{"id":1181,"date":"2016-09-06T12:47:57","date_gmt":"2016-09-06T12:47:57","guid":{"rendered":"http:\/\/blogs.cardiff.ac.uk\/bioimaging\/?p=1181"},"modified":"2022-04-13T16:27:04","modified_gmt":"2022-04-13T16:27:04","slug":"in-focus-better-to-burn-bright-than-to-fade-away-reducing-photo-bleaching-in-fluorescence-microscopy","status":"publish","type":"post","link":"https:\/\/blogs.cardiff.ac.uk\/bioimaging\/in-focus-better-to-burn-bright-than-to-fade-away-reducing-photo-bleaching-in-fluorescence-microscopy\/","title":{"rendered":"IN-FOCUS: Better To Burn Bright Than To Fade Away: Reducing Photo-bleaching in Fluorescence Microscopy."},"content":{"rendered":"<p><a href=\"http:\/\/blogs.cardiff.ac.uk\/bioimaging\/wp-content\/uploads\/sites\/492\/2016\/09\/Photobleach.jpg\"><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter wp-image-1265\" src=\"http:\/\/blogs.cardiff.ac.uk\/bioimaging\/wp-content\/uploads\/sites\/492\/2016\/09\/Photobleach-1024x509.jpg\" alt=\"[Parameter-Settings] FileVersion = 2000 Date\/Time = 0000:00:00 00:00:00 Date\/Time + ms = 0000:00:00,00:00:00:000 User Name = TCS User Width = 1032 Length = 1032 Bits per Sample = 8 Used Bits per Sample = 8 Samples per Pixel = 3 ScanMode = xy Series Name = demo2.lei\" width=\"1000\" height=\"497\" srcset=\"https:\/\/blogs.cardiff.ac.uk\/bioimaging\/wp-content\/uploads\/sites\/492\/2016\/09\/Photobleach-1024x509.jpg 1024w, https:\/\/blogs.cardiff.ac.uk\/bioimaging\/wp-content\/uploads\/sites\/492\/2016\/09\/Photobleach-300x149.jpg 300w, https:\/\/blogs.cardiff.ac.uk\/bioimaging\/wp-content\/uploads\/sites\/492\/2016\/09\/Photobleach-768x382.jpg 768w\" sizes=\"auto, (max-width: 1000px) 100vw, 1000px\" \/><\/a><\/p>\n<p><em>Above: Photo-bleaching (fading)&nbsp;occurs when a fluorochrome permanently loses the ability to fluoresce due to photon-induced chemical damage and covalent modification.&nbsp;<\/em><\/p>\n<hr>\n<p style=\"text-align: justify\">Hands up if you&#8217;ve&nbsp;spent hours preparing a sample for fluorescence microscopy only to see the&nbsp;signal disappear&nbsp;before your eyes upon excitation? Frustrating eh (unless, of course, <a href=\"http:\/\/blogs.cardiff.ac.uk\/bioimaging\/fluorescence-recovery-after-photobleaching-frap\/\">FRAP <\/a>is&nbsp;your objective)? Well here&#8217;s some simple and sound advice on how you can&nbsp;minimise photo-bleaching&nbsp;and get the best out of your samples under the&nbsp;fluorescence microscope.<\/p>\n<p style=\"text-align: justify\"><strong>1. Visualise your samples immediately after fluorescent labelling &#8211;&nbsp;this is when they&nbsp;are at their brightest.<\/strong><\/p>\n<ul>\n<li style=\"text-align: justify\">If this is not possible then loosely&nbsp;wrap your samples&nbsp;in aluminium foil and keep them in the dark at 4oC until you get the opportunity to image them.<\/li>\n<\/ul>\n<p style=\"text-align: justify\"><strong>2. Minimise their&nbsp;exposure to light in order to reduce photo-bleaching.<\/strong><\/p>\n<ul>\n<li>visualise your samples under low light conditions.<\/li>\n<li>use transmitted light to find a region of interest (ROI) and then switch to epifluorescence observation &#8211;&nbsp;avoid dwelling too long on the ROI.<\/li>\n<li>step down the intensity level of excitation&nbsp;light or insert a&nbsp;neutral density filter into the&nbsp;light path.<\/li>\n<li>set up imaging parameters on a neighbouring region and then return to the ROI for image capture.<\/li>\n<li>use image binning to reduce exposure time.<\/li>\n<li>use the microscope shutter to switch&nbsp;off the&nbsp;light source between images.<\/li>\n<li>create a photo-bleach curve from&nbsp;a timed series of images. This can be used to normalise for loss of fluorescence intensity.<\/li>\n<\/ul>\n<p><strong>3. Switch to&nbsp;a mounting medium with anti-fade protection <\/strong>e.g. <a href=\"https:\/\/vectorlabs.com\/uk\/vectashield-mounting-medium.html\">Vectashield<\/a>, <a href=\"https:\/\/www.thermofisher.com\/uk\/en\/home\/life-science\/cell-analysis\/cellular-imaging\/fluorescence-microscopy-and-immunofluorescence-if\/mounting-medium-antifades.html\">Prolong Gold\/Diamond, SlowFade Gold\/Diamond<\/a>. These work by reducing the oxygen available for photo-oxidation reactions, thus reducing photo-bleaching. N.B. Many of these&nbsp;are available with a nuclear counterstain (e.g. Dapi) included in the formulation. Alternatively, make your own anti-fade reagent (instructions below).<\/p>\n<p><strong>4. Switch to brighter, more photo-stable&nbsp;fluorochromes.<\/strong>&nbsp;First generation fluorochromes such as FITC and TRITC photo-bleach readily (and are pH sensitive) thus&nbsp;should be replaced with modern dyes such as the <a href=\"https:\/\/www.thermofisher.com\/uk\/en\/home\/brands\/molecular-probes\/key-molecular-probes-products\/alexa-fluor\/alexa-fluor-dyes-across-the-spectrum.html\">Alexa Fluor<\/a>, <a href=\"https:\/\/www.thermofisher.com\/uk\/en\/home\/life-science\/protein-biology\/protein-labeling-crosslinking\/protein-labeling\/fluorescent-protein-labeling\/dylight-fluors-technology-product-guide.html?gclid=CN7LmKDt984CFcQV0woddOsD9g&amp;s_kwcid=AL!3652!3!85589465054!b!!g!!_cat:protein%20labeling&amp;ef_id=VZvdewAAAUmczc8l:20160905085430:s\">Dylight<\/a>, or <a href=\"http:\/\/www.sigmaaldrich.com\/life-science\/cell-biology\/detection\/learning-center\/atto.html\">Atto <\/a>&nbsp;range&nbsp;of fluorochromes, which are much&nbsp;brighter and far more photo-stable.<\/p>\n<p>Good luck!<\/p>\n<p>AJH<\/p>\n<p>&nbsp;<\/p>\n<p>Further reading<\/p>\n<ul>\n<li>Wright Cell Imaging Facility: <a href=\"http:\/\/wwwfacilities.uhnresearch.ca\/wcif\/PDF\/Mountants.pdf\">Mounting Media and Anti-fade Reagents<\/a><\/li>\n<li><a href=\"http:\/\/wwwuser.gwdg.de\/~uboehm\/images\/39.pdf\">Photobleaching: a review<\/a><\/li>\n<\/ul>\n","protected":false},"excerpt":{"rendered":"<p>Above: Photo-bleaching (fading)&nbsp;occurs when a fluorochrome permanently loses the ability to fluoresce due to photon-induced chemical damage and covalent modification.&nbsp; Hands up if you&#8217;ve&nbsp;spent hours preparing a sample for fluorescence microscopy only to see the&nbsp;signal disappear&nbsp;before your eyes upon excitation? Frustrating eh (unless, of course, FRAP is&nbsp;your objective)? Well here&#8217;s some simple and sound advice [&hellip;]<\/p>\n","protected":false},"author":908,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[15,10],"tags":[],"class_list":["post-1181","post","type-post","status-publish","format-standard","hentry","category-in-focus","category-techniques"],"meta_box":[],"_links":{"self":[{"href":"https:\/\/blogs.cardiff.ac.uk\/bioimaging\/wp-json\/wp\/v2\/posts\/1181","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/blogs.cardiff.ac.uk\/bioimaging\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/blogs.cardiff.ac.uk\/bioimaging\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/blogs.cardiff.ac.uk\/bioimaging\/wp-json\/wp\/v2\/users\/908"}],"replies":[{"embeddable":true,"href":"https:\/\/blogs.cardiff.ac.uk\/bioimaging\/wp-json\/wp\/v2\/comments?post=1181"}],"version-history":[{"count":27,"href":"https:\/\/blogs.cardiff.ac.uk\/bioimaging\/wp-json\/wp\/v2\/posts\/1181\/revisions"}],"predecessor-version":[{"id":2028,"href":"https:\/\/blogs.cardiff.ac.uk\/bioimaging\/wp-json\/wp\/v2\/posts\/1181\/revisions\/2028"}],"wp:attachment":[{"href":"https:\/\/blogs.cardiff.ac.uk\/bioimaging\/wp-json\/wp\/v2\/media?parent=1181"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/blogs.cardiff.ac.uk\/bioimaging\/wp-json\/wp\/v2\/categories?post=1181"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/blogs.cardiff.ac.uk\/bioimaging\/wp-json\/wp\/v2\/tags?post=1181"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}