scholarly journals Rapid Ensemble Measurement of Protein Diffusion, Probe Blinking and Photobleaching Dynamics in the Complex Cellular Space

2021 ◽  
Author(s):  
Simon Sehayek ◽  
Xiyu Yi ◽  
Shimon Weiss ◽  
Paul W. Wiseman
Keyword(s):  
Hela Cells ◽  
Super Resolution ◽  
Excitation Power ◽  
Image Correlation ◽  
Protein Diffusion ◽  
Cellular Space ◽  
Transport Dynamics ◽  
Correlation Analysis Method ◽  
Nih 3T3 ◽  
Fluorescent Molecules

We present a fluorescence fluctuation image correlation analysis method that can rapidly and simultaneously measure the diffusion coefficient, photoblinking rates, and fraction of diffusing particles of fluorescent molecules in cells. Unlike other image correlation techniques, we demonstrated that our method could be applied irrespective of a non-uniformly distributed, immobile blinking fluorophore population. This allows us to measure blinking and transport dynamics in complex cell morphologies, a benefit for a range of super-resolution fluorescence imaging approaches that rely on probe emission blinking. Furthermore, we showed that our technique could be applied without directly accounting for photobleaching. We successfully employed our technique on several simulations with realistic EMCCD noise and photobleaching models, as well as on Dronpa-C12 labeled beta-actin in living NIH/3T3 and HeLa cells. We found that the diffusion coefficients measured using our method were consistent with previous literature values. We further found that photoblinking rates measured in the live HeLa cells varied as expected with changing excitation power.

scholarly journals Super Resolution Digital Image Correlation (SR-DIC): an Alternative to Image Stitching at High Magnifications

2021 ◽  
Author(s):  
R. S. Hansen ◽  
D. W. Waldram ◽  
T. Q. Thai ◽  
R. B. Berke
Keyword(s):  
High Resolution ◽  
Digital Image Correlation ◽  
Spatial Resolution ◽  
Digital Image ◽  
Super Resolution ◽  
Image Correlation ◽  
Low Resolution ◽  
Strain Measurements ◽  
Resolution Image ◽  
High Resolution Image

Abstract Background High-resolution Digital Image Correlation (DIC) measurements have previously been produced by stitching of neighboring images, which often requires short working distances. Separately, the image processing community has developed super resolution (SR) imaging techniques, which improve resolution by combining multiple overlapping images. Objective This work investigates the novel pairing of super resolution with digital image correlation, as an alternative method to produce high-resolution full-field strain measurements. Methods First, an image reconstruction test is performed, comparing the ability of three previously published SR algorithms to replicate a high-resolution image. Second, an applied translation is compared against DIC measurement using both low- and super-resolution images. Third, a ring sample is mechanically deformed and DIC strain measurements from low- and super-resolution images are compared. Results SR measurements show improvements compared to low-resolution images, although they do not perfectly replicate the high-resolution image. SR-DIC demonstrates reduced error and improved confidence in measuring rigid body translation when compared to low resolution alternatives, and it also shows improvement in spatial resolution for strain measurements of ring deformation. Conclusions Super resolution imaging can be effectively paired with Digital Image Correlation, offering improved spatial resolution, reduced error, and increased measurement confidence.

scholarly journals Axonemal Lumen Dominates Cytosolic Protein Diffusion inside the Primary Cilium

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Wangxi Luo ◽  
Andrew Ruba ◽  
Daisuke Takao ◽  
Ludovit P. Zweifel ◽  
Roderick Y. H. Lim ◽  
...  
Keyword(s):  
High Speed ◽  
Primary Cilia ◽  
Protein Transport ◽  
Genetic Diseases ◽  
Super Resolution ◽  
Intraflagellar Transport ◽  
Protein Diffusion ◽  
Spatiotemporal Resolution ◽  
Cytosolic Proteins ◽  
Relative Contribution

AbstractTransport of membrane and cytosolic proteins in primary cilia is thought to depend on intraflagellar transport (IFT) and diffusion. However, the relative contribution and spatial routes of each transport mechanism are largely unknown. Although challenging to decipher, the details of these routes are essential for our understanding of protein transport in primary cilia, a critically affected process in many genetic diseases. By using a high-speed virtual 3D super-resolution microscopy, we have mapped the 3D spatial locations of transport routes for various cytosolic proteins in the 250-nm-wide shaft of live primary cilia with a spatiotemporal resolution of 2 ms and <16 nm. Our data reveal two spatially distinguishable transport routes for cytosolic proteins: an IFT-dependent path along the axoneme, and a passive-diffusion route in the axonemal lumen that escaped previous studies. While all cytosolic proteins tested primarily utilize the IFT path in the anterograde direction, differences are observed in the retrograde direction where IFT20 only utilizes IFT, and approximately half of KIF17 and one third of α–tubulin utilizes diffusion besides IFT.

scholarly journals Cell-specific regulation of oncogene-responsive sequences of the c-fos promoter

1991 ◽  
Vol 11 (10) ◽  
pp. 5381-5387
Author(s):  
A Gutman ◽  
C Wasylyk ◽  
B Wasylyk
Keyword(s):  
Hela Cells ◽  
Serum Response Factor ◽  
Tumor Promoter ◽  
3T3 Cells ◽  
Carg Box ◽  
Specific Regulation ◽  
Nih 3T3 ◽  
Serum Response ◽  
Nih 3T3 Cells

We have identified oncogene-responsive sequences in the human c-fos promoter that mediate induction of transcription by several nonnuclear oncoproteins and the tumor promoter TPA. These sequences are regulated in a cell-specific manner. (i) In NIH 3T3 cells, the CArG box of the c-fos promoter is sufficient to mediate activation by oncogenes. (ii) In contrast, in HeLa cells, additional flanking sequences are also required, including the outer arm of the serum response element and the FAP site. We also show that the serum response factor, which binds to the CArG box, activates transcription in vivo in NIH 3T3 cells but not in HeLa cells. Finally, we present evidence that the intracellular level of the c-Fos protein could be a major determinant of cell-specific regulation of these oncogene-responsive elements of the c-fos promoter.

scholarly journals Measurement of Protein 53 Diffusion Coefficient in Live HeLa Cells Using Raster Image Correlation Spectroscopy (RICS)

2010 ◽  
Vol 01 (01) ◽  
pp. 31-36 ◽  
Author(s):  
Sungmin Hong ◽  
Ying-Nai Wang ◽  
Hirohito Yamaguchi ◽  
Harinibytaraya Sreenivasappa ◽  
Chao-Kai Chou ◽  
...  
Keyword(s):  
Diffusion Coefficient ◽  
Hela Cells ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Raster Image ◽  
Image Correlation Spectroscopy

scholarly journals Microtubules Regulate Spatial Localization and Availability of Insulin Granules in Pancreatic Beta Cells

2019 ◽  
Author(s):  
Kai M. Bracey ◽  
Kung-Hsien Ho ◽  
Dmitry Yampolsky ◽  
Guoqiang Gu ◽  
Irina Kaverina ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Single Cells ◽  
Critical Role ◽  
Super Resolution ◽  
Transport Dynamics ◽  
Insulin Granules ◽  
Resolution Imaging ◽  
Glucose Stimulated Insulin Secretion

AbstractTwo key prerequisites for glucose stimulated insulin secretion (GSIS) in Beta cells are the proximity of insulin granules to the plasma membrane and their anchoring or docking to the plasma membrane (PM). While recent evidence has indicated that both of these factors are altered in the context of diabetes, it is unclear what regulates localization of insulin and its interactions with the PM within single cells. Here we demonstrate that microtubule (MT) motor mediated transport dynamics have a critical role in regulating both factors. Super-resolution imaging shows that while the MT cytoskeleton resembles a random meshwork in the cells’ interior, MTs near the cells surface are preferentially aligned with the PM. Computational modeling demonstrates two consequences of this alignment. First, this structured MT network preferentially withdraws granules from the PM. Second, the binding and transport of insulin granules by MT motors prevents their stable anchoring to the PM. The MT cytoskeleton thus negatively regulates GSIS by both limiting the amount of insulin proximal to the PM and preventing/breaking interactions between the PM and the remaining nearby insulin. These results predict that altering MT structure in beta cells can be used to tune GSIS. Thus, our study points to a potential of an alternative therapeutic strategy for diabetes by targeting specific MT regulators.

scholarly journals Evaluation of sted super-resolution image quality by image correlation spectroscopy (QuICS)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Elena Cerutti ◽  
Morgana D’Amico ◽  
Isotta Cainero ◽  
Gaetano Ivan Dellino ◽  
Mario Faretta ◽  
...  
Keyword(s):  
Autocorrelation Function ◽  
Signal To Noise Ratio ◽  
Super Resolution ◽  
Image Contrast ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Signal To Noise ◽  
Image Correlation Spectroscopy ◽  
Noise Ratio

AbstractQuantifying the imaging performances in an unbiased way is of outmost importance in super-resolution microscopy. Here, we describe an algorithm based on image correlation spectroscopy (ICS) that can be used to assess the quality of super-resolution images. The algorithm is based on the calculation of an autocorrelation function and provides three different parameters: the width of the autocorrelation function, related to the spatial resolution; the brightness, related to the image contrast; the relative noise variance, related to the signal-to-noise ratio of the image. We use this algorithm to evaluate the quality of stimulated emission depletion (STED) images of DNA replication foci in U937 cells acquired under different imaging conditions. Increasing the STED depletion power improves the resolution but may reduce the image contrast. Increasing the number of line averages improves the signal-to-noise ratio but facilitates the onset of photobleaching and subsequent reduction of the image contrast. Finally, we evaluate the performances of two different separation of photons by lifetime tuning (SPLIT) approaches: the method of tunable STED depletion power and the commercially available Leica Tau-STED. We find that SPLIT provides an efficient way to improve the resolution and contrast in STED microscopy.

An Alchemy of DNA: Exploring the Chemistry of Biology Through BioArt

Leonardo ◽  
2019 ◽  
pp. 1-2
Author(s):  
Anna Dumitriu ◽  
Robert K. Neely
Keyword(s):  
Chemical Nature ◽  
Bacterial Genome ◽  
Super Resolution ◽  
Laser Imaging ◽  
Imaging Technologies ◽  
The Creation ◽  
The University ◽  
Fluorescent Molecules

This article is a reflection by artist Anna Dumitriu on her residency biochemist Robert K Neely at the University of Birmingham which led to the creation of “The Chemistry of Biology: An Alchemy of DNA”, a sculptural and bio-digital installation which premiered at BOM (Birmingham Open Media) in October 2017. Their project ex-plored the chemical nature of DNA, the enigmatic ‘instruction book of life’, through new super-resolution laser imaging technologies us-ing fluorescent molecules enabling them to physically observe a re-gion of DNA containing a scarless CRISPR edit to a bacterial genome, building on an earlier project “Make Do and Mend”.

scholarly journals Regulatory role of MEF2D in serum induction of the c-jun promoter.

1995 ◽  
Vol 15 (6) ◽  
pp. 2907-2915 ◽  
Author(s):  
T H Han ◽  
R Prywes
Keyword(s):  
Dna Binding ◽  
Reporter Gene ◽  
Hela Cells ◽  
Serum Response Factor ◽  
Common Mechanism ◽  
Response Factor ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Serum Response ◽  
Nih 3T3 Cells

Serum induction of c-jun expression in HeLa cells requires a MEF2 site at -59 in the c-jun promoter. MEF2 sites, found in many muscle-specific enhancers, are bound by a family of transcription factors, MEF2A through -D, which are related to serum response factor in their DNA binding domains. We have found that MEF2D is the predominant protein in HeLa cells that binds to the c-jun MEF2 site. Serum induction of a MEF2 reporter gene was not observed in a line of NIH 3T3 cells which contain low MEF2 site binding activity. Transfection of MEF2D into NIH 3T3 cells reconstituted serum induction, demonstrating that MEF2D is required for the serum response. Deletion analysis of MEF2D showed that its DNA binding domain, when fused to a heterologous transcriptional activation domain, was sufficient for serum induction of a MEF2 reporter gene. This is the domain homologous to that in the serum response factor which is required for serum induction of the c-fos serum response element, suggesting that serum regulation of c-fos and c-jun may share a common mechanism.

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