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Innovations in Single-Molecule Imaging Techniques – Nanotechnology
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id="primary"> <main class="site-main" id="main"> <article id="post-91" class="post-91 post type-post status-publish format-standard has-post-thumbnail hentry category-single-molecule-imaging tag-dna-origami tag-heavy-water tag-high-resolution-colocalization tag-live-cell-imaging tag-localization-microscopy tag-minflux-nanoscopy tag-nanometer-localized-microscopy tag-photoactivatable-fluorescent-proteins tag-photobleaching tag-quantum-dots tag-single-molecule-imaging tag-sted-microscopy" itemtype="https://schema.org/CreativeWork" itemscope> <div class="inside-article"> <div class="featured-image page-header-image-single "> <img width="800" height="419" src="https://nanotechnology.blog/archive/wp-content/uploads/2024/09/image-37-min.jpg" class="attachment-full size-full" alt="" itemprop="image" decoding="async" fetchpriority="high" srcset="https://nanotechnology.blog/archive/wp-content/uploads/2024/09/image-37-min.jpg 800w, https://nanotechnology.blog/archive/wp-content/uploads/2024/09/image-37-min-300x157.jpg 300w, https://nanotechnology.blog/archive/wp-content/uploads/2024/09/image-37-min-768x402.jpg 768w" sizes="(max-width: 800px) 100vw, 800px" /> </div> <header class="entry-header"> <h1 class="entry-title" itemprop="headline">Innovations in Single-Molecule Imaging Techniques</h1> <div class="entry-meta"> <span class="posted-on"><time class="entry-date published" datetime="2024-09-24T18:02:02+05:30" itemprop="datePublished">September 24, 2024</time></span> <span class="byline">by <span class="author vcard" itemprop="author" itemtype="https://schema.org/Person" itemscope><a class="url fn n" href="https://nanotechnology.blog/archive/author/nanotechnology/" title="View all posts by nanotechnology" rel="author" itemprop="url"><span class="author-name" itemprop="name">nanotechnology</span></a></span></span> </div> </header> <div class="entry-content" itemprop="text"> <p><span style="font-weight: 400;">Microscopy as a field has been expanded and grown greatly through the use of single-molecule imaging methods. These improvements have moved beyond the problems that traditional microscopy posed, wherein nowadays it is possible to gain a greater understanding of biological processes and to investigate them at the molecular level. Through some sort of molecular model, one can observe the detailed topography, behavior, and functioning of numerous biomolecular structures at the atomic level. In this article, the author explores some of the remarkable points about single-molecule imaging and the methods and uses that are defining a new frontier for the exploration of biology.</span></p> <h3><b>High-Resolution Colocalization Techniques</b></h3> <p><span style="font-weight: 400;">High-resolution colocalization techniques are one of the most significant advancements in single-molecule imaging. These methods can provide quantitative data on distances between fluorescent molecules and exceed the resolution of optical microscopy. Single-molecule high-resolution colocalization (SHREC) can be described as one of these techniques, in which two fluorophores with different chromatic resolutions are used as the probes. This technique allows one to determine the distance between two fluorophores in a macromolecule with an accuracy of better than 10 nm. Thus, overcoming the limitations set by the Rayleigh criterion, SHREC offers rich information on the spatial structure, arrangement, and interactions within molecular complexes.</span></p> <h3><b>Super-Resolution Imaging by Photobleaching</b></h3> <p><span style="font-weight: 400;">Another great leap forward is a technique called super-resolution imaging using photobleaching. Otherwise, conventional light microscopy has certain weaknesses, which are defined by Rayleigh’s limit, and the maximal possible resolution is about 200 nM. But single-molecule imaging with photobleaching can locate a single dye molecule with a high precision of a few nanometers. This is accomplished by the method of fitting before and after photobleaching one of the dyes, where the precise differential separation between the fluorophores can be calculated. It can be used to complement FRET-based measurements and diffraction-limited microscopy, resulting in the ability to map the structures of biomolecules on the nanoscale level.</span></p> <p></div></div> <div style="background: #f7f7f7;border: 1px solid rgba(0, 0, 0, 0.07);"> <div style="padding: 30px;"><div class="Adblock-main"> <div class="Adblock-head"> <h2>Yearwise Publication Trend on <b>“<a href="https://nanotechnology.blog/publication-trends/index/single molecule imaging" target="_blank" title="single molecule imaging - yearwise publication trends">single molecule imaging</a>”</b></h2> </div> </div><div class="results-container"><div class="chart-block" style="padding:15px;"> <div class="left"> <div id="results" class="results"></div> </div> <div class="right"> <div class="chart-container"><canvas id="publicationChart"></canvas></div> </div> <div class="keywordsdiv"> <div style="text-align:center;"><b>Find publication trends on relevant topics</b> </div> <span class="gp-icon icon-tags"><svg viewBox="0 0 512 512" aria-hidden="true" xmlns="http://www.w3.org/2000/svg" width="1em" height="1em"><path d="M20 39.5c-8.836 0-16 7.163-16 16v176c0 4.243 1.686 8.313 4.687 11.314l224 224c6.248 6.248 16.378 6.248 22.626 0l176-176c6.244-6.244 6.25-16.364.013-22.615l-223.5-224A15.999 15.999 0 00196.5 39.5H20zm56 96c0-13.255 10.745-24 24-24s24 10.745 24 24-10.745 24-24 24-24-10.745-24-24z"></path><path d="M259.515 43.015c4.686-4.687 12.284-4.687 16.97 0l228 228c4.686 4.686 4.686 12.284 0 16.97l-180 180c-4.686 4.687-12.284 4.687-16.97 0-4.686-4.686-4.686-12.284 0-16.97L479.029 279.5 259.515 59.985c-4.686-4.686-4.686-12.284 0-16.97z"></path></svg></span> <span id="keyword-stats"></span> </div> </div></div></div><div class="inside-article"><style> table { margin: 0 0 1.5em; 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These nanoscale semiconductor particles with sizes of 1-10 nm have a unique photophysical property termed blinking and have applications in superresolution. As for the blinking statistics, according to the experimental results, normal blinking behavior can be described effectively by first-order kinetics, and the blinking statistics are localized with high accuracy with the help of techniques such as Independent Component Analysis (ICA). This method means the possibility of alignment of the closely spaced quantum dots and, thus, an improved understanding of the complex molecular system and their relations.</span></p> <h3><b>Nanometer-Localized Multiple Single-Molecule Fluorescence Microscopy</b></h3> <p><span style="font-weight: 400;">NALMS fluorescence microscopy is an approach that uses centroid localization and photobleaching that the eye uses to illuminate individual single molecules with diffraction-limited spots with the precision of a nanometer. Optical two-color fluorescence microscopy is very appropriate for investigating biological systems at this scale and closes the methodological gap between FRET and diffraction-limited microscopy. The high-resolution potential of NALMS microscopy has been checked using short duplex DNA strands as nanoscale rulers.</span></p> <h3><b>Photoactivatable Fluorescent Proteins</b></h3> <p><span style="font-weight: 400;">PA-FPs, photoactivatable fluorescent proteins, have been of immense importance in single-molecule imaging because they allow the activation of fluorescence. An example is the green fluorescent protein photoactivatable (GFP-Pa), which enhances the fluorescence after exposure to specific wavelengths of light. This property enables the researcher to follow up on an individual molecule at very enhanced spatial and temporal resolution. PA-FPs enable clustering and imaging of freely diffusing proteins within living cells without significantly affecting them; they are useful for studying cellular processes.</span></p> <h3><b>Single-Molecule Kinetics on DNA Origami</b></h3> <p><span style="font-weight: 400;">Thanks to the method of DNA origami to build molecular structures on the nanoscale, single-molecule kinetics and dynamic processes have been investigated with high spatial resolution. Thus, by applying binding and unbinding kinetics to DNA origami structures, researchers can reach sub-30 nm resolution with the help of PAINT imaging. The use of this technique promotes the study of the interaction of the molecules or the changes in conformation in real-time, therefore describing the function of the molecules.</span></p> <h3><b>Fluorescence Imaging with Stimulated Emission</b></h3> <p><span style="font-weight: 400;">STED microscopy is a fluorescent technique with intense applied southern capability that goes over the diffraction constraint of light by eliminating fluorescence on the circle of the focal area. This method allows resolutions of up to tens of nanometers, enabling one to see even the smallest structures of a cell. Thus, the integration of STED microscopy and endogenous protein labeling has perfect yields in vivo, which enables the investigation of the nanoscale localization of synaptic proteins and other biomolecules within living organisms.</span></p> <p></div></div> <div style="background: #f7f7f7;border: 1px solid rgba(0, 0, 0, 0.07);"> <div style="padding: 30px;"><div class="Adblock-main"> <div class="Adblock-head"> <h2>Recent Publications on <b>“<a href="https://nanotechnology.blog/recent-publications/index/single molecule imaging" target="_blank" rel="noopener" title="single molecule imaging - yearwise publication list">single molecule imaging</a>”</b></h2> </div> </div> <div class="pb-main"><div class="article-scroll"><div id="results_recent" class="results"></div></div><div class="keywordsdiv" style="margin: 0px 15px;margin-top:20px;"> <div style="text-align:center;"><b>Find publications on relevant topics</b> </div> <span class="gp-icon icon-tags"><svg viewBox="0 0 512 512" aria-hidden="true" xmlns="http://www.w3.org/2000/svg" width="1em" height="1em"><path d="M20 39.5c-8.836 0-16 7.163-16 16v176c0 4.243 1.686 8.313 4.687 11.314l224 224c6.248 6.248 16.378 6.248 22.626 0l176-176c6.244-6.244 6.25-16.364.013-22.615l-223.5-224A15.999 15.999 0 00196.5 39.5H20zm56 96c0-13.255 10.745-24 24-24s24 10.745 24 24-10.745 24-24 24-24-10.745-24-24z"></path><path d="M259.515 43.015c4.686-4.687 12.284-4.687 16.97 0l228 228c4.686 4.686 4.686 12.284 0 16.97l-180 180c-4.686 4.687-12.284 4.687-16.97 0-4.686-4.686-4.686-12.284 0-16.97L479.029 279.5 259.515 59.985c-4.686-4.686-4.686-12.284 0-16.97z"></path></svg></span> <span id="keyword-papers"></span> </div></div></div><div class="inside-article"> <style> .pb-main{ border: solid 1px #ccc; border-top: none; margin-bottom: 20px; padding-bottom: 25px; background:#fff; } .author-main { border: solid 1px #ccc; border-top: none; margin-bottom: 20px; padding-bottom: 25px; background:#fff; } .publication-block { padding: 10px; margin-bottom: 10px; background-color: #f9f9f9; text-align: left; background: #FFF; border-bottom: solid 1px #ccc; margin-left: 15px; margin-right: 15px; } .publication-block h3 { margin: 0 0 10px; color: #000!important; } .publication-block a { font-size: 16px !important; line-height: 1em; font-weight: 600; text-transform: none; color: #000; padding: 0px; } .publication-block a:hover{ color: #227cdc; text-decoration:underline; } .article-scroll { max-height: 445px; overflow-y: auto; overflow-x: hidden; } ::-webkit-scrollbar-track { -webkit-box-shadow: inset 0 0 6px rgba(0,0,0,0.3); background-color: #efefef; border-radius:30px; } ::-webkit-scrollbar { width: 6px; background-color: #efefef; border-radius:30px; } ::-webkit-scrollbar-thumb { background-color: #ababab; 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publicationBlock.innerHTML = publicationHTML; resultsContainer.appendChild(publicationBlock); }); } function displayKeywordPapers(keywords) { var resultsContainer = document.getElementById('keyword-papers'); resultsContainer.innerHTML = ''; if (!keywords || keywords.length === 0) { resultsContainer.innerHTML = '<p>No data found.</p>'; return; } var keywordHTML = ''; keywords.forEach((key, index) => { let key_replace = key.replace(/ /g, '-'); key_replace = key_replace.toLowerCase(); keywordHTML += `<a href="https://nanotechnology.blog/recent-publications/index/${key_replace}" target="_blank" title="${key} - publication list">${key}</a>`; if (index < keywords.length - 1) { keywordHTML += ', '; } }); resultsContainer.innerHTML = keywordHTML; } // Call the function with the PHP data var recent_papers = [ { "title": "Machine Learning for Single-Molecule Localization Microscopy: From Data Analysis to Quantification.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/38946062", "publishedDate": "2024" }, { "title": "Protocol for matching protein localization to synapse morphology in primary rat neurons by correlative super-resolution microscopy.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/38943646", "publishedDate": "2024" }, { "title": "Highly inclined light sheet allows volumetric super-resolution imaging of efflux pumps distribution in bacterial biofilms.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/38839922", "publishedDate": "2024" }, { "title": "Ratiometric fluorescence nanoscopy and lifetime imaging of novel Nile Red analogs for analysis of membrane packing in living cells.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/38877068", "publishedDate": "2024" }, { "title": "In situ reprogramming of cardiac fibroblasts into cardiomyocytes in mouse heart with chemicals.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/38890526", "publishedDate": "2024" }, { "title": "The condensation of HP1\u03b1\/Swi6 imparts nuclear stiffness.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/38900638", "publishedDate": "2024" }, { "title": "Single-Molecule RNA Imaging in Live Cells with an Avidity-Based Fluorescent Light-Up Aptamer biRhoBAST.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/38907914", "publishedDate": "2024" }, { "title": "An extracellular vesicle microRNA-initiated 3D DNAzyme motor for colorectal cancer diagnosis.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/38910520", "publishedDate": "2024" }, { "title": "When Force Met Fluorescence: Single-Molecule Manipulation and Visualization of Protein-DNA Interactions.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/38237015", "publishedDate": "2024" }, { "title": "Super-resolution Multispectral Imaging Nanoimmunosensor for Simultaneous Detection of Diverse Early Cancer Biomarkers.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/38960915", "publishedDate": "2024" }, { "title": "Functional role of carbohydrate-binding modules in multi-modular chitinase OfChtII.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/39098522", "publishedDate": "2024" }, { "title": "Single-Molecule Localization Microscopy and Tracking with a Fluorescent Mechanosensitive Probe.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/39119910", "publishedDate": "2024" }, { "title": "Ultrasensitive Detection of Cancer Biomarkers Using Photonic-Crystal-Enhanced Single-Molecule Imaging.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/39120618", "publishedDate": "2024" }, { "title": "Nanoscale single-vesicle analysis: High-throughput approaches through AI-enhanced super-resolution image analysis.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/39106689", "publishedDate": "2024" }, { "title": "The DNA-PAINT palette: a comprehensive performance analysis of fluorescent dyes.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/39112798", "publishedDate": "2024" }, { "title": "Single molecule visualization of tropomyosin isoform organization in the mammalian actin cytoskeleton.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/38872463", "publishedDate": "2024" }, { "title": "Relationship of Dose and Signal Enhancement Properties of Gadoquatrane, a New Tetrameric, Macrocyclic Gadolinium-Based Contrast Agent, Compared With Gadobutrol: A Randomized Crossover Study in Healthy Adults.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/38904771", "publishedDate": "2024" }, { "title": "Cell-Impermeable Buffering Fluorogenic Probes for Live-Cell Super-Resolution Imaging of Plasma Membrane Morphology Dynamics.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/38859630", "publishedDate": "2024" }, { "title": "Imaging the Architecture of Granulomas Induced by Mycobacterium tuberculosis Infection with Single-molecule Fluorescence In Situ Hybridization.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/38912840", "publishedDate": "2024" }, { "title": "Single-molecule characterization of salivary protein aggregates from Parkinson\\\\\\'s disease patients: a pilot study.", "url": "https:\/\/pubmed.ncbi.nlm.nih.gov\/38863577", "publishedDate": "2024" } ]; var keywordsArray = ["Single-molecule imaging","High-resolution colocalization","Photobleaching","Quantum dots","Nanometer-localized microscopy","Photoactivatable fluorescent proteins","DNA origami","STED microscopy","MINFLUX nanoscopy","Localization microscopy","Heavy water","Live-cell imaging"]; displayResults_recent(recent_papers); displayKeywordPapers(keywordsArray); // function stripslashes(str) { // if (typeof str === 'string') { // return str.replace(/\/g, ''); // } // } </script></p> <h3><b>MINFLUX Nanoscopy</b></h3> <p><span style="font-weight: 400;">MINFLUX is a new approach to single-molecule localization nanoscopy for minimal photon fluxes. This technique localizes photon emitters with minimum excitation light intensity probes that allow localization precision with a fraction of the fluorescence photons. MINFLUX reaches down to a single-digit nanometer precision, also in the sub-millisecond timescale, with high enough sensitivity to detect molecular encounters and conformational changes. This advancement gives a fresh outlook on the dynamics of single molecules in living cells.</span></p> <h3><b>Pointillism and Localization Microscopy</b></h3> <p><span style="font-weight: 400;">Super-resolution microscopy techniques like STORM or PALM work by obtaining a high-resolution image of the specimen through the localization of many sparse subsets of single photoactivatable fluorescent protein molecules. Such methods ensure the ability to have nanometer spatial resolution in that the position information of all subsets is incorporated in the super-resolution image. This technology has been employed to image specific target proteins in different cellular structures and organelles, thereby depicting the precise dispositions of molecules and their interactions.</span></p> <h3><b>Heavy Water to Enhance Fluorescent Protein Brightness</b></h3> <p><span style="font-weight: 400;">The New BSS has revealed that heavy water (D2O) has the potential to improve the brightness of photoactivatable fluorescent proteins compared to regular water (H2O). PA-FPs produce many more photons in heavy water to enhance localization accuracy in super-resolution imaging. This alteration proves to be very effective in the design and specification of fluorescent proteins and increases our chances of studying single-molecule dynamics and interactions in systems biology.</span></p> <h3><b>Applications in Live-Cell Imaging</b></h3> <p><span style="font-weight: 400;">Live-cell imaging could not have been enhanced without single-molecule imaging approaches. Visualization and tracking of particular molecules within cells allow for obtaining critical information about cellular processes and molecular effects. Strategies such as SHREK, NALMS, and MINFLUX allow people to investigate the motions, associations, and signaling processes of proteins at a microscopic level and in real time. These concepts are useful in cell biology, neuroscience, and biomedical research in general for the study of intricate biological organizations.</span></p> <h3><b>Future Directions and Challenges</b></h3> <p><span style="font-weight: 400;">However, several issues are still prevalent, even with the current advancements in single-molecule imaging methods. Optimization of the photostability of the fluorescent probes, reducing the possibility of photobleaching, and increasing the signal-to-noise ratio are topics to be discussed. Also, in parallel with single-molecule imaging, it is possible to use other techniques, for example, cryo-electron microscopy and mass spectrometry, which would give more detailed information about biological systems.</span></p> <p><span style="font-weight: 400;">The future of single-molecule imaging appears to be extremely bright, with the possibility to establish new measures of drug design, diagnostic tools, and even customized and targeted treatments for various diseases. Moreover, as technology progresses in the future, researchers will be able to expand the frontiers of molecular and cellular biology by deciphering complex living processes at the individual molecules’ level.</span></p> <h3><b>Conclusion</b></h3> <p><span style="font-weight: 400;">New proteins and tags for imaging at the single-molecule level have brought significant changes in the field of microscopy, leading to effective methods for studying biological processes. High-resolution colocalization and photobleaching methods, quantum dot blinking, and MINFLUX nanoscopy are some of the developments that have led to these possibilities for studying molecular dynamics and interactions. As these techniques are refined, they will certainly result in countless breakthroughs and improvements in the understanding of life at a molecular level.</span></p> <p></p> <h3><b>References</b></h3> <ol> <li>Churchman, L.S., Ökten, Z., Rock, R.S., Dawson, J.F. and Spudich, J.A., 2005. <a href="https://www.pnas.org/doi/abs/10.1073/pnas.0409487102">Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time.</a> <i>Proceedings of the National Academy of Sciences</i>, <i>102</i>(5), pp.1419-1423.</li> <li>Gordon, M.P., Ha, T. and Selvin, P.R., 2004. <a href="https://www.pnas.org/doi/abs/10.1073/pnas.0401638101">Single-molecule high-resolution imaging with photobleaching.</a> <i>Proceedings of the National Academy of Sciences</i>, <i>101</i>(17), pp.6462-6465.</li> <li>Lidke, K.A., Rieger, B., Jovin, T.M. and Heintzmann, R., 2005. <a href="https://opg.optica.org/oe/abstract.cfm?uri=OE-13-18-7052">Superresolution by localization of quantum dots using blinking statistics.</a> <i>Optics express</i>, <i>13</i>(18), pp.7052-7062.</li> <li>Qu, X., Wu, D., Mets, L. and Scherer, N.F., 2004. <a href="https://www.pnas.org/doi/abs/10.1073/pnas.0402155101">Nanometer-localized multiple single-molecule fluorescence microscopy.</a> <i>Proceedings of the national academy of sciences</i>, <i>101</i>(31), pp.11298-11303.</li> <li>Patterson, G.H. and Lippincott-Schwartz, J., 2002. <a href="https://www.science.org/doi/abs/10.1126/science.1074952">A photoactivatable GFP for selective photolabeling of proteins and cells.</a> <i>Science</i>, <i>297</i>(5588), pp.1873-1877.</li> <li>Jungmann, R., Steinhauer, C., Scheible, M., Kuzyk, A., Tinnefeld, P. and Simmel, F.C., 2010. <a href="https://pubs.acs.org/doi/abs/10.1021/nl103427w">Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami.</a> <i>Nano letters</i>, <i>10</i>(11), pp.4756-4761.</li> <li>Masch, J.M., Steffens, H., Fischer, J., Engelhardt, J., Hubrich, J., Keller-Findeisen, J., D’Este, E., Urban, N.T., Grant, S.G., Sahl, S.J. and Kamin, D., 2018. <a href="https://www.pnas.org/doi/abs/10.1073/pnas.1807104115">Robust nanoscopy of a synaptic protein in living mice by organic-fluorophore labeling.</a> <i>Proceedings of the National Academy of Sciences</i>, <i>115</i>(34), pp.E8047-E8056.</li> </ol> <p></div></div> <div style="background: #f7f7f7;border: 1px solid rgba(0, 0, 0, 0.07);"> <div style="padding: 30px;"><div class="Adblock-main"> <div class="Adblock-head"> <h2>Top Experts on “<b style="color:#000;font-size:22px;">single molecule imaging</b>“</h2> </div> </div><div class="author-main"><div id="results_author"></div><div style="text-align: center;"><a class="register-button" href="https://nanotechnology.blog/expert-search" target="_blank" rel="noopener">Find experts on any field</a></div></div><div class="inside-article" style="background: none;border: none;box-shadow: none;margin-top: -70px;"> <style> .author-block { padding: 15px; 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