{"id":3414,"date":"2021-09-27T09:48:21","date_gmt":"2021-09-27T09:48:21","guid":{"rendered":"https:\/\/www.embl.org\/about\/info\/imaging-centre\/?page_id=3414"},"modified":"2022-11-27T22:06:45","modified_gmt":"2022-11-27T22:06:45","slug":"single-molecule-localisation-microscopy-smlm","status":"publish","type":"page","link":"https:\/\/www.embl.org\/about\/info\/imaging-centre\/single-molecule-localisation-microscopy-smlm\/","title":{"rendered":"Single Molecule Localisation Microscopy (SMLM)"},"content":{"rendered":"\n<div class=\"vf-grid | vf-grid__col-3\"><div class=\"vf-grid__col--span-2\"><!--[vf\/content]-->\n<div class=\"vf-content\">\n\n<p>If you want to know how SMLM works in comparison to other super-resolution technologies we offer &#8211; read more <a href=\"https:\/\/www.embl.org\/about\/info\/imaging-centre\/super-resolution-microscopy\/\">here<\/a>.<\/p>\n\n<\/div>\n<\/div>\n\n\n<div class=\"\"><!--[vf\/content]-->\n<div class=\"vf-content\">\n\n<\/div>\n<\/div>\n<\/div>\n\n\n\n<div class=\"vf-grid | vf-grid__col-1\"><div class=\"\"><!--[vf\/content]-->\n<div class=\"vf-content\">\n\n<div class=\"vf-tabs\"><ul class=\"vf-tabs__list\" data-vf-js-tabs=\"true\"><li class=\"vf-tabs__item\"><a class=\"vf-tabs__link\" href=\"#vf-tabs__section-97af3868-1fc7-4270-b4b8-e45d2014bdd4\" data-vf-js-location-nearest-activation-target=\"\">3D-SMLM<\/a><\/li><\/ul><div class=\"vf-tabs-content\" data-vf-js-tabs-content=\"true\">\n<section class=\"vf-tabs__section\" id=\"vf-tabs__section-97af3868-1fc7-4270-b4b8-e45d2014bdd4\"><h2>3D-SMLM<\/h2>\n<div class=\"vf-grid | vf-grid__col-3\"><div class=\"vf-grid__col--span-2\"><!--[vf\/content]-->\n<div class=\"vf-content\">\n\n<p>Three-dimensional single-molecule localisation microscopy (3D-SMLM) is an advanced light microscopy methodology based on localisation of sparse single-molecule emitters for computational reconstruction of a super-resolved image. The 3D-SMLM at the EMBL IC has been developed in collaboration with the lab of\u00a0<a href=\"https:\/\/www.embl.org\/groups\/ries\/\" target=\"_blank\" rel=\"noreferrer noopener\">Jonas Ries<\/a>\u00a0at EMBL. At the core of the microscope is an extremely stable inverted microscope. (f)PALM\/(d)STORM workflows can be accommodated and additional functionalities will be added dependent on user need (e.g. support for live-cell imaging\/PAINT\/SOFI and others).<\/p>\n\n\n\n<hr class=\"vf-divider\">\n\n\n\n<p><strong>Features<\/strong><\/p>\n\n\n\n<ul class=\"wp-block-list\"><li>Ratiometric multi-colour imaging (e.g. simultaneous multi-colour imaging of spectrally overlapping fluorophores)<\/li><li>Homogenised Epi-\/HILO\/TIRF illumination with variable illuminated field ca. \u230020 &#8211; 70 microns, Super-resolution over large field of views (up to \u230070 microns)<\/li><li>Option for 3D imaging via astigmatism<\/li><li>&lt; 10 nm localisation precision (in x,y, dependent on fluorophore, imaging mode, &lt; 20 nm in z)<\/li><li>Automated high-throughput imaging (e.g. multiple field of views)<\/li><\/ul>\n\n\n\n<p><strong>Specifications<\/strong><\/p>\n\n\n\n<ul class=\"wp-block-list\"><li>4 lasers generating ca. 100 mW at source (Toptica iChrome MLE, 405, 488, 561, 640 nm), additional booster laser at 640 nm (Toptica iBeam Smart 200 mW).<\/li><li>Back-thinned sCMOS camera (Hamamatsu Orca Fusion BT &gt;95% QE, &lt;0.7 e- read noise)<\/li><li>Focus locking via reflection of NIR laser (Toptica iBeam Smart, 785 nm)<\/li><li>Motorized XY nanopositioning stage (SmarAct), Piezo actuator (PI PIFOC, P-726) for objective.<\/li><li>Olympus 100x\/1.45 (oil), 100x\/1.35 (silicone oil) objective lenses<\/li><li>The microscope can be configured for the needs of the experiment via a range of switchable optical elements. 3D imaging is achieved using the astigmatic method and analysis\/reconstruction is achieved using the SMAP toolset<\/li><\/ul>\n\n<\/div>\n<\/div>\n\n\n<div class=\"\"><!--[vf\/content]-->\n<div class=\"vf-content\">\n\n<figure class=\"vf-figure wp-block-image size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"1005\" height=\"607\" class=\"vf-figure__image\" src=\"https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-content\/uploads\/2022\/11\/3D-SMLM-B.jpg\" alt=\"Credit: Merle Hantsche-Grininger\/EMBL\" class=\"wp-image-15062\" srcset=\"https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-content\/uploads\/2022\/11\/3D-SMLM-B.jpg 1005w, https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-content\/uploads\/2022\/11\/3D-SMLM-B-300x181.jpg 300w, https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-content\/uploads\/2022\/11\/3D-SMLM-B-768x464.jpg 768w\" sizes=\"auto, (max-width: 1005px) 100vw, 1005px\" \/><figcaption class=\"vf-figure__caption\"><meta charset=\"utf-8\">3D-SMLM images of U2OS Nup96-mEGFP cells stained with Q nanobody AF647 (orange) and WGA-CF680 (blue). Left: Zoom in on several nuclear pores. Top right: an XY view of a single nuclear pore showing the ring structure and separation of the two dyes. Bottom right: an XZ view of the same nuclear pore, showing<br>that the two stacked rings can be resolved. Scale bars, 100 nm. Credit: Merle Hantsche-Grininger\/EMBL.<\/figcaption><\/figure>\n\n\n\n<figure class=\"vf-figure wp-block-image size-large is-style-default\"><a href=\"https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-content\/uploads\/2021\/12\/IC_Equipment_137-HDR-Edit.jpg\"><img loading=\"lazy\" decoding=\"async\" width=\"1024\" height=\"683\" class=\"vf-figure__image\" src=\"https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-content\/uploads\/2021\/12\/IC_Equipment_137-HDR-Edit-1024x683.jpg\" alt=\"Credit: Stuart Ingham\/EMBL.\" class=\"wp-image-4434\" srcset=\"https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-content\/uploads\/2021\/12\/IC_Equipment_137-HDR-Edit-1024x683.jpg 1024w, https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-content\/uploads\/2021\/12\/IC_Equipment_137-HDR-Edit-300x200.jpg 300w, https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-content\/uploads\/2021\/12\/IC_Equipment_137-HDR-Edit-768x512.jpg 768w, https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-content\/uploads\/2021\/12\/IC_Equipment_137-HDR-Edit.jpg 1200w\" sizes=\"auto, (max-width: 1024px) 100vw, 1024px\" \/><\/a><figcaption class=\"vf-figure__caption\">3D-SMLM at IC. Credit: Stuart Ingham\/EMBL.<\/figcaption><\/figure>\n\n<\/div>\n<\/div>\n<\/div>\n<\/section>\n<\/div><\/div>\n\n<\/div>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":4,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"template-title-left-aligned.php","meta":{"_acf_changed":false,"footnotes":""},"embl_taxonomy":[],"class_list":["post-3414","page","type-page","status-publish","hentry"],"acf":[],"embl_taxonomy_terms":[],"_links":{"self":[{"href":"https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-json\/wp\/v2\/pages\/3414","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-json\/wp\/v2\/users\/4"}],"replies":[{"embeddable":true,"href":"https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-json\/wp\/v2\/comments?post=3414"}],"version-history":[{"count":14,"href":"https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-json\/wp\/v2\/pages\/3414\/revisions"}],"predecessor-version":[{"id":44817,"href":"https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-json\/wp\/v2\/pages\/3414\/revisions\/44817"}],"wp:attachment":[{"href":"https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-json\/wp\/v2\/media?parent=3414"}],"wp:term":[{"taxonomy":"embl_taxonomy","embeddable":true,"href":"https:\/\/www.embl.org\/about\/info\/imaging-centre\/wp-json\/wp\/v2\/embl_taxonomy?post=3414"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}