{"id":10337,"date":"2023-07-25T05:52:03","date_gmt":"2023-07-25T09:52:03","guid":{"rendered":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/?p=10337"},"modified":"2023-07-25T05:52:35","modified_gmt":"2023-07-25T09:52:35","slug":"super_resolution_microscopy_intro","status":"publish","type":"post","link":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/immuno-techniques\/super_resolution_microscopy_intro\/","title":{"rendered":"Super-Resolution Microscopy: Principles, Technologies, and Considerations for Reagent Selection"},"content":{"rendered":"\n\n\n\t<div class=\"dkpdf-button-container\" style=\" text-align:right \">\n\n\t\t<a class=\"dkpdf-button\" href=\"\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/10337?pdf=10337\" target=\"_blank\"><span class=\"dkpdf-button-icon\"><i class=\"fa fa-file-pdf-o\"><\/i><\/span> Download PDF<\/a>\n\n\t<\/div>\n\n\n\n\n\n<p><style>.entry p, .entry ol{font-size: 1rem;}.entry h3{color:#009fe3;margin-top:0}.entry h4{color:#009453;margin-top:1.5rem}.entry h5{color: #003a5c; font-size: 1.05rem;margin-top:1.5rem;}.entry figure{margin:32px auto;border:1px solid #ccc}.entry figure img{width:100%;display:block;margin:0 auto}.entry figcaption{font-size:.875rem;line-height:1.35rem;padding:10px;color:#222}.entry .blog-tbl{margin:1rem auto;caption-side:bottom}.entry .blog-tbl td,.entry .blog-tbl th{padding:10px 9px;border:1px solid #00172b;text-align:left}.entry .blog-tbl td{border-color:#003a5c}.entry .blog-tbl th{background-color:#00172b;color:#fff;border-left-color:#fff;border-right-color:#fff}.entry .blog-tbl th p{color:#fff}.entry .blog-tbl th:first-of-type{border-left-color:#00172b}.entry .blog-tbl th:last-of-type{border-right-color:#00172b}.entry .blog-tbl p{text-align:left;margin:0}.entry .box-note{border:2px solid #009453;padding:12px;margin:1rem 0}.entry .box-note p{margin:0;padding:0}.entry .styled-list{list-style-type:none}.entry .styled-list li{margin-top:1rem;line-height:1rem;font-size:1rem;}.entry .styled-list li::before{font-family:\"Font Awesome 5 Pro\";display:inline-block;content:\"\\f3c5\";-webkit-transform:rotate(-90deg);transform:rotate(-90deg);margin-left:-20px;margin-right:11px;font-size:.75rem;color:#ed7004;font-weight:600}.entry .styled-list ol li::before{display:none}.entry .styled-list li>ul li::before{font-weight:200}.entry .overview{width:-webkit-fit-content;width:-moz-fit-content;width:fit-content;padding:16px;margin:1rem auto;border:1px solid #eee}.entry .overview hr{margin-top:14px}.entry .overview-text{text-align:center;font-size:1rem;margin:0}.entry .btn-sq{color:#fff;font-size:.9rem;font-weight:600;border:2px solid rgb(237, 112, 4);padding:7px 13px;background:rgb(237, 112, 4);cursor:pointer;text-align:center;line-height:1.5rem;margin:0 auto;}.entry .btn-sq:hover{text-decoration:none;color:rgb(237, 112, 4);background:#fff;}.entry .btn-container{display:flex;width:100%;margin:1.3rem 0;}.entry .btn-container .fa-solid, .entry .btn-container .fa-duotone{margin-right:10px;}@media(max-width: 768px){.entry .btn-sq{font-size:1.05rem;}}<\/style> <style>.entry .styled-list li::before{display:none} .entry .styled-list li{list-style-type:decimal;line-height:24px}.entry .styled-list li::marker{font-size:75%}<\/style> <style>.entry figure {max-width:450px}<\/style><\/p>\n<p><script src=\"https:\/\/kit.fontawesome.com\/904923013f.js\" crossorigin=\"anonymous\"><\/script><\/p>\n<h2>While conventional microscopy techniques are still widely used for scientific research, super-resolution microscopy (SRM) is increasingly popular for the greater level of detail it provides. Specifically, by overcoming the optical resolution limit of established microscopy methods, super-resolution microscopy allows researchers to observe sub-cellular processes such as mitochondrial function, synaptic vesicle release, and the formation of microtubule protofilaments, and can reveal how these essential functions are impacted by aging or disease. The field of super-resolution microscopy is evolving rapidly with advances in instrumentation and the development of novel reagents such as monovalent antibody fragments and brighter, more photostable fluorescent dyes. Here, we explain the underlying principles of super-resolution microscopy and highlight some of the many different methods that have been reported within the last 30 years.<\/h2>\n<p><a href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/company-news\/webinar-nanobodies-for-super-resolution-microscopy-3\/\"><img decoding=\"async\" loading=\"lazy\" class=\"aligncenter wp-image-10345 size-full\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Abberior-JIR-Hero.jpg\" alt=\"\" width=\"700\" height=\"200\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Abberior-JIR-Hero.jpg 700w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Abberior-JIR-Hero-300x86.jpg 300w\" sizes=\"(max-width: 700px) 100vw, 700px\" \/><\/a><\/p>\n<h4>What is super-resolution microscopy?<\/h4>\n<p>Super-resolution microscopy encompasses a growing number of techniques that overcome the optical resolution limit of conventional microscopy methods. This limit is imposed by the wavelength (\u03bb) of light used to illuminate the sample and the numerical aperture (NA) of the microscope objective, and is defined by Abbe\u2019s law, which states that the lateral resolution is equal to \u03bb\/(2NA) and the axial resolution is equal to 2\u03bb\/NA<sup>2<\/sup>. In practice, this means that, with conventional microscopy, researchers can only attain a resolution of around 250 nm in the x and y directions and 470-670 nm in the z direction<sup>1<\/sup>. Super-resolution microscopy is capable of much lower resolutions, in some cases down to just a few nanometers, although the exact value will vary depending on the method being used.<\/p>\n<hr \/>\n<h4>How are SRM techniques categorized?<\/h4>\n<p>Super-resolution microscopy techniques can be broadly divided into two main categories. The first of these, known as ensemble techniques, reduce the diameter of the point spread function (PSF), which is the fixed size of the spread of a single point of light following its diffraction through a microscope, commonly defined by the Rayleigh criterion (R=0.61\u03bb\/NA). To do this, ensemble techniques restrict the production of fluorescent signals to a single focal point within the PSF by forcing any peripheral fluorophores to immediately return to a ground state following excitation<sup>1,2<\/sup>. Contemporary ensemble techniques include Stimulated Emission Depletion Microscopy (STED), Ground-State Depletion Microscopy (GSD), and Structured Illumination Microscopy (SIM), which were respectively reported to resolve 35 nm, 15 nm, and ~100 nm when they were first described in the literature<sup>3,4,5<\/sup>.<\/p>\n<figure><img decoding=\"async\" loading=\"lazy\" class=\"wp-image-10350 size-full\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/sted-1.jpg\" alt=\"\" width=\"500\" height=\"194\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/sted-1.jpg 500w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/sted-1-300x116.jpg 300w\" sizes=\"(max-width: 500px) 100vw, 500px\" \/>\n<figcaption><strong>Figure 1:<\/strong> In optical microscopy, imaging is achieved by light rays from a point source converging to a single point at the image plane. Beyond the limits of light diffraction exact convergence of the rays is prevented, leading to the image of the object blurring. The resolution of a microscope is determined by the size of the point spread function (PSF), or three-dimensional intensity distribution of the object at a point. In STED a donut-shaped depletion laser is applied with the zero point overlapping the maximum of the excitation laser focus. The STED laser causes \u201csaturated depletion\u201d of fluorescence, whereby \u201cfluorescence from regions near the zero point are suppressed, leading to a decreased size of the effective PSF\u201d. Reproduced from Huang et al. (2009).<\/figcaption>\n<\/figure>\n<p>Single-molecule localization microscopy (SMLM) techniques instead work by detecting small numbers of individual fluorophores at different points in time before combining all of the data to construct an image. They rely on the use of photoswitchable fluorophores that can be interchanged between a long-lived dark off-state and a bright on-state, and allow for 3-dimensional (3D) imaging by analyzing both the position of each fluorophore and the shape of its PSF<sup>6<\/sup>. Examples of SMLM techniques include Photo-Activated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM), as well as direct STORM (dSTORM), which improves on the original STORM method by replacing matched pairs of activator and reporter dyes with a single fluorophore<sup>7,8,9<\/sup>. Resolutions in the region of 20 nm have been reported for SMLM, although this value looks set to become lower.<\/p>\n<div class=\"btn-container\"><a class=\"btn-sq\" href=\"https:\/\/www.jacksonimmuno.com\/technical\/products\/applications\/super-resolution-microscopy\"><i class=\"fa-solid fa-graduation-cap\"><\/i>Learn more about STORM and STED<\/a><\/div>\n<hr \/>\n<h4>Fluorophore considerations for super-resolution microscopy<\/h4>\n<p>When performing ensemble techniques such as STED, GSD, and SIM, it is recommended that researchers select fluorophores which are resistant to photobleaching, have high quantum yields, and are small enough to provide a sufficient labeling density for detecting the target of interest. Dyes that are useful for these methods include Alexa Fluor\u00ae 488, FITC, and Alexa Fluor\u00ae 594, as well as the abberior STAR dyes, which are especially valuable for STED. For SMLM techniques like PALM, STORM, and dSTORM, fluorophores should again be very bright (have high quantum yields) and should generate enough photons to reliably produce tight Gaussian distributions (narrow emission spectra) and ensure high precision. Examples of dyes that can be used for SMLM include many of the Alexa Fluor\u00ae dyes (e.g., Alexa Fluor\u00ae 488, 647, and 680), as well as Cy\u2122 dyes such as Cy\u21225.<\/p>\n<div class=\"btn-container\"><a class=\"btn-sq\" href=\"https:\/\/www.jacksonimmuno.com\/technical\/products\/applications\/super-resolution-microscopy\"><i class=\"fa-solid fa-graduation-cap\"><\/i>Learn more about JIR conjugates for SRM<\/a><\/div>\n<hr \/>\n<h4>How can antibody fragments benefit super-resolution microscopy?<\/h4>\n<p>Most super-resolution microscopy techniques were developed using fluorophore-labeled antibodies. However, the large size of whole antibody molecules (10\u201315 nm) results in fluorophores being distant from the target, while antibody multivalency risks clustering, which could complicate downstream analysis<sup>10<\/sup>. Smaller, monovalent alternatives such as antibody fragments (Fabs) and single-domain (VHH) antibodies offer many advantages for high-resolution imaging, including higher density labeling and better sample penetration, and are being used for novel research applications such as live-cell STED<sup>11<\/sup>.<\/p>\n<div id=\"attachment_10253\" style=\"width: 622px\" class=\"wp-caption aligncenter\"><img aria-describedby=\"caption-attachment-10253\" decoding=\"async\" loading=\"lazy\" class=\"wp-image-10253 size-full\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/IF2Artboard-5.png\" alt=\"\" width=\"612\" height=\"397\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/IF2Artboard-5.png 612w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/IF2Artboard-5-300x195.png 300w\" sizes=\"(max-width: 612px) 100vw, 612px\" \/><p id=\"caption-attachment-10253\" class=\"wp-caption-text\">Figure 2: Single color microscopy. Indirect immunostaining was performed with JIR AffiniPure VHH\u2122 Anti-Mouse antibodies conjugated to Abberior STAR RED. Clear improvements in resolution can be seen when comparing confocal to STED microscopy.<\/p><\/div>\n<hr \/>\n<h4>Supporting super-resolution microscopy<\/h4>\n<p>Jackson ImmunoResearch specializes in producing secondary antibodies for life science applications. We offer an extensive selection of secondary antibodies that are conjugated to fluorophores, including dyes that have been literature-cited for super-resolution microscopy, as well as our range of AffiniPure-VHH\u2122 secondaries, which are now available from Abberior conjugated to their super bright STAR dyes making them an excellent choice for SRM.\u00a0<\/p>\n<p>Learn more about VHH fragment antibodies for super-resolution microscopy <a href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/company-news\/webinar-nanobodies-for-super-resolution-microscopy-3\/\">here<\/a><\/p>\n<p><a href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/company-news\/webinar-nanobodies-for-super-resolution-microscopy\/\"><img decoding=\"async\" loading=\"lazy\" class=\"aligncenter wp-image-10410 size-full\" style=\"width: 100%;\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Email-sig-Abberior-collab-3.jpg\" alt=\"\" width=\"600\" height=\"107\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Email-sig-Abberior-collab-3.jpg 600w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Email-sig-Abberior-collab-3-300x54.jpg 300w\" sizes=\"(max-width: 600px) 100vw, 600px\" \/><\/a><\/p>\n<hr \/>\n<ol class=\"styled-list\">\n<li><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/34015334\/\">Valli J, Garcia-Burgos A, Rooney LM, Vale de Melo E Oliveira B, Duncan RR, Rickman C. Seeing beyond the limit: A guide to choosing the right super-resolution microscopy technique.\u00a0<i>J Biol Chem<\/i>. 2021;297(1):100791. doi:10.1016\/j.jbc.2021.100791\/<\/a><\/li>\n<li><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/21536831\/\">Galbraith CG, Galbraith JA. Super-resolution microscopy at a glance.\u00a0<i>J Cell Sci<\/i>. 2011;124(Pt 10):1607-1611. doi:10.1242\/jcs.080085\/<\/a><\/li>\n<li><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/19844443\/\">Hell SW, Wichmann J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy.\u00a0<i>Opt Lett<\/i>. 1994;19(11):780-782. doi:10.1364\/ol.19.000780\/<\/a><\/li>\n<li><a href=\"https:\/\/link.springer.com\/article\/10.1007\/BF01081333\">Hell, S.W., Kroug, M. Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit.\u00a0<i>Appl. Phys. B<\/i>\u00a0<b>60<\/b>, 495\u2013497 (1995). https:\/\/doi.org\/10.1007\/BF01081333<\/a><\/li>\n<li><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/10810003\/\">Gustafsson MG. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.\u00a0<i>J Microsc<\/i>. 2000;198(Pt 2):82-87. doi:10.1046\/j.1365-2818.2000.00710.x<\/a><\/li>\n<li><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/35119945\/\">Liu S, Hoess P, Ries J. Super-Resolution Microscopy for Structural Cell Biology.\u00a0<i>Annu Rev Biophys<\/i>. 2022;51:301-326. doi:10.1146\/annurev-biophys-102521-112912\/<\/a><\/li>\n<li><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/16902090\/\">Betzig E, Patterson GH, Sougrat R, et al. Imaging intracellular fluorescent proteins at nanometer resolution.\u00a0<i>Science<\/i>. 2006;313(5793):1642-1645. doi:10.1126\/science.1127344<\/a><\/li>\n<li><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/23734025\/\">Bates M, Jones SA, Zhuang X. Stochastic optical reconstruction microscopy (STORM): a method for superresolution fluorescence imaging.\u00a0<i>Cold Spring Harb Protoc<\/i>. 2013;2013(6):498-520. Published 2013 Jun 1. doi:10.1101\/pdb.top075143<\/a><\/li>\n<li><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/18646237\/\">Heilemann M, van de Linde S, Sch\u00fcttpelz M, et al. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes.\u00a0<i>Angew Chem Int Ed Engl<\/i>. 2008;47(33):6172-6176. doi:10.1002\/anie.200802376<\/a><\/li>\n<li><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/34861482\/\">Hassdenteufel S, Schuldiner M. Show your true color: Mammalian cell surface staining for tracking cellular identity in multiplexing and beyond.\u00a0<i>Curr Opin Chem Biol<\/i>. 2022;66:102102. doi:10.1016\/j.cbpa.2021.102102<\/a><\/li>\n<li><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/34288299\/\">Schneider AFL, Benz LS, Lehmann M, Hackenberger CPR. Cell-Permeable Nanobodies Allow Dual-Color Super-Resolution Microscopy in Untransfected Living Cells.\u00a0<i>Angew Chem Int Ed Engl<\/i>. 2021;60(40):22075-22080. doi:10.1002\/anie.202103068<\/a><\/li>\n<li>Huang, B., Bates, M., &amp; Zhuang, X. (2009). Super-resolution fluorescence microscopy. Annual Review of Biochemistry, 78 , 993\u20131016.<\/li>\n<\/ol>\n<p><span style=\"font-weight: 400;\">\u00a0 <\/span><\/p>\n<p>\u00a0<\/p>\n<table class=\"table blogLinks\">\n<thead>\n<tr>\n<th class=\"span6\">Learn more:<\/th>\n<th class=\"span6\">Do more:<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td class=\"span6\"><a href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/immuno-techniques\/directandindirectwesternblotting\/\">Indirect and direct Western blotting<\/a><\/td>\n<td class=\"span6\"><a href=\"https:\/\/jireurope.com\/home\/exhibitions\">Exhibition Schedule<\/a><\/td>\n<\/tr>\n<tr>\n<td class=\"span6\"><a href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/immuno-techniques\/chemiluminescent-western-blotting\/\">Chemiluminescence western blotting<\/a><\/td>\n<td class=\"span6\"><a href=\"https:\/\/jireurope.com\/home\/offers\/citeandwin\">Cite and Win<\/a><\/td>\n<\/tr>\n<tr>\n<td class=\"span6\"><a class=\"row-title\" href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-admin\/post.php?post=3642&amp;action=edit\" aria-label=\"\u201cAn Introduction to Expansion Microscopy\u201d (Edit)\">An Introduction to Expansion Microscopy<\/a><\/td>\n<td class=\"span6\"><a href=\"https:\/\/jireurope.com\/home\/offers\/previous-winners\">Past Competition Winners<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n<p><\/p>\n<!-- AddThis Advanced Settings generic via filter on the_content --><!-- AddThis Share Buttons generic via filter on the_content --><!-- AddThis Related Posts generic via filter on the_content -->","protected":false},"excerpt":{"rendered":"<p>Download PDF While conventional microscopy techniques are still widely used for scientific research, super-resolution microscopy (SRM) is increasingly popular for the greater level of detail it provides. Specifically, by overcoming the optical resolution limit of established microscopy methods, super-resolution microscopy allows researchers to observe sub-cellular processes such as mitochondrial function, synaptic vesicle release, and the [&hellip;]<!-- AddThis Advanced Settings generic via filter on get_the_excerpt --><!-- AddThis Share Buttons generic via filter on get_the_excerpt --><!-- AddThis Related Posts generic via filter on get_the_excerpt --><\/p>\n","protected":false},"author":14,"featured_media":10239,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"content-type":""},"categories":[3,20],"tags":[],"acf":[],"_links":{"self":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/10337"}],"collection":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/users\/14"}],"replies":[{"embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/comments?post=10337"}],"version-history":[{"count":21,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/10337\/revisions"}],"predecessor-version":[{"id":10593,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/10337\/revisions\/10593"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/media\/10239"}],"wp:attachment":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/media?parent=10337"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/categories?post=10337"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/tags?post=10337"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}