The influence of optical aberrations in large field-of-view single-molecule localization microscopy

2021 ◽  
Author(s):  
Lei Xu ◽  
Jun Lu ◽  
Biqin Dong
Keyword(s):  
Single Molecule ◽  
Field Of View ◽  
Large Field ◽  
Optical Aberrations ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

scholarly journals Maximizing the field of view and accuracy in 3D Single Molecule Localization Microscopy

2018 ◽  
Vol 26 (4) ◽  
pp. 4631 ◽  
Author(s):  
Sohaib Abdul Rehman ◽  
Alexander R. Carr ◽  
Martin O. Lenz ◽  
Steven F. Lee ◽  
Kevin O’Holleran
Keyword(s):  
Single Molecule ◽  
Field Of View ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

scholarly journals Photon count estimation in single-molecule localization microscopy

2018 ◽  
Author(s):  
Rasmus Ø. Thorsen ◽  
Christiaan N. Hulleman ◽  
Mathias Hammer ◽  
David Grünwald ◽  
Sjoerd Stallinga ◽  
...  
Keyword(s):  
Single Molecule ◽  
Axial Position ◽  
Optical Aberrations ◽  
Intensity Estimation ◽  
Localization Microscopy ◽  
Photon Count ◽  
Point Spread ◽  
Spread Function ◽  
Gaussian Point ◽  
Single Molecule Localization Microscopy

Recently, Franke, Sauer and van de Linde1 introduced a way to estimate the axial position of single-molecules (TRABI). To this end, they compared the detected photon count from a temporal radial-aperture-based intensity estimation to the estimated count from Gaussian point-spread function (PSF) fitting to the data. Empirically they found this photometric ratio to be around 0.7-0.8 close to focus and decreasing away from it. Here, we explain this reported but unexplained discrepancy and furthermore show that the photometric ratio as indicator for axial position is susceptible even to typical optical aberrations.

scholarly journals Hessian single molecule localization microscopy using sCMOS camera

2018 ◽  
Author(s):  
Fudong Xue ◽  
Wenting He ◽  
Fan Xu ◽  
Mingshu Zhang ◽  
Liangyi Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Signal To Noise Ratio ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Fast Imaging ◽  
Large Field ◽  
Localization Microscopy ◽  
Readout Noise ◽  
Single Pixel ◽  
Single Molecule Localization Microscopy

AbstractSingle-molecule localization microscopy (SMLM) has the highest spatial resolution among the existing super-resolution (SR) imaging techniques, but its temporal resolution needs further improvement. An sCMOS camera can effectively increase the imaging rate due to its large field of view and fast imaging speed. Using an sCMOS camera for SMLM imaging can significantly improve the imaging time resolution, but the unique single pixel-dependent readout noise of sCMOS cameras severely limits their application in SMLM imaging. This paper develops a Hessian-based SMLM (Hessian-SMLM) method that can correct the variance, gain and offset of a single pixel of a camera and effectively eliminate the pixel-dependent readout noise of sCMOS cameras, especially when the signal-to-noise ratio is low. Using Hessian SMLM to image mEos3.2-labeled actin was able to significantly reduce the artifacts due to camera noise.

scholarly journals Computational Correction of Spatially-Variant Optical Aberrations in 3D Single Molecule Localization Microscopy

2018 ◽  
Author(s):  
Ting Yan ◽  
Charles J. Richardson ◽  
Mingxing Zhang ◽  
Andreas Gahlmann
Keyword(s):  
Single Molecule ◽  
Phase Retrieval ◽  
Wide Field ◽  
Optical Aberrations ◽  
Point Spread Functions ◽  
Localization Microscopy ◽  
Accuracy And Precision ◽  
Spatially Variant ◽  
Emitter Localization ◽  
Single Molecule Localization Microscopy

3D single-molecule localization microscopy relies on fitting the shape of point-spread-functions (PSFs) recorded on a wide-field detector. However, optical aberrations distort those shapes, which compromise the accuracy and precision of single-molecule localization microscopy. Here we employ a computational phase retrieval based on a vectorial PSF model to quantify the spatially-variance of optical aberrations in a two-channel ultrawide-field single-molecule localization microscope. The use of a spatially-variant PSF model enables accurate and precise emitter localization in x, y- and z- directions throughout the entire field-of-view.

Single-Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo-Crosslinking Fluorescent Sensor

2019 ◽  
Author(s):  
Zacharias Thiel ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Subcellular Localization ◽  
Fluorescent Probe ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Fluorescent Sensor ◽  
Biological Functions ◽  
Localization Microscopy ◽  
Drug Activation ◽  
Nitroreductase Activity ◽  
Single Molecule Localization Microscopy

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, threedimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.<br>

Single-Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo-Crosslinking Fluorescent Sensor

2019 ◽  
Author(s):  
Zacharias Thiel ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Subcellular Localization ◽  
Fluorescent Probe ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Fluorescent Sensor ◽  
Biological Functions ◽  
Localization Microscopy ◽  
Drug Activation ◽  
Nitroreductase Activity ◽  
Single Molecule Localization Microscopy

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, threedimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.<br>

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