{"id":10859,"date":"2024-08-31T07:00:29","date_gmt":"2024-08-31T11:00:29","guid":{"rendered":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/?p=10859"},"modified":"2025-02-13T06:51:59","modified_gmt":"2025-02-13T11:51:59","slug":"far-red-and-near-infrared-dyes","status":"publish","type":"post","link":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/immuno-techniques\/far-red-and-near-infrared-dyes\/","title":{"rendered":"Far-Red and Near Infrared Dyes"},"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\/10859?pdf=10859\" 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<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:22px;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>\n<style>.entry .vertical-btns {display: block;}.entry .vertical-btns .btn-sq{margin: 1rem auto;max-width:420px;}.entry .styled-list:first-of-type li{line-height:1.6rem;}<\/style>\n<p><script src=\"https:\/\/kit.fontawesome.com\/904923013f.js\" crossorigin=\"anonymous\"><\/script><\/p>\n<div class=\"entry\">\n<h2>Antibodies labeled with fluorescent dyes are essential tools for a broad range of research techniques, especially those requiring multiplexed detection. In recent years, dyes that emit in the far-red and near-infrared (NIR) regions have seen increased use due to the advantages that they offer.<\/h2>\n<p><img decoding=\"async\" loading=\"lazy\" width=\"512\" height=\"109\" class=\"aligncenter wp-image-9830 size-full\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/hero-fluoro.jpg\" style=\"width:100%;\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/hero-fluoro.jpg 512w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/hero-fluoro-300x64.jpg 300w\" sizes=\"(max-width: 512px) 100vw, 512px\" \/><\/p>\n<h4>A brief history of antibody labeling<\/h4>\n<p>The labeling of an antibody with a dye was first reported in 1934, when John Marrack coupled antibodies to tetrazotized benzidine<sup>1<\/sup>. However, because only a faint color was produced when the antibodies precipitated in solution, his method was subsequently adapted by Coons <em>et al. <\/em>to produce a fluorescent signal.<\/p>\n<p>By coupling anthracene isocyanate to anti-pneumococcal antibodies and using these to agglutinate pneumococcal bacteria in solution, Coons <em>et al. <\/em>were able to observe visible fluorescence when the sample was excited with ultraviolet light<sup>2<\/sup>. However, because autofluorescence prohibited similar results in infected tissue sections, they later switched to labeling with fluorescein isocyanate.<\/p>\n<p>Today, a vast array of fluorescent labels is available to researchers, with products spanning the violet region of the spectrum (~400 nm) through to emissions. In addition, various tandem dyes have been developed to increase flexibility for experimental design.<\/p>\n<hr>\n<h4>Principles of fluorescence<\/h4>\n<p>Fluorescence is produced when specialized molecules called fluorophores absorb photons of light within a specific range of wavelengths and emit them at another, longer range of wavelengths. During this process, the fluorophore\u2019s electrons are excited from a resting state to a higher energy state, and it is only by releasing energy that they can return to a stable condition (Figure 1). Because the energy is released as both heat and light, the resultant fluorescence has a higher wavelength than that which was used for excitation. When fluorophores are attached to antibodies, the emitted light can be used for analyte detection with techniques including flow cytometry, fluorescence microscopy, and western blot.<\/p>\n<figure style=\"max-width: 280px;\">\n        <img decoding=\"async\" loading=\"lazy\" class=\"aligncenter size-medium wp-image-8633\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/emission-and-ecitation-247x300.jpg\" alt=\"\" width=\"247\" height=\"300\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/emission-and-ecitation-247x300.jpg 247w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/emission-and-ecitation.jpg 458w\" sizes=\"(max-width: 247px) 100vw, 247px\" \/><figcaption><strong>Figure 1:<\/strong> The Jablonski diagram illustrates the change in energy states the electron passes through on releasing a photon of light. As the electron returns to its ground state, the fluorophore emits energy in the form of a photon of light at a lower energy than the excitation wavelength.<\/figcaption><\/figure>\n<hr>\n<h4>Key fluorophore properties<\/h4>\n<p>Fluorophores are characterized by several key properties. The excitation maximum and emission maximum are, respectively, the wavelengths at which light is absorbed and emitted most efficiently, while the Stokes shift is the difference between these two values. The molar extinction coefficient, which is the amount of light absorbed at a given wavelength, and the quantum yield, which is the number of photons emitted for every photon absorbed, govern fluorophore brightness.<\/p>\n<p>Often, fluorophore properties are influenced by the assay environment. For example, Propidium Iodide (PI) is only weakly fluorescent in water, but produces bright fluorescence upon intercalating with DNA due to its hydrophobic surroundings. And Acridine Orange (AO) emits green fluorescence when bound to dsDNA and red fluorescence when bound to ssDNA or RNA, owing to the differing nature of the interactions.<\/p>\n<hr>\n<h4>Reasons to consider far-red and NIR dyes<\/h4>\n<p>One of the main reasons for using far-red and NIR dyes is to avoid non-specific background signal due to autofluorescence when performing microscopy experiments. Sources of autofluorescence include common endogenous sample components such as collagen, riboflavin, and the reduced form of nicotinamide adenine dinucleotide (NADH), as well as aldehyde fixatives such as formalin and glutaraldehyde, all of which typically emit in the blue to green spectrum.<\/p>\n<p>Far-red and NIR dyes also offer a way to minimize the risk of photobleaching during live-cell imaging studies. Because far-red and NIR dyes are excited with lower energy lasers than other dyes, they are less likely to photobleach. In addition, far-red and NIR dyes are popular for imaging 3-dimensional (3D) cell cultures such as spheroids and organoids, and for imaging living organisms, due to their deeper sample penetration depth.<\/p>\n<hr>\n<h4>Tips for setting up a dye panel<\/h4>\n<p>Fluorescently labeled antibodies are widely used for multiplexed experiments, which today cover everything from simple 2 to 3 color western blots through to sophisticated 20+ parameter flow cytometry assays. Although the complexity of panel design will vary with the type of experiment being performed, the following are some general tips for fluorophore selection:<\/p>\n<ul class=\"styled-list\">\n<li>Choose dyes that are well resolved from one another to avoid spectral overlap (the spillover of fluorescent signal into other detectors than the primary detector)<\/li>\n<li>Assign bright fluorophores to analytes with low expression and dim fluorophores to more abundant targets<\/li>\n<li>Consider using far-red and NIR dyes to avoid non-specific background signal from autofluorescence or for performing live-cell imaging studies<\/li>\n<li>Think about introducing tandem dyes into your staining panel if the number of available lasers is likely to become restrictive<\/li>\n<\/ul>\n<div class=\"btn-container vertical-btns\">\n        <a class=\"btn-sq\" href=\"\/technical\/products\/conjugate-selection\/fluorophore-selection\"><i class=\"fa-solid fa-graduation-cap\"><\/i>Learn more about choosing fluorescent conjugates<\/a><a class=\"btn-sq\" href=\"\/technical\/products\/protocols\/multiple-labeling\"><i class=\"fa-solid fa-graduation-cap\"><\/i>Learn more about multiple labeling<\/a><\/div>\n<hr>\n<p><!-- Spectra viewer --><br \/>\n<!-- Spectra Viewer -->\r\n\r\n<style>\r\n#viewer {\r\n    margin-bottom: 2rem;\r\n    \r\n}\r\n\r\n#viewer #ffsv {\r\n    width: 100%;\r\n    border: none;\r\n  }\r\n\r\n#viewer #ffsv.spectra-border{\r\n    border: 1px solid #f0f2f5;\r\n}\r\n  \r\n  #viewer,\r\n  #viewer #ffsv {\r\n    -webkit-transition: height .5s linear;\r\n    transition: height .5s linear;\r\n  }\r\n  \r\n  .spectra-viewer {\r\n    height: 900px;\r\n  }\r\n  \r\n  .spectra-hidden {\r\n    height: 0;\r\n  }\r\n  \r\n  .spectra-open-container {\r\n    display: -webkit-box;\r\n    display: -ms-flexbox;\r\n    display: flex;\r\n    -webkit-box-pack: justify;\r\n        -ms-flex-pack: justify;\r\n            justify-content: space-between;\r\n    width: 100%;\r\n  }\r\n  \r\n  .spectra-open-container > span {\r\n    margin: auto;\r\n  }\r\n  \r\n  .spectra-open-container > span:hover {\r\n    color: #555555;\r\n    cursor: pointer;\r\n  }\r\n  \r\n  .spectra-open-container hr {\r\n    -ms-flex-preferred-size: 26%;\r\n        flex-basis: 26%;\r\n    height: 0px;\r\n    margin-left: 0;\r\n    margin-right: 0;\r\n    border-top-color: #000;\r\n  }\r\n  \r\n  #spectra-btn-container + #viewer iframe {\r\n    margin-top: 1rem;\r\n  }\r\n  \r\n  @media (max-width: 980px) {\r\n    .spectra-open-container hr {\r\n      -ms-flex-preferred-size: 20%;\r\n          flex-basis: 20%;\r\n    }\r\n  }\r\n  \r\n  @media (max-width: 580px) {\r\n    .spectra-open-container hr {\r\n      -ms-flex-preferred-size: 14%;\r\n          flex-basis: 14%;\r\n    }\r\n  }\r\n  \r\n  @media (max-width: 465px) {\r\n    .spectra-open-container hr {\r\n      -ms-flex-preferred-size: 11%;\r\n          flex-basis: 11%;\r\n    }\r\n  }\r\n  \r\n  @media (max-width: 430px) {\r\n    .spectra-open-container hr {\r\n      -ms-flex-preferred-size: 6%;\r\n          flex-basis: 6%;\r\n    }\r\n  }\r\n  \r\n  @media (max-width: 400px) {\r\n    .spectra-open-container hr {\r\n      -ms-flex-preferred-size: 4%;\r\n          flex-basis: 4%;\r\n    }\r\n  }\r\n  \r\n  @media (max-width: 360px) {\r\n    .spectra-open-container hr {\r\n      display: none;\r\n    }\r\n  }\r\n  \r\n  @media (max-width: 340px) {\r\n    #spectra-btn {\r\n      font-size: 13px;\r\n    }\r\n  }<\/style>\r\n\r\n<h4>Spectra Viewer<\/h4>\r\n<p>Use the spectra viewer below to compare fluorophores.<\/p>\r\n\r\n<div class=\"btn-container\">\r\n    <a class=\"btn-sq\" href=\"\/technical\/products\/conjugate-selection\/fluorophores\"><i class=\"fad fa-book-reader\"><\/i>Read Our Blog About Selecting Fluorophores Using the Spectra Viewer<\/a>\r\n<\/div>\r\n\r\n<div id=\"spectra-btn-container\">\r\n    <div class=\"spectra-open-container\">\r\n        <hr \/> \r\n        <span id=\"spectra-btn\"><i class=\"fal fa-chevron-double-down\"><\/i>&nbsp;&nbsp;<span class=\"toggle-text\">Show<\/span> Excitation and Emission Spectra&nbsp;&nbsp;<i class=\"fal fa-chevron-double-down\"><\/i><\/span>\r\n        <hr \/>\r\n    <\/div>\r\n<\/div>\r\n\r\n<div id=\"viewer\"><\/div>\r\n\r\n<script>\r\n    const spectraViewer = document.getElementById('viewer');\r\n    let openTrack = 0;\r\n    const fluoroNum = '9,132,423';\r\n    const iFrame = document.createElement('iframe');\r\n    iFrame.setAttribute('id', 'ffsv');\r\n    iFrame.setAttribute('class', 'spectra-viewer');\r\n    iFrame.setAttribute('src', `https:\/\/app.fluorofinder.com\/jackson\/spectra_viewers?wmode=transparent&cids=${fluoroNum}`);\r\n\r\n    const spectraBtn = document.getElementById('spectra-btn');\r\n    const toggleText = document.querySelector('.toggle-text');\r\n\r\n    spectraBtn.addEventListener('click', function() {\r\n        this.children[0].classList.toggle('fa-chevron-double-down');\r\n        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spectraViewer.classList.remove('spectra-hidden');\r\n            spectraViewer.classList.add('spectra-viewer');\r\n            spectraViewer.children[0].classList.remove('spectra-hidden');\r\n            spectraViewer.children[0].classList.add('spectra-viewer');\r\n            toggleText.innerText = 'Hide';\r\n            iFrame.classList.add('spectra-border');\r\n            openTrack = true;\r\n        }\r\n        \r\n    });\r\n<\/script><\/p>\n<p>Jackson ImmunoResearch specializes in producing secondary antibodies for life science applications and offers an extensive selection of fluorescent conjugates, including far-red and NIR dyes.<\/p>\n<hr>\n<h4>References<\/h4>\n<ul class=\"styled-list\">\n<li>R. Marrack. (Medical Research Council, Special Report Series No. 230.) Pp. 194. (London: H.M. Stationery Office, 1938.) 3<em>s<\/em>. net. The Chemistry of Antigens and Antibodies.&nbsp;<em>Nature<\/em><strong>142<\/strong>, 316 (1938). <a href=\"https:\/\/doi.org\/10.1038\/142316c0\">https:\/\/doi.org\/10.1038\/142316c0<\/a> <a href=\"https:\/\/www.nature.com\/articles\/142316c0\">https:\/\/www.nature.com\/articles\/142316c0<\/a><\/li>\n<li>Albert H. Coons,&nbsp;Hugh J. Creech,&nbsp; Norman Jones,&nbsp;Ernst Berliner; The Demonstration of Pneumococcal Antigen in Tissues by the Use of Fluorescent Antibody<sup>1<\/sup>.&nbsp;<em>J Immunol<\/em>1 November 1942; 45 (3): 159\u2013170.&nbsp;<a href=\"https:\/\/doi.org\/10.4049\/jimmunol.45.3.159\">https:\/\/doi.org\/10.4049\/jimmunol.45.3.159<\/a> <a href=\"https:\/\/journals.aai.org\/jimmunol\/article-abstract\/45\/3\/159\/63211\/\">https:\/\/journals.aai.org\/jimmunol\/article-abstract\/45\/3\/159\/63211\/<\/a><\/li>\n<\/ul>\n<a style=\"display:block;margin:1rem auto 10px;\" href=\"https:\/\/www.jacksonimmuno.com\/technical\/products\/AffiniPure-VHH-secondary-antibodies\"><img class=\"aligncenter wp-image-9850 size-full\" style=\"width: 100%;\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/VHH-affinipure-Q4-push_creatives_GIF_email_600x107_01.gif\" alt=\"\"><\/a>\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:\/\/jacksonimmuno.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:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/immuno-techniques\/western-blotting-guide\/\">Western blotting guide<\/a><\/td>\n<\/tr>\n<tr>\n<td class=\"span6\"><a class=\"row-title\" href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/technical-tips\/expansionmicroscopy\/\">An Introduction to Expansion Microscopy<\/a><\/td>\n<td class=\"span6\"><a href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/immuno-techniques\/elisa-guide\/\">ELISA guide<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/div>\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 Antibodies labeled with fluorescent dyes are essential tools for a broad range of research techniques, especially those requiring multiplexed detection. In recent years, dyes that emit in the far-red and near-infrared (NIR) regions have seen increased use due to the advantages that they offer. A brief history of antibody labeling The labeling of [&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":11,"featured_media":1515,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"content-type":""},"categories":[3,16],"tags":[],"acf":[],"_links":{"self":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/10859"}],"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\/11"}],"replies":[{"embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/comments?post=10859"}],"version-history":[{"count":13,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/10859\/revisions"}],"predecessor-version":[{"id":10926,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/10859\/revisions\/10926"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/media\/1515"}],"wp:attachment":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/media?parent=10859"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/categories?post=10859"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/tags?post=10859"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}