scholarly journals Two-color super-resolution localization microscopy via joint encoding of emitter location and color

2021 ◽  
Author(s):  
Yujie Wang ◽  
Weibing Kuang ◽  
Mingtao Shang ◽  
Zhen-Li Huang
Keyword(s):  
Near Infrared ◽  
Super Resolution ◽  
Encoding Scheme ◽  
Localization Microscopy ◽  
Color Recognition ◽  
Emitter Location ◽  
Repeated Pattern ◽  
Set Up ◽  
Joint Encoding ◽  
20 Nm

AbstractMulti-color super-resolution localization microscopy (SRLM) provides great opportunities for studying the structural and functional details of biological samples. However, current multi-color SRLM methods either suffer from medium to high crosstalk, or require a dedicated optical system and a complicated image analysis procedure. To address these problems, here we propose a completely different method to realize multi-color SRLM. This method is built upon a customized RGBW camera with a repeated pattern of filtered (Red, Green, Blue and Near-infrared) and unfiltered (White) pixels. With a new insight that RGBW camera is advantageous for color recognition instead of color reproduction, we developed a joint encoding scheme of emitter location and color. By combing this RGBW camera with the joint encoding scheme and a simple optical set-up, we demonstrated two-color SRLM with ∼20 nm resolution and < 2% crosstalk (which is comparable to the best reported values). This study significantly reduces the complexity of two-color SRLM (and potentially multi-color SRLM), and thus offers good opportunities for general biomedical research laboratories to use multi-color SRLM, which is currently mastered only by well-trained researchers.

scholarly journals Single-molecule Photoswitching and Localization

2011 ◽  
Vol 64 (5) ◽  
pp. 503 ◽  
Author(s):  
Sebastian van de Linde ◽  
Steve Wolter ◽  
Markus Sauer
Keyword(s):  
Single Molecule ◽  
Optical Resolution ◽  
Super Resolution ◽  
Image Plane ◽  
Localization Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Spatio Temporal ◽  
Optical Reconstruction ◽  
20 Nm

Within only a few years super-resolution fluorescence imaging based on single-molecule localization and image reconstruction has attracted considerable interest because it offers a comparatively simple way to achieve a substantially improved optical resolution down to ∼20 nm in the image plane. Since super-resolution imaging methods such as photoactivated localization microscopy, fluorescence photoactivation localization microscopy, stochastic optical reconstruction microscopy, and direct stochastic optical reconstruction microscopy rely critically on exact fitting of the centre of mass and the shape of the point-spread-function of isolated emitters unaffected by neighbouring fluorophores, controlled photoswitching or photoactivation of fluorophores is the key parameter for resolution improvement. This review will explain the principles and requirements of single-molecule based localization microscopy, and compare different super-resolution imaging concepts and highlight their strengths and limitations with respect to applications in fixed and living cells with high spatio-temporal resolution.

scholarly journals Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

2021 ◽  
Vol 22 (4) ◽  
pp. 1903
Author(s):  
Ivona Kubalová ◽  
Alžběta Němečková ◽  
Klaus Weisshart ◽  
Eva Hřibová ◽  
Veit Schubert
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Chromatin Condensation ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Wide Field ◽  
Structured Illumination Microscopy ◽  
Metaphase Chromosomes ◽  
Localization Microscopy ◽  
Super Resolution Microscopy

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

scholarly journals Camera-based localization microscopy optimized with calibrated structured illumination

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Martin Schmidt ◽  
Adam C. Hundahl ◽  
Henrik Flyvbjerg ◽  
Rodolphe Marie ◽  
Kim I. Mortensen
Keyword(s):  
Data Analysis ◽  
Super Resolution ◽  
Wide Field ◽  
Structured Illumination ◽  
Uniform Illumination ◽  
Localization Microscopy ◽  
Localization And Tracking ◽  
Field Imaging ◽  
Wide Field Imaging ◽  
Localization Information

AbstractUntil very recently, super-resolution localization and tracking of fluorescent particles used camera-based wide-field imaging with uniform illumination. Then it was demonstrated that structured illuminations encode additional localization information in images. The first demonstration of this uses scanning and hence suffers from limited throughput. This limitation was mitigated by fusing camera-based localization with wide-field structured illumination. Current implementations, however, use effectively only half the localization information that they encode in images. Here we demonstrate how all of this information may be exploited by careful calibration of the structured illumination. Our approach achieves maximal resolution for given structured illumination, has a simple data analysis, and applies to any structured illumination in principle. We demonstrate this with an only slightly modified wide-field microscope. Our protocol should boost the emerging field of high-precision localization with structured illumination.

Effect of Sample Heterogeneity on near Infrared Meat Analysis: The Use of the RMS Statistic

1998 ◽  
Vol 6 (A) ◽  
pp. A313-A320 ◽  
Author(s):  
M.L. Martínez ◽  
Ana Garrido-Varo ◽  
E. De Pedro ◽  
L. Sánchez
Keyword(s):  
Near Infrared ◽  
Fiber Optic ◽  
Iberian Pig ◽  
Set Up ◽  
Sample Heterogeneity ◽  
Meat Analysis

Ground and emulsified samples from Iberian pig hams were analysed by reflectance and interactance reflectance mode. Spectral errors due to intra-sample variations were calculated for both analysis modes. The spectral errors were calculated by means of the STD RMS statistic included on the ISI software. The results obtained show that a mean STD RMS value as low as 4200, could be obtained for paired subsamples of the same sample and that an STD limit of 4374 could be fixed at the instrument set-up program in order to ensure that a representative spectrum has been obtained from two subsamples readings of the same sample. That procedure avoids the need to take numerous subsamples, as is traditional in NIR/NIT meat analysis. The results also show that the spectral repeatability using fiber optic is worse than for spinning cups and it has been concluded that effort should be made to avoid moisture variations during scanning in order to improve spectral repeatability.

scholarly journals Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy

PLoS ONE ◽  
2016 ◽  
Vol 11 (7) ◽  
pp. e0158884 ◽  
Author(s):  
Leila Nahidiazar ◽  
Alexandra V. Agronskaia ◽  
Jorrit Broertjes ◽  
Bram van den Broek ◽  
Kees Jalink
Keyword(s):  
Super Resolution ◽  
Localization Microscopy ◽  
Imaging Conditions

scholarly journals Optimum three-point linkage set up for improving the quality of soil spectra and the accuracy of soil phosphorus measured using an on-line visible and near infrared sensor

2009 ◽  
Vol 103 (1) ◽  
pp. 144-152 ◽  
Author(s):  
A.M. Mouazen ◽  
M.R. Maleki ◽  
L. Cockx ◽  
M. Van Meirvenne ◽  
L.H.J. Van Holm ◽  
...  
Keyword(s):  
Near Infrared ◽  
Soil Phosphorus ◽  
Infrared Sensor ◽  
On Line ◽  
Set Up

Near Infrared Photothermoelectric Effect in Transparent AZO/ITO/Ag/ITO Thin Films

2021 ◽  
Author(s):  
Catarina Bianchi ◽  
Ana Marques ◽  
Rui Silva ◽  
Tomas Calmeiro ◽  
Isabel Ferreira
Keyword(s):  
Indium Tin Oxide ◽  
Near Infrared ◽  
Thermal Control ◽  
High Energy ◽  
Visible Range ◽  
Azo Thin Film ◽  
Ito Thin Films ◽  
Aluminum Doped Zinc Oxide ◽  
Temperature Constant ◽  
20 Nm

Abstract A new concept of oxide-metal-oxide structures that combine photothermoelectric effect with high reflectance (~80%) at wavelengths in the infrared (> 1100 nm) and high transmittance in the visible range is reported here. This was observed in optimized ITO/Ag/ITO structure, 20 nm of Siver (Ag) and 40 nm of Indium Tin Oxide (ITO), deposited on Aluminum doped Zinc Oxide (AZO) thin film. These layers show high energy saving efficiency by keeping the temperature constant inside a glazed compartment under solar radiation, but additionally they also show a photothermoelectric effect. Under uniform heating of the sample a thermoelectric effect is observed (S = 40 μV/K), but when irradiated, a potential proportional to the intensity of the radiation is also observed. Therefore, in addition to thermal control in windows, these low emission coatings can be applied as transparent photothermoelectric devices.

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.

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