scholarly journals Super-resolution microscopy based on parallel detection

2019 ◽  
Vol 12 (06) ◽  
pp. 1950023
Author(s):  
Zhimin Zhang ◽  
Shaocong Liu ◽  
Liang Xu ◽  
Yubing Han ◽  
Cuifang Kuang ◽  
...  
Keyword(s):  
Cross Correlation ◽  
Signal To Noise Ratio ◽  
3D Structure ◽  
Super Resolution ◽  
Confocal Imaging ◽  
Scanning Microscopy ◽  
Resolution Limit ◽  
Image Scanning ◽  
Parallel Detection ◽  
Super Resolution Microscopy

Image scanning microscopy based on pixel reassignment can improve the confocal resolution limit without losing the image signal-to-noise ratio (SNR) greatly [C. J. R. Sheppard, “Super-resolution in confocal imaging,” Optik 80(2) 53–54 (1988). C. B. Müller, E. Jörg, “Image scanning microscopy, “Phys. Rev. Lett. 104(19) 198101 (2010). C. J. R. Sheppard, S. B. Mehta, R. Heintzmann, “Superresolution by image scanning microscopy using pixel reassignment,” Opt. Lett. 38(15) 2889–2892 (2013)]. Here, we use a tailor-made optical fiber and 19 avalanche photodiodes (APDs) as parallel detectors to upgrade our existing confocal microscopy, termed as parallel-detection super-resolution (PDSR) microscopy. In order to obtain the correct shift value, we use the normalized 2D cross correlation to calculate the shifting value of each image. We characterized our system performance by imaging fluorescence beads and applied this system to observing the 3D structure of biological specimen.

scholarly journals The Development of Microscopy for Super-Resolution: Confocal Microscopy, and Image Scanning Microscopy

2021 ◽  
Vol 11 (19) ◽  
pp. 8981
Author(s):  
Colin J. R. Sheppard
Keyword(s):  
Confocal Microscopy ◽  
Biological Samples ◽  
Super Resolution ◽  
Scanning Microscopy ◽  
Optical Methods ◽  
Nonlinear Microscopy ◽  
Structured Illumination ◽  
Basic Principles ◽  
Image Scanning ◽  
Super Resolution Microscopy

Optical methods of super-resolution microscopy, such as confocal microscopy, structured illumination, nonlinear microscopy, and image scanning microscopy are reviewed. These methods avoid strong invasive interaction with a sample, allowing the observation of delicate biological samples. The meaning of resolution and the basic principles and different approaches to superresolution are discussed.

scholarly journals On cross-correlations, averages and noise in electron microscopy

2019 ◽  
Vol 75 (1) ◽  
pp. 12-18 ◽  
Author(s):  
Michael Radermacher ◽  
Teresa Ruiz
Keyword(s):  
High Resolution ◽  
Cross Correlation ◽  
Signal To Noise Ratio ◽  
3D Structure ◽  
Signal To Noise ◽  
High Resolution Structure ◽  
Multiple Copies ◽  
High Level ◽  
2D Images ◽  
Resolution Structure

Biological samples are radiation-sensitive and require imaging under low-dose conditions to minimize damage. As a result, images contain a high level of noise and exhibit signal-to-noise ratios that are typically significantly smaller than 1. Averaging techniques, either implicit or explicit, are used to overcome the limitations imposed by the high level of noise. Averaging of 2D images showing the same molecule in the same orientation results in highly significant projections. A high-resolution structure can be obtained by combining the information from many single-particle images to determine a 3D structure. Similarly, averaging of multiple copies of macromolecular assembly subvolumes extracted from tomographic reconstructions can lead to a virtually noise-free high-resolution structure. Cross-correlation methods are often used in the alignment and classification steps of averaging processes for both 2D images and 3D volumes. However, the high noise level can bias alignment and certain classification results. While other approaches may be implicitly affected, sensitivity to noise is most apparent in multireference alignments, 3D reference-based projection alignments and projection-based volume alignments. Here, the influence of the image signal-to-noise ratio on the value of the cross-correlation coefficient is analyzed and a method for compensating for this effect is provided.

Practical aspects of super-resolution optical fluctuation image scanning microscopy (SOFISM)

2020 ◽  
Author(s):  
Adrian Makowski ◽  
Gur Lubin ◽  
Ron Tenne ◽  
Aleksandra Sroda ◽  
Uri Rossman ◽  
...  
Keyword(s):  
Super Resolution ◽  
Scanning Microscopy ◽  
Image Scanning

scholarly journals Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy

2018 ◽  
Author(s):  
Gerti Beliu ◽  
Andreas Kurz ◽  
Alexander Kuhlemann ◽  
Lisa Behringer-Pliess ◽  
Natalia Wolf ◽  
...  
Keyword(s):  
Single Molecule ◽  
Signal To Noise Ratio ◽  
Super Resolution ◽  
Alder Reaction ◽  
Cell Permeability ◽  
High Signal ◽  
Noncanonical Amino Acids ◽  
Single Molecule Level ◽  
Genetic Code Expansion ◽  
Super Resolution Microscopy

Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine as the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.

scholarly journals A robust and versatile platform for image scanning microscopy enabling super-resolution FLIM

2019 ◽  
Vol 16 (2) ◽  
pp. 175-178 ◽  
Author(s):  
Marco Castello ◽  
Giorgio Tortarolo ◽  
Mauro Buttafava ◽  
Takahiro Deguchi ◽  
Federica Villa ◽  
...  
Keyword(s):  
Super Resolution ◽  
Scanning Microscopy ◽  
Image Scanning

scholarly journals Super-resolution enhancement by quantum image scanning microscopy

2018 ◽  
Vol 13 (2) ◽  
pp. 116-122 ◽  
Author(s):  
Ron Tenne ◽  
Uri Rossman ◽  
Batel Rephael ◽  
Yonatan Israel ◽  
Alexander Krupinski-Ptaszek ◽  
...  
Keyword(s):  
Resolution Enhancement ◽  
Super Resolution ◽  
Scanning Microscopy ◽  
Quantum Image ◽  
Image Scanning

scholarly journals Fluorescent Probes for STED Optical Nanoscopy

Nanomaterials ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 21
Author(s):  
Sejoo Jeong ◽  
Jerker Widengren ◽  
Jong-Chan Lee
Keyword(s):  
Fluorescent Probes ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Photophysical Properties ◽  
Optical Resolution ◽  
Super Resolution ◽  
Fluorescent Nanoparticles ◽  
Resolution Limit ◽  
Photochemical Properties ◽  
Super Resolution Microscopy

Progress in developing fluorescent probes, such as fluorescent proteins, organic dyes, and fluorescent nanoparticles, is inseparable from the advancement in optical fluorescence microscopy. Super-resolution microscopy, or optical nanoscopy, overcame the far-field optical resolution limit, known as Abbe’s diffraction limit, by taking advantage of the photophysical properties of fluorescent probes. Therefore, fluorescent probes for super-resolution microscopy should meet the new requirements in the probes’ photophysical and photochemical properties. STED optical nanoscopy achieves super-resolution by depleting excited fluorophores at the periphery of an excitation laser beam using a depletion beam with a hollow core. An ideal fluorescent probe for STED nanoscopy must meet specific photophysical and photochemical properties, including high photostability, depletability at the depletion wavelength, low adverse excitability, and biocompatibility. This review introduces the requirements of fluorescent probes for STED nanoscopy and discusses the recent progress in the development of fluorescent probes, such as fluorescent proteins, organic dyes, and fluorescent nanoparticles, for the STED nanoscopy. The strengths and the limitations of the fluorescent probes are analyzed in detail.

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