scholarly journals Nanometric axial localization of single fluorescent molecules with modulated excitation

2019 ◽  
Author(s):  
Pierre Jouchet ◽  
Clément Cabriel ◽  
Nicolas Bourg ◽  
Marion Bardou ◽  
Christian Poüs ◽  
...  
Keyword(s):  
Super Resolution ◽  
Acquisition Time ◽  
Distance Measurements ◽  
Time Modulation ◽  
Fluorescent Response ◽  
Capture Range ◽  
Localization Precision ◽  
Excitation Pattern ◽  
Sparse Samples ◽  
Lateral Localization

AbstractStrategies have been developed in LIDAR to perform distance measurements for non-coherent emission in sparse samples based on excitation modulation. Super-resolution fluorescence microscopy is also striving to perform axial localization but through entirely different approaches. Here we revisit the amplitude modulated LIDAR approach to reach nanometric localization precision and we successfully adapt it to bring distinct advantages to super-resolution microscopy. The excitation pattern is performed by interference enabling the decoupling between spatial and time modulation. The localization of a single emitter is performed by measuring the relative phase of its linear fluorescent response to the known shifting excitation field. Taking advantage of a tilted interfering configuration, we obtain a typical axial localization precision of 7.5 nm over the entire field of view and the axial capture range, without compromising on the acquisition time, the emitter density or the lateral localization precision. The interfering pattern being robust to optical aberrations, this modulated localization (ModLoc) strategy is particularly well suited for observations deep in the samples. Images performed on various biological samples show that the localization precision remains nearly constant up to several micrometers.

scholarly journals MINSTED fluorescence localization and nanoscopy

2021 ◽  
Author(s):  
Michael Weber ◽  
Marcel Leutenegger ◽  
Stefan Stoldt ◽  
Stefan Jakobs ◽  
Tiberiu S. Mihaila ◽  
...  
Keyword(s):  
Standard Deviation ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stimulated Emission ◽  
Far Field ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Molecular Scale ◽  
Localization Precision ◽  
Super Resolution Microscopy

AbstractWe introduce MINSTED, a fluorophore localization and super-resolution microscopy concept based on stimulated emission depletion (STED) that provides spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED doughnut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background and the required number of fluorescence detections are low compared with most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200–1,000 detections per fluorophore provide a localization precision of 1–3 nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a ~100-fold improvement in far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.

scholarly journals Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors

2018 ◽  
Author(s):  
Shama Sograte-Idrissi ◽  
Nazar Oleksiievets ◽  
Sebastian Isbaner ◽  
Mariana Eggert-Martinez ◽  
Jörg Enderlein ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Super Resolution ◽  
Resolving Power ◽  
Acquisition Time ◽  
Efficient Manner ◽  
Target Dna ◽  
Multiplexed Imaging ◽  
Dna Strands ◽  
Linkage Error ◽  
20 Nm

AbstractDNA-PAINT is a rapidly developing fluorescence super-resolution technique which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20-25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using 3 nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the 3 targets with 20 nm resolution, and within only 35 minutes acquisition time.

Super Spatio-Temporal Resolution, Digital PIV System for Multi-Phase Flows With Phase Differentiation and Simultaneous Shape and Size Quantification

2002 ◽  
Author(s):  
Claude Abiven ◽  
Pavlos P. Vlachos
Keyword(s):  
Temporal Resolution ◽  
Cross Correlation ◽  
Super Resolution ◽  
Quantitative Information ◽  
Acquisition Time ◽  
Correlation Algorithm ◽  
Image Velocimetry ◽  
Spatio Temporal ◽  
Multi Phase ◽  
Shape And Size

A unique, super spatio-temporal resolution Digital Particle Image Velocimetry (DPIV) system for the analysis of time-dependent multiphase flows has been developed. The system delivers a sampling frequency between 1KHz and 10KHz, with continuous total acquisition time up to 4 secs and resolution 1Kx1K pixels down to 256×256 pixels. The hardware is integrated with sophisticated image processing algorithms that allow direct image segmentation in order to resolve the multiple phases present in the flow and provides quantitative information about the shape and size of droplets or bubbles present. Finally, the in-plane velocities are measured by a super-resolution, dynamically-adaptive cross-correlation algorithm which is coupled with a particle-tracking scheme. Each individual phase present in the flow is resolved with mean spatial resolution in the order of 3–4 pixels, and accuracy in the order of 0.01–0.1 pixels, while the spatial averaging effects of cross correlation are eliminated.

scholarly journals Boosting the Localization Precision in Super-Resolution Microscopy: booSTORM

2018 ◽  
Vol 114 (3) ◽  
pp. 530a
Author(s):  
Hannah S. Heil ◽  
Benjamin Schreiber ◽  
Marie-Christine Dabauvalle ◽  
Georg Krohne ◽  
Sven Höfling ◽  
...  
Keyword(s):  
Super Resolution ◽  
Localization Precision ◽  
Super Resolution Microscopy

scholarly journals Characterizing Locus Specific Chromatin Structure and Dynamics with Correlative Conventional and Super Resolution imaging in living cells

2021 ◽  
Author(s):  
Dushyant Mehra ◽  
Santosh Adhikari ◽  
Chiranjib Banerjee ◽  
Elias M. Puchner
Keyword(s):  
Single Molecule ◽  
Chromatin Structure ◽  
Spatial Organization ◽  
Super Resolution ◽  
Living Cells ◽  
Diffraction Limit ◽  
Optical Diffraction ◽  
Acquisition Time ◽  
Structure And Dynamics ◽  
Chromatin Structure And Dynamics

The dynamic rearrangement of chromatin is critical for gene regulation, but mapping both the spatial organization of chromatin and its dynamics remains a challenge. Many structural conformations are too small to be resolved via conventional fluorescence microscopy and the long acquisition time of super-resolution PALM imaging precludes the structural characterization of chromatin below the optical diffraction limit in living cells due to chromatin motion. Here we develop a correlative conventional fluorescence and PALM imaging approach to quantitatively map time-averaged chromatin structure and dynamics below the optical diffraction limit in living cells. By assigning localizations to a locus as it moves, we reliably discriminate between bound and searching dCas9 molecules, whose mobility overlap. Our approach accounts for changes in DNA mobility and relates local chromatin motion to larger scale domain movement. In our experimental system, we show that compacted telomeres have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Correlative conventional and PALM imaging therefore improves the ability to analyze the mobility and time-averaged nanoscopic structural features of locus specific chromatin with single molecule precision and yields unprecedented insights across length and time scales.

Magnetic-field-enabled resolution enhancement in super-resolution imaging

2015 ◽  
Vol 17 (10) ◽  
pp. 6722-6727 ◽  
Author(s):  
Min Zhang ◽  
Junling Chen ◽  
Jing Gao ◽  
Zhiyong Wang ◽  
Haijiao Xu ◽  
...  
Keyword(s):  
Magnetic Field ◽  
Fluorescence Intensity ◽  
Resolution Enhancement ◽  
Super Resolution ◽  
Resolution Imaging ◽  
Localization Precision

Magnetic field could increase dye's fluorescence intensity and number of photons, thus better localization precision of super-resolution imaging was achieved.

scholarly journals Heavy water: a simple solution to increasing the brightness of fluorescent proteins in super-resolution imaging

2015 ◽  
Vol 51 (70) ◽  
pp. 13451-13453 ◽  
Author(s):  
Wei Qiang Ong ◽  
Y. Rose Citron ◽  
Joerg Schnitzbauer ◽  
Daichi Kamiyama ◽  
Bo Huang
Keyword(s):  
Heavy Water ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Simple Solution ◽  
Photon Yield ◽  
Resolution Imaging ◽  
Localization Precision ◽  
Super Resolution Microscopy

D2O improves the photon yield of photoactivatable fluorescent proteins and thus the localization precision for super-resolution microscopy.

scholarly journals Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells

2020 ◽  
Vol 6 (16) ◽  
pp. eaay8271 ◽  
Author(s):  
Simao Coelho ◽  
Jongho Baek ◽  
Matthew S. Graus ◽  
James M. Halstead ◽  
Philip R. Nicovich ◽  
...  
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Distance Measurements ◽  
Intact Cells ◽  
Localization Microscopy ◽  
Direct Distance ◽  
Time Drift ◽  
Localization Precision ◽  
20 Nm ◽  
Single Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) has the potential to quantify the diversity in spatial arrangements of molecules in intact cells. However, this requires that the single-molecule emitters are localized with ultrahigh precision irrespective of the sample format and the length of the data acquisition. We advance SMLM to enable direct distance measurements between molecules in intact cells on the scale between 1 and 20 nm. Our actively stabilized microscope combines three-dimensional real-time drift corrections and achieves a stabilization of <1 nm and localization precision of ~1 nm. To demonstrate the biological applicability of the new microscope, we show a 4- to 7-nm difference in spatial separations between signaling T cell receptors and phosphatases (CD45) in active and resting T cells. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.

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