Solving the boundary artifact for the enhanced deconvolution algorithm SUPPOSe applied to fluorescence microscopy

The SUPPOSe enhanced deconvolution algorithm relies in assuming that the image source can be described by an incoherent superposition of virtual point sources of equal intensity and finding the number and position of such virtual sources. In this work we describe the recent advances in the implementation of the method to gain resolution and remove artifacts due to the presence of fluorescent molecules close enough to the image frame boundary. The method was modified removing the invariant used before given by the product of the flux of the virtual sources times the number of virtual sources, and replacing it by a new invariant given by the total flux within the frame, thus allowing the location of virtual sources outside the frame but contributing to the signal inside the frame.

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Recent advances in the standardization of fluorescence microscopy for quantitative image analysis

Biophysical Reviews ◽

10.1007/s12551-021-00871-0 ◽

2021 ◽

Author(s):

Akira Sasaki

Keyword(s):

Image Analysis ◽

Fluorescence Microscopy ◽

Quantitative Image Analysis ◽

Recent Advances ◽

Quantitative Image

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Methods For Calculating Radio Holograms Of Volumetric Objects

Radioelectronics Nanosystems Information Technologies ◽

10.17725/rensit.2020.12.429 ◽

2020 ◽

Vol 12 (4) ◽

pp. 429-436

Author(s):

Valery A. Golunov ◽

◽

Vadim A. Korotkov ◽

Keyword(s):

Point Sources ◽

Real Point ◽

Two Dimensional ◽

Frequency Range ◽

Satisfactory Accuracy ◽

Phase Errors ◽

Sphere Radius ◽

Virtual Point ◽

Volumetric Objects

A method for calculating holograms for volumetric objects based on the representation of objects in the form of ensembles of virtual point sources distributed on a set of parallel planes has been proposed. The proposed method is the development of the well-known method in which objects are represented as ensemble of real point scatterers. The possibilities of the proposed method are demonstrated by calculating a hologram of a fragment of a sphere, on which 1000 points are randomly selected, at which radiation emanating from the center of the sphere is scattered. The choice of a fragment of a sphere as an object under study is due to the fact that when calculating its hologram, phase errors inherent in approximate calculations are most pronounced. The calculations were performed for the frequency range of 2...100 GHz, the sphere radius of 0.5 m, a two-dimensional hologram size of 0.65×0.65 m, and a pixel count of 512×512. It is shown that, in comparison with the known method, the proposed method makes it possible to calculate the amplitude of a hologram with satisfactory accuracy if virtual sources are placed on parallel planes in an amount of more than 64 pieces. In the case of objects that require representation in the form of an ensemble of point scatterers in the amount of more than 1000 pieces, the calculation of their holograms by the proposed method turns out to be much more efficient than the known method.

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Two-Photon Fluorescence Microscopy: A Review of Recent Advances in Deep-Tissue Imaging

Alternative Toxicological Methods ◽

10.1201/9780203008799-44 ◽

2003 ◽

pp. 385-397

Keyword(s):

Fluorescence Microscopy ◽

Tissue Imaging ◽

Deep Tissue ◽

Two Photon ◽

Recent Advances ◽

Deep Tissue Imaging ◽

Two Photon Fluorescence ◽

Photon Fluorescence

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Recent Advances in Biological Single-Molecule Applications of Optical Tweezers and Fluorescence Microscopy

Methods in Enzymology - Single-Molecule Enzymology: Nanomechanical Manipulation and Hybrid Methods ◽

10.1016/bs.mie.2016.09.047 ◽

2017 ◽

pp. 85-119 ◽

Cited By ~ 29

Author(s):

M. Hashemi Shabestari ◽

A.E.C. Meijering ◽

W.H. Roos ◽

G.J.L. Wuite ◽

E.J.G. Peterman

Keyword(s):

Fluorescence Microscopy ◽

Optical Tweezers ◽

Single Molecule ◽

Recent Advances

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Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1621495114 ◽

2017 ◽

Vol 114 (9) ◽

pp. 2125-2130 ◽

Cited By ~ 61

Author(s):

Fabian Göttfert ◽

Tino Pleiner ◽

Jörn Heine ◽

Volker Westphal ◽

Dirk Görlich ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Stimulated Emission ◽

Limiting Factor ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Initial State ◽

Intensity Pattern ◽

Optical Fluorescence ◽

Pore Complexes ◽

Fluorescent Molecules

Photobleaching remains a limiting factor in superresolution fluorescence microscopy. This is particularly true for stimulated emission depletion (STED) and reversible saturable/switchable optical fluorescence transitions (RESOLFT) microscopy, where adjacent fluorescent molecules are distinguished by sequentially turning them off (or on) using a pattern of light formed as a doughnut or a standing wave. In sample regions where the pattern intensity reaches or exceeds a certain threshold, the molecules are essentially off (or on), whereas in areas where the intensity is lower, that is, around the intensity minima, the molecules remain in the initial state. Unfortunately, the creation of on/off state differences on subdiffraction scales requires the maxima of the intensity pattern to exceed the threshold intensity by a large factor that scales with the resolution. Hence, when recording an image by scanning the pattern across the sample, each molecule in the sample is repeatedly exposed to the maxima, which exacerbates bleaching. Here, we introduce MINFIELD, a strategy for fundamentally reducing bleaching in STED/RESOLFT nanoscopy through restricting the scanning to subdiffraction-sized regions. By safeguarding the molecules from the intensity of the maxima and exposing them only to the lower intensities (around the minima) needed for the off-switching (on-switching), MINFIELD largely avoids detrimental transitions to higher molecular states. A bleaching reduction by up to 100-fold is demonstrated. Recording nanobody-labeled nuclear pore complexes in Xenopus laevis cells showed that MINFIELD-STED microscopy resolved details separated by <25 nm where conventional scanning failed to acquire sufficient signal.

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Accuracy and precision in quantitative fluorescence microscopy

The Journal of Cell Biology ◽

10.1083/jcb.200903097 ◽

2009 ◽

Vol 185 (7) ◽

pp. 1135-1148 ◽

Cited By ~ 357

Author(s):

Jennifer C. Waters

Keyword(s):

Fluorescence Microscopy ◽

Cell Biology ◽

Imaging System ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Digital Cameras ◽

Accuracy And Precision ◽

Fluorescent Molecules ◽

Quantitative Fluorescence ◽

Biology Research

The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Förster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.

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