scholarly journals Addressing systematic errors in axial distance measurements in single-emitter localization microscopy

2020 ◽  
Vol 28 (13) ◽  
pp. 18616 ◽  
Author(s):  
Petar N. Petrov ◽  
W. E. Moerner
Keyword(s):  
Systematic Errors ◽  
Axial Distance ◽  
Distance Measurements ◽  
Localization Microscopy ◽  
Single Emitter ◽  
Emitter Localization

A literature review of localization methods and emergence of deep learning in single-molecule localization microscopy

2020 ◽  
Author(s):  
Anish Mukherjee
Keyword(s):  
Deep Learning ◽  
Single Molecule ◽  
Super Resolution ◽  
Point Sources ◽  
Learning Approaches ◽  
Localization Algorithm ◽  
Localization Microscopy ◽  
Trade Offs ◽  
Emitter Localization ◽  
Single Molecule Localization Microscopy

The quality of super-resolution images largely depends on the performance of the emitter localization algorithm used to localize point sources. In this article, an overview of the various techniques which are used to localize point sources in single-molecule localization microscopy are discussed and their performances are compared. This overview can help readers to select a localization technique for their application. Also, an overview is presented about the emergence of deep learning methods that are becoming popular in various stages of single-molecule localization microscopy. The state of the art deep learning approaches are compared to the traditional approaches and the trade-offs of selecting an algorithm for localization are discussed.

scholarly journals High precision wavefront control in point spread function engineering for single emitter localization

2018 ◽  
Vol 26 (7) ◽  
pp. 8397 ◽  
Author(s):  
M. Siemons ◽  
C. N. Hulleman ◽  
R. Ø. Thorsen ◽  
C. S. Smith ◽  
S. Stallinga
Keyword(s):  
High Precision ◽  
Point Spread Function ◽  
Wavefront Control ◽  
Point Spread ◽  
Spread Function ◽  
Point Spread Function Engineering ◽  
Single Emitter ◽  
Emitter Localization

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.

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.

scholarly journals High precision wavefront control in point spread function engineering for single emitter localization

2018 ◽  
Author(s):  
M. Siemons ◽  
C. N. Hulleman ◽  
R. Ø. Thorsen ◽  
C. S. Smith ◽  
S. Stallinga
Keyword(s):  
High Precision ◽  
Point Spread Function ◽  
Time Window ◽  
Wavefront Aberration ◽  
Wavefront Control ◽  
Point Spread ◽  
Spread Function ◽  
Wavefront Error ◽  
Single Emitter ◽  
Emitter Localization

AbstractPoint spread function (PSF) engineering is used in single emitter localization to measure the emitter position in 3D and possibly other parameters such as the emission color or dipole orientation as well. Advanced PSF models such as spline fits to experimental PSFs or the vectorial PSF model can be used in the corresponding localization algorithms in order to model the intricate spot shape and deformations correctly. The complexity of the optical architecture and fit model makes PSF engineering approaches particularly sensitive to optical aberrations. Here, we present a calibration and alignment protocol for fluorescence microscopes equipped with a spatial light modulator (SLM) with the goal of establishing a wavefront error well below the diffraction limit for optimum application of complex engineered PSFs. We achieve high-precision wavefront control, to a level below 20 mλ wavefront aberration over a 30 minute time window after the calibration procedure, using a separate light path for calibrating the pixel-to-pixel variations of the SLM, and alignment of the SLM with respect to the optical axis and Fourier plane within 3 µm (x/y) and 100 µm (z) error. Aberrations are retrieved from a fit of the vectorial PSF model to a bead z-stack and compensated with a residual wavefront error comparable to the error of the SLM calibration step. This well-calibrated and corrected setup makes it possible to create complex ‘3D+λ’ PSFs that fit very well to the vectorial PSF model. Proof-of-principle bead experiments show precisions below 10 nm in x, y, and λ, and below 20 nm in z over an axial range of 1 µm with 2000 signal photons and 12 background photons.

scholarly journals EXPERIMENTAL ASSESSMENT OF THE QUANERGY M8 LIDAR SENSOR

2016 ◽  
Vol XLI-B5 ◽  
pp. 527-531 ◽  
Author(s):  
M.-A. Mitteta ◽  
H. Nouira ◽  
X. Roynard ◽  
F. Goulette ◽  
J.-E. Deschaud
Keyword(s):  
Measurement Errors ◽  
Temporal Stability ◽  
Systematic Errors ◽  
Distance Measurement ◽  
Lidar System ◽  
Distance Measurements ◽  
Scanning Lidar ◽  
Different Sources ◽  
Indoor And Outdoor

In this paper, some experiments with the Quanergy M8 scanning LIDAR system are related. The distance measurement obtained with the Quanergy M8 can be influenced by different factors. Moreover, measurement errors can originate from different sources. The environment in which the measurements are performed has an influence (temperature, light, humidity, etc.). Errors can also arise from the system itself. Then, it is necessary to determine the influence of these parameters on the quality of the distance measurements. For this purpose different studies are presented and analyzed. First, we studied the temporal stability of the sensor by analyzing observations during time. Secondly, the assessment of the distance measurement quality has been conducted. The aim of this step is to detect systematic errors in measurements regarding the range. Differents series of measurements have been conducted : at different range and in diffrent conditions (indoor and outdoor). Finally, we studied the consistency between the differents beam of the LIDAR.

scholarly journals Single molecule localization microscopy with autonomous feedback loops for ultrahigh precision

2018 ◽  
Author(s):  
Simao Coelho ◽  
Jongho Baek ◽  
Matthew S. Graus ◽  
James M. Halstead ◽  
Philip R. Nicovich ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Separation Distance ◽  
Distance Measurements ◽  
Intact Cells ◽  
Localization Accuracy ◽  
Localization Microscopy ◽  
Signaling Receptors ◽  
Single Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) promises to provide truly molecular scale images of biological specimens1–5. However, mechanical instabilities in the instrument, readout errors and sample drift constitute significant challenges and severely limit both the useable data acquisition length and the localization accuracy of single molecule emitters6. Here, we developed an actively stabilized total internal fluorescence (TIRF) microscope that performs 3D real-time drift corrections and achieves a stability of ≤1 nm. Self-alignment of the emission light path and corrections of readout errors of the camera automate channel alignment and ensure localization precisions of 1-4 nm in DNA origami structures and cells for different labels. We used Feedback SMLM to measure the separation distance of signaling receptors and phosphatases in T cells. Thus, an improved SMLM enables direct distance measurements between molecules in intact cells on the scale between 1-20 nm, potentially replacing Förster resonance energy transfer (FRET) to quantify molecular interactions7. 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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