Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time

We present a single-molecule tool called the CoPro (concentration of proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrate its utility experimentally on modelEscherichia colibacteria andSaccharomyces cerevisiaebudding yeast cells, and use it to address the biological question of how signals are transduced in cells. Cells in all domains of life dynamically sense their environment through signal transduction mechanisms, many involving gene regulation. The glucose sensing mechanism ofS. cerevisiaeis a model system for studying gene regulatory signal transduction. It uses the multi-copy expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in the presence of elevated extracellular glucose concentrations. We fluorescently labelled Mig1 molecules with green fluorescent protein (GFP)viachromosomal integration at physiological expression levels in livingS. cerevisiaecells, in addition to the RNA polymerase protein Nrd1 with the fluorescent protein reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1 protein concentrations in the cytoplasm and nucleus compartments on a cell-by-cell basis under physiological conditions. These estimates indicate a ∼4-fold shift towards higher values in the concentration of diffusive Mig1 in the nucleus if the external glucose concentration is raised, whereas equivalent levels in the cytoplasm shift to smaller values with a relative change an order of magnitude smaller. This compares with Nrd1 which is not involved directly in glucose sensing, and which is almost exclusively localized in the nucleus under high and low external glucose levels. CoPro facilitates time-resolved quantification of protein concentrations in single functional cells, and enables the distributions of concentrations across a cell population to be measured. This could be useful in investigating several cellular processes that are mediated by proteins, especially where changes in protein concentration in a single cell in response to changes in the extracellular chemical environment are subtle and rapid and may be smaller than the variability across a cell population.

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Combining single-molecule super-resolved localization microscopy with fluorescence polarization imaging to study cellular processes

10.1101/2021.01.06.425576 ◽

2021 ◽

Author(s):

Jack W Shepherd ◽

Alex L Payne-Dwyer ◽

Mark C Leake

Keyword(s):

Single Molecule ◽

Fluorescence Polarization ◽

Fluorescent Protein ◽

Imaging Modality ◽

Simultaneous Detection ◽

Optical Resolution ◽

Biological Processes ◽

Localization Microscopy ◽

Cellular Processes ◽

Wide Range

AbstractSuper-resolution microscopy has enabled valuable insights into the subcellular, mechanistic details of many different biological processes across a wide range of cell types. Fluorescence polarization spectroscopy tools have also enabled important insights into cellular processes through identifying orientational changes of biological molecules typically at an ensemble level. Here, we combine these two biophysical methodologies in a single home-made instrument to enable the simultaneous detection of orthogonal fluorescence polarization signals from single fluorescent protein molecules used as common reporters on the localization on biomolecules in cellular processes, whose spatial location can be pinpointed to a super-resolved precision better than the diffraction-limited optical resolution. In this small innovation we have adapted a millisecond timescale “Slimfield” microscope used for single-molecule detection to enable spitting of the fluorescence polarization emissions into two separate imaging channels for s- and p- polarization signals that are imaged onto separate halves of the same high sensitivity back-illuminated CMOS camera detector. We applied this fluorescence polarization super-resolved imaging modality to a range of test fluorescent samples relevant to the study of biological processes, including purified monomeric green fluorescent protein (mGFP). Our findings are largely qualitative but demonstrate promise in showing how fluorescence polarization and Slimfield-mediated super-resolved localization microscopy can be combined on the same sample to enable new biological insights.

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Enhanced UnaG With Minimal Labeling Artifact for Single-Molecule Localization Microscopy

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.647590 ◽

2021 ◽

Vol 8 ◽

Author(s):

Sangyoon Ko ◽

Jiwoong Kwon ◽

Sang-Hee Shim

Keyword(s):

Single Molecule ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Super Resolution ◽

Green Emission ◽

Switching Behavior ◽

Light Illumination ◽

High Concentration ◽

Localization Microscopy ◽

Single Molecule Localization Microscopy

We introduced enhanced UnaG (eUnaG), a ligand-activatable fluorescent protein, for conventional and super-resolution imaging of subcellular structures in the mammalian cells. eUnaG is a V2L mutant of UnaG with twice brighter bulk fluorescence. We previously discovered the reversible fluorescence switching behavior of UnaG and demonstrated the high photon outputs and high localization numbers in single-molecule localization microscopy (SMLM). In this study, we showed that the fluorescence of eUnaG can be switched off under blue-light illumination, while a high concentration of fluorogenic ligands in the buffer can efficiently restore the fluorescence, as in UnaG. We demonstrated the capacity of eUnaG as an efficient protein label in mammalian cells, as well as for SMLM by utilizing its photoswitchable nature. While cytosolic UnaG proteins showed aggregated patches and fluorescence reduction at high expression levels, eUnaG-labeled protein targets successfully formed their proper structures in mammalian cells without notable distortion from the endogenous structure in the majority of transiently expressing cells. In particular, eUnaG preserved the vimentin filament structures much better than the UnaG. eUnaG provided similarly high single-molecule photon count distribution to UnaG, thus also similarly high resolution in the super-resolution images of various subcellular structures. The sampling coverage analysis of vimentin filaments in SMLM images showed the improvement of labeling efficiency of eUnaG. eUnaG is a high-performance fluorescent protein for fluorescence and single-molecule localization imaging in green emission with minimal labeling artifact.

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Using Persistent Homology as a New Approach for Super-Resolution Localization Microscopy Data Analysis and Classification of γH2AX Foci/Clusters

International Journal of Molecular Sciences ◽

10.3390/ijms19082263 ◽

2018 ◽

Vol 19 (8) ◽

pp. 2263 ◽

Cited By ~ 14

Author(s):

Andreas Hofmann ◽

Matthias Krufczik ◽

Dieter Heermann ◽

Michael Hausmann

Keyword(s):

Single Molecule ◽

Spatial Organization ◽

Genome Structure ◽

Super Resolution ◽

Analytical Methodology ◽

Damage Site ◽

New Approach ◽

Localization Microscopy ◽

Γh2ax Foci ◽

A Cell

DNA double strand breaks (DSB) are the most severe damages in chromatin induced by ionizing radiation. In response to such environmentally determined stress situations, cells have developed repair mechanisms. Although many investigations have contributed to a detailed understanding of repair processes, e.g., homologous recombination repair or non-homologous end-joining, the question is not sufficiently answered, how a cell decides to apply a certain repair process at a certain damage site, since all different repair pathways could simultaneously occur in the same cell nucleus. One of the first processes after DSB induction is phosphorylation of the histone variant H2AX to γH2AX in the given surroundings of the damaged locus. Since the spatial organization of chromatin is not random, it may be conclusive that the spatial organization of γH2AX foci is also not random, and rather, contributes to accessibility of special repair proteins to the damaged site, and thus, to the following repair pathway at this given site. The aim of this article is to demonstrate a new approach to analyze repair foci by their topology in order to obtain a cell independent method of categorization. During the last decade, novel super-resolution fluorescence light microscopic techniques have enabled new insights into genome structure and spatial organization on the nano-scale in the order of 10 nm. One of these techniques is single molecule localization microscopy (SMLM) with which the spatial coordinates of single fluorescence molecules can precisely be determined and density and distance distributions can be calculated. This method is an appropriate tool to quantify complex changes of chromatin and to describe repair foci on the single molecule level. Based on the pointillist information obtained by SMLM from specifically labeled heterochromatin and γH2AX foci reflecting the chromatin morphology and repair foci topology, we have developed a new analytical methodology of foci or foci cluster characterization, respectively, by means of persistence homology. This method allows, for the first time, a cell independent comparison of two point distributions (here the point distributions of two γH2AX clusters) with each other of a selected ensample and to give a mathematical measure of their similarity. In order to demonstrate the feasibility of this approach, cells were irradiated by low LET (linear energy transfer) radiation with different doses and the heterochromatin and γH2AX foci were fluorescently labeled by antibodies for SMLM. By means of our new analysis method, we were able to show that the topology of clusters of γH2AX foci can be categorized depending on the distance to heterochromatin. This method opens up new possibilities to categorize spatial organization of point patterns by parameterization of topological similarity.

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Site-Specific Protein Covalent Attachment to Nanotubes and Its Electronic Impact on Single Molecule Function

10.26434/chemrxiv.7798973.v1 ◽

2019 ◽

Author(s):

Adam Beachey ◽

Harley Worthy ◽

William David Jamieson ◽

Suzanne Thomas ◽

Benjamin Bowen ◽

...

Keyword(s):

Single Molecule ◽

Fluorescent Protein ◽

Functional Integration ◽

Attachment Site ◽

Covalent Attachment ◽

Great Promise ◽

Functional Coupling ◽

Phenyl Azide ◽

Electronic Impact ◽

Protein Attachment

<p>Functional integration of proteins with carbon-based nanomaterials such as nanotubes holds great promise in emerging electronic and optoelectronic applications. Control over protein attachment poses a major challenge for consistent and useful device fabrication, especially when utilizing single/few molecule properties. Here, we exploit genetically encoded phenyl azide photochemistry to define the direct covalent attachment of three different proteins, including the fluorescent protein GFP, to carbon nanotube side walls. Single molecule fluorescence revealed that on attachment to SWCNTs GFP’s fluorescence changed in terms of intensity and improved resistance to photobleaching; essentially GFP is fluorescent for much longer on attachment. The site of attachment proved important in terms of electronic impact on GFP function, with the attachment site furthest from the functional center having the larger effect on fluorescence. Our approach provides a versatile and general method for generating intimate protein-CNT hybrid bioconjugates. It can be potentially applied easily to any protein of choice; attachment position and thus interface characteristics with the CNT can easily be changed by simply placing the phenyl azide chemistry at different residues by gene mutagenesis. Thus, our approach will allow consistent construction and modulate functional coupling through changing the protein attachment position.</p>

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

10.26434/chemrxiv.7992980.v1 ◽

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

10.26434/chemrxiv.7992980 ◽

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

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Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

International Journal of Molecular Sciences ◽

10.3390/ijms22041903 ◽

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.

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