
Pengcheng Zhang ◽  
Xi Chen ◽  
Hui Yang
Keyword(s):  
Self Assembly ◽  
Large Scale ◽  
Super Resolution ◽  
Light Signal ◽  
Practical Applications ◽  
Photonic Nanojet ◽  
Detection Technology ◽  
Size Uniformity ◽  
Resolution Imaging ◽  
Photonic Nanojets

A large-scale homogenized photonic nanojet array with defined pattern and spacing facilitates practical applications in super-resolution imaging, subwavelength-resolution nanopatterning, nano objects trapping and detection technology. In this paper, we present the fabrication of a large-scale photonic nanojet array via the template-assisted self-assembly (TASA) approach. Templates of two-dimensional (2D) large-scale microwell array with defined pattern and spacing are fabricated. Melamine microspheres with excellent size uniformity are utilized to pattern on the template. It is found that microwells can be filled at a yield up to 95%. These arrayed microspheres on the template serve as microlenses and can be excited to generate large-scale photonic nanojets. The uniformly-sized melamine spheres are beneficial for the generation of a homogenized photonic nanojet array. The intensity of the photonic nanojets in water is as high as ~2 fold the background light signal. Our work shows a simple, robust, and fast means for the fabrication of a large-scale homogenized photonic nanojet array.

scholarly journals Demonstration of nanoimprinted hyperlens array for high-throughput sub-diffraction imaging

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Minseop Byun ◽  
Dasol Lee ◽  
Minkyung Kim ◽  
Yangdoo Kim ◽  
Kwan Kim ◽  
...  
Keyword(s):  
Real Time ◽  
Large Scale ◽  
Medical Science ◽  
Super Resolution ◽  
Fabrication Process ◽  
Far Field ◽  
Resolution Limit ◽  
Imaging Science ◽  
Diffraction Imaging ◽  
Resolution Imaging

Abstract Overcoming the resolution limit of conventional optics is regarded as the most important issue in optical imaging science and technology. Although hyperlenses, super-resolution imaging devices based on highly anisotropic dispersion relations that allow the access of high-wavevector components, have recently achieved far-field sub-diffraction imaging in real-time, the previously demonstrated devices have suffered from the extreme difficulties of both the fabrication process and the non-artificial objects placement. This results in restrictions on the practical applications of the hyperlens devices. While implementing large-scale hyperlens arrays in conventional microscopy is desirable to solve such issues, it has not been feasible to fabricate such large-scale hyperlens array with the previously used nanofabrication methods. Here, we suggest a scalable and reliable fabrication process of a large-scale hyperlens device based on direct pattern transfer techniques. We fabricate a 5 cm × 5 cm size hyperlenses array and experimentally demonstrate that it can resolve sub-diffraction features down to 160 nm under 410 nm wavelength visible light. The array-based hyperlens device will provide a simple solution for much more practical far-field and real-time super-resolution imaging which can be widely used in optics, biology, medical science, nanotechnology and other closely related interdisciplinary fields.

scholarly journals Super-resolution light-sheet fluorescence microscopy by SOFI

2020 ◽  
Author(s):  
Judith Mizrachi ◽  
Arun Narasimhan ◽  
Xiaoli Qi ◽  
Rhonda Drewes ◽  
Ramesh Palaniswamy ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Ampa Receptors ◽  
Super Resolution ◽  
Light Sheet ◽  
Cytoskeletal Proteins ◽  
The Body ◽  
Synaptic Proteins ◽  
Resolution Analysis ◽  
Resolution Imaging ◽  
Light Sheet Fluorescence Microscopy

Here we describe a new method, named LS-SOFI, that combines light-sheet fluorescence microscopy and super-resolution optical fluctuation imaging to achieve fast nanoscale-resolution imaging over large fields of view in native 3D tissues. We demonstrate the use of LS-SOFI in super-resolution analysis of neuronal structures and synaptic proteins, including cortical axons, dendritic spines, pre- and postsynaptic cytoskeletal proteins and postsynaptic AMPA receptors, in thick mouse brain sections. We also introduce an algorithm to determine the number of active fluorophore emitters detected, allowing the localization of individual molecules in LS-SOFI images. We conclude that LS-SOFI is a versatile method for fast super-resolution imaging from any tissue of the body using both commercial and custom LSFM instruments.

scholarly journals Lightsheet localization microscopy enables fast, large-scale, and three-dimensional super-resolution imaging

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Chieh-Han Lu ◽  
Wei-Chun Tang ◽  
Yen-Ting Liu ◽  
Shu-Wei Chang ◽  
Frances Camille M. Wu ◽  
...  
Keyword(s):  
Large Scale ◽  
Three Dimensional ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Resolution Imaging

scholarly journals Metrology of DNA arrays by super-resolution microscopy

Nanoscale ◽  
2017 ◽  
Vol 9 (29) ◽  
pp. 10205-10211 ◽  
Author(s):  
Christopher M. Green ◽  
Kelly Schutt ◽  
Noah Morris ◽  
Reza M. Zadegan ◽  
William L. Hughes ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
High Throughput ◽  
Self Assembly ◽  
Super Resolution ◽  
Dna Nanostructures ◽  
Dna Arrays ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

Crystal-PAINT super-resolution imaging enables high-throughput metrology of DNA nanostructures for quantitative analysis of arrays formed through self-assembly.

scholarly journals Super-resolution Imaging of Synaptic and Extra-synaptic Pools of AMPA Receptors with Different-sized Fluorescent Probes

2016 ◽  
Author(s):  
Sang Hak Lee ◽  
Chaoyi Jin ◽  
En Cai ◽  
Pinghua Ge ◽  
Yuji Ishitsuka ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Ampa Receptors ◽  
Fluorescent Dyes ◽  
Super Resolution ◽  
Free Access ◽  
Neuronal Synapses ◽  
Small Quantum ◽  
Probe Size ◽  
Resolution Imaging ◽  
20 Nm

AbstractWhether AMPA receptors (AMPARs) enter into neuronal synapses, by exocytosis from an internal pool, or by diffusion from an external membrane-bound pool, is hotly contested. 3D super-resolution fluorescent nanoscopy to measure the dynamics and placement of AMPAR is a powerful method for addressing this issue. However, probe size and accessibility to tightly packed spaces can be limiting. We have therefore labeled AMPARs with differently sized fluorophores: small organic fluorescent dyes (~ 4 nm), small quantum dots (sQD, ~10 nm in diameter), or big (commercial) quantum dots (bQD, ~ 20 nm in diameter). We then compared their diffusion rate, trajectories, and placement with respect to a postsynaptic density (PSD) protein, Homer 1c. Labeled with the small probes of sQDs or organic fluorophores, we find that AMPARs are located largely within PSDs (~73-93%), and generally reside in “nanodomains” with constrained diffusion. In contrast, when labeled with bQDs, only 5-10% of AMPARs are within PSDs. The results can be explained by relatively free access, or lack thereof, to synaptic clefts of the AMPARs when labeled with small or big probes, respectively. This implies that AMPARs primarily enter PSDs soon after their exocytosis and not from a large diffusive pool of extrasynaptic AMPARs.

scholarly journals Adhering interacting cells to two opposing coverslips allows super-resolution imaging of cell-cell interfaces

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Julia Sajman ◽  
Yair Razvag ◽  
Shachar Schidorsky ◽  
Seon Kinrot ◽  
Kobi Hermon ◽  
...  
Keyword(s):  
Large Scale ◽  
Super Resolution ◽  
Molecular Organization ◽  
Chemical Information ◽  
Cell Trapping ◽  
Immune Synapses ◽  
Resolution Imaging ◽  
Orthogonal Orientation ◽  
Antigen Presenting ◽  
Cell Cell

AbstractCell-cell interfaces convey mechanical and chemical information in multicellular systems. Microscopy has revealed intricate structure of such interfaces, yet typically with limited resolution due to diffraction and unfavourable orthogonal orientation of the interface to the coverslip. We present a simple and robust way to align cell-cell interfaces in parallel to the coverslip by adhering the interacting cells to two opposing coverslips. We demonstrate high-quality diffraction-limited and super-resolution imaging of interfaces (immune-synapses) between fixed and live CD8+ T-cells and either antigen presenting cells or melanoma cells. Imaging methods include bright-field, confocal, STED, dSTORM, SOFI, SRRF and large-scale tiled images. The low background, lack of aberrations and enhanced spatial stability of our method relative to existing cell-trapping techniques allow use of these methods. We expect that the simplicity and wide-compatibility of our approach will allow its wide dissemination for super-resolving the intricate structure and molecular organization in a variety of cell-cell interfaces.

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