{"id":4879,"date":"2026-01-14T09:40:43","date_gmt":"2026-01-14T08:40:43","guid":{"rendered":"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/?p=4879"},"modified":"2026-04-14T09:23:37","modified_gmt":"2026-04-14T07:23:37","slug":"cycif-alignment","status":"publish","type":"post","link":"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/cycif-alignment\/","title":{"rendered":"Manual Spatial Proteomics: Fusing Cycles into OME-TIF was Never Easier"},"content":{"rendered":"\n<p class=\"wp-block-paragraph\"><strong>Spatial Proteomics<\/strong> was named the <a href=\"https:\/\/www.nature.com\/articles\/s41592-024-02565-3\">Nature Method of the Year 2024<\/a>. One predominant method is <strong>Cyclic Immunofluorescence<\/strong> (CycIF). Here, a tissue sample is stained and scanned during a first cycle. Next, the stain is bleached away, and the sample is restained for other antigens before being scanned again. This process can be repeated in multiple cycles. Typically, a nuclear stain like DAPI is used in each cycle to facilitate high-accuracy alignment of the cycles. Ultimately, the resulting low-plex scans &#8212; one per cycle &#8212; are aligned (aka co-registered) and fused into a single high-plex scan, e.g., in OME-TIFF format.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">This process is automated by instruments such as PhenoCycler by Akoya Biosciences, Comet by Lunaphore, MACSima by Miltenyi Biotec, and others (see free download of <a href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/spatial-biology-vendor-list\/\">List of Commercial Spatial Biology Instruments, Assays &amp; Softwares<\/a>). These instruments come with software that carries out the alignment automatically. The resulting slides can be directly opened and analyzed using MIKAIA<sup>\u00ae<\/sup>. For more, refer to our <a href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/overview-of-spatial-analysis-apps-for-mif-slides\/\">Overview of MIKAIA\u2019s Spatial Analysis Apps for Multiplexed Immunofluorescence Slides<\/a>.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">However, if no such instruments are available, the process can also be carried out manually by using a standard low-plex fluorescent scanner like the Zeiss AxioScan or the Olympus VS100. In this case, the last step of fusing the low-plexes into a single high-plex has been a technical challenge, but it is now easily possible with <strong>MIKAIA&#8217;s new Slide Align module<\/strong>, powered by the <a href=\"https:\/\/www.mevis.fraunhofer.de\/en\/portfolio\/solutions\/histokatfusion.html\">HistokatFusion technology<\/a> developed by Fraunhofer MEVIS.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Here is a step-by-step guide on how to align 16 markers scanned in three cycles.<\/p>\n\n\n\n\n\n\n<div id=\"ez-toc-container\" class=\"ez-toc-v2_0_84 counter-hierarchy ez-toc-counter ez-toc-grey ez-toc-container-direction\">\n<div class=\"ez-toc-title-container\">\n<p class=\"ez-toc-title\" style=\"cursor:inherit\">Table of Contents<\/p>\n<span class=\"ez-toc-title-toggle\"><a href=\"#\" class=\"ez-toc-pull-right ez-toc-btn ez-toc-btn-xs ez-toc-btn-default ez-toc-toggle\" aria-label=\"Toggle Table of Content\"><span class=\"ez-toc-js-icon-con\"><span class=\"\"><span class=\"eztoc-hide\" style=\"display:none;\">Toggle<\/span><span class=\"ez-toc-icon-toggle-span\"><svg style=\"fill: #999;color:#999\" xmlns=\"http:\/\/www.w3.org\/2000\/svg\" class=\"list-377408\" width=\"20px\" height=\"20px\" viewBox=\"0 0 24 24\" fill=\"none\"><path d=\"M6 6H4v2h2V6zm14 0H8v2h12V6zM4 11h2v2H4v-2zm16 0H8v2h12v-2zM4 16h2v2H4v-2zm16 0H8v2h12v-2z\" fill=\"currentColor\"><\/path><\/svg><svg style=\"fill: #999;color:#999\" class=\"arrow-unsorted-368013\" xmlns=\"http:\/\/www.w3.org\/2000\/svg\" width=\"10px\" height=\"10px\" viewBox=\"0 0 24 24\" version=\"1.2\" baseProfile=\"tiny\"><path d=\"M18.2 9.3l-6.2-6.3-6.2 6.3c-.2.2-.3.4-.3.7s.1.5.3.7c.2.2.4.3.7.3h11c.3 0 .5-.1.7-.3.2-.2.3-.5.3-.7s-.1-.5-.3-.7zM5.8 14.7l6.2 6.3 6.2-6.3c.2-.2.3-.5.3-.7s-.1-.5-.3-.7c-.2-.2-.4-.3-.7-.3h-11c-.3 0-.5.1-.7.3-.2.2-.3.5-.3.7s.1.5.3.7z\"\/><\/svg><\/span><\/span><\/span><\/a><\/span><\/div>\n<nav><ul class='ez-toc-list ez-toc-list-level-1 ' ><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-1\" href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/cycif-alignment\/#Load_slides_and_start_Slide_Align_Wizard\" >Load slides and start Slide Align Wizard<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-2\" href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/cycif-alignment\/#Structure_of_image_sources\" >Structure of image sources<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-3\" href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/cycif-alignment\/#Grouping_slides_into_cases_Composing_alignment_jobs\" >Grouping slides into cases: Composing alignment jobs<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-4\" href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/cycif-alignment\/#Output_channel_configuration_when_creating_high-plexes\" >Output channel configuration when creating high-plexes<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-5\" href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/cycif-alignment\/#Export_configuration\" >Export configuration<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-6\" href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/cycif-alignment\/#Reviewing_alignment_result_before_exporting\" >Reviewing alignment result before exporting<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-7\" href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/cycif-alignment\/#Exporting_aligned_fused_OME-TIF\" >Exporting aligned &amp; fused OME-TIF<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-8\" href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/cycif-alignment\/#Viewing_and_analyzing_generated_High-plex_OME-TIF_in_MIKAIA%C2%AE\" >Viewing and analyzing generated High-plex OME-TIF in MIKAIA\u00ae<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-9\" href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/cycif-alignment\/#MIKAIA%C2%AE_Slide_Align_is_powered_by_award-winning_science_HistokatFusion_by_Fraunhofer_MEVIS\" >MIKAIA\u00ae&nbsp;Slide Align&nbsp;is powered by award-winning science: HistokatFusion by Fraunhofer MEVIS<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-10\" href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/cycif-alignment\/#Trying_out_MIKAIA_Slide_Align\" >Trying out MIKAIA Slide Align<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-11\" href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/cycif-alignment\/#Data_source\" >Data source<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-12\" href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/cycif-alignment\/#Funding\" >Funding<\/a><\/li><\/ul><\/nav><\/div>\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"Load_slides_and_start_Slide_Align_Wizard\"><\/span>Load slides and start Slide Align Wizard <span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">Load the slides into MIKAIA<sup>\u00ae<\/sup> by either dragging them into the &#8220;Slides&#8221; workspace or by adding the enclosing folder as a workspace folder to include all contained slides. In the toolbar of the &#8220;Slides&#8221; panel, click the <strong>&#8220;Align&#8230;&#8221; button<\/strong> to start the alignment wizard:<\/p>\n\n\n\n<figure class=\"wp-block-image size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"1002\" height=\"667\" src=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Welcome-1.png\" alt=\"Screenshot of Slide Align Wizard\" class=\"wp-image-4882\" srcset=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Welcome-1.png 1002w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Welcome-1-300x200.png 300w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Welcome-1-768x511.png 768w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Welcome-1-370x246.png 370w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Welcome-1-270x180.png 270w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Welcome-1-570x379.png 570w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Welcome-1-740x493.png 740w\" sizes=\"auto, (max-width: 1002px) 100vw, 1002px\" \/><figcaption class=\"wp-element-caption\">MIKAIA<strong><sup>\u00ae<\/sup><\/strong> Slide Align Wizard &#8212; Page 1: Welcome &amp; Explanation (<a href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Welcome-1.png\" target=\"_blank\" rel=\"noreferrer noopener\">full res<\/a>)<\/figcaption><\/figure>\n\n\n\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"Structure_of_image_sources\"><\/span>Structure of image sources<span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<div class=\"wp-block-columns is-layout-flex wp-container-core-columns-is-layout-8f761849 wp-block-columns-is-layout-flex\">\n<div class=\"wp-block-column is-vertically-aligned-center is-layout-flow wp-block-column-is-layout-flow\" style=\"flex-basis:33.34%\">\n<figure class=\"wp-block-image size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"260\" height=\"175\" src=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/image-1.png\" alt=\"\" class=\"wp-image-4895\"\/><\/figure>\n<\/div>\n\n\n\n<div class=\"wp-block-column is-layout-flow wp-block-column-is-layout-flow\" style=\"flex-basis:33.34%\">\n<p class=\"wp-block-paragraph\"><\/p>\n\n\n\n<figure class=\"wp-block-image\"><img loading=\"lazy\" decoding=\"async\" width=\"308\" height=\"109\" src=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/image.png\" alt=\"\" class=\"wp-image-4894\" srcset=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/image.png 308w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/image-300x106.png 300w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/image-270x96.png 270w\" sizes=\"auto, (max-width: 308px) 100vw, 308px\" \/><\/figure>\n\n\n\n<p class=\"wp-block-paragraph\">*.multifile.csv companion file &#8220;ties&#8221; multiple single-channel files together. Contents of the *.multifile.csv file: Path, Name, Color<\/p>\n<\/div>\n\n\n\n<div class=\"wp-block-column is-vertically-aligned-center is-layout-flow wp-block-column-is-layout-flow\" style=\"flex-basis:33.33%\">\n<p class=\"wp-block-paragraph\">Each cycle input scan is comprised of one *.ome.tif file per channel. They are tied together by a *.multifile.csv companion file, which MIKAIA<sup>\u00ae<\/sup> treats like a single multi-channel scan.<\/p>\n<\/div>\n<\/div>\n\n\n\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"Grouping_slides_into_cases_Composing_alignment_jobs\"><\/span>Grouping slides into cases: Composing alignment jobs<span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">Each cycle is represented as a single multi-channel file in the job compositor page, which is designed to group files into cases. Here, all three files belong to the same case. Since the case name is not coded into the file names, the easiest approach is to click the &#8220;All to same case&#8221; button and enter a name (e.g., &#8220;case A&#8221;). If the case name is included in the file path, it can be automatically extracted by entering delimiters that inform how to extract the Case ID (and, in the case of brightfield scans, also the stain name), from the source paths.<\/p>\n\n\n\n<figure class=\"wp-block-image size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"1002\" height=\"667\" src=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Job-Compositor-2.png\" alt=\"Screenshot of Slide Align Wizard\" class=\"wp-image-4885\" srcset=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Job-Compositor-2.png 1002w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Job-Compositor-2-300x200.png 300w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Job-Compositor-2-768x511.png 768w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Job-Compositor-2-370x246.png 370w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Job-Compositor-2-270x180.png 270w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Job-Compositor-2-570x379.png 570w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Job-Compositor-2-740x493.png 740w\" sizes=\"auto, (max-width: 1002px) 100vw, 1002px\" \/><figcaption class=\"wp-element-caption\">MIKAIA<strong><sup>\u00ae<\/sup><\/strong> Slide Align Wizard &#8212; Page 2: Composition into cases (filled) (<a href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Job-Compositor-2.png\" target=\"_blank\" rel=\"noreferrer noopener\">full res<\/a>)<\/figcaption><\/figure>\n\n\n\n<p class=\"wp-block-paragraph\">The three rows need to be <strong>included<\/strong> by checking the box in the first column (in case you have further slides in the workspace that are not part of any alignment job). You also need to select a <strong>reference<\/strong> cycle needs. The DAPI channel is automatically pre-selected as the alignment channel. Optionally, you can select multiple channels. The resulting transform and deformation field will then be applied to the remaining channels accordingly.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Click the &#8220;Proceed&#8221; button to move to the next wizard step.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"Output_channel_configuration_when_creating_high-plexes\"><\/span>Output channel configuration when creating high-plexes<span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">It is recognized that at least one case contains fluorescence data, which brings up the &#8220;Channel Setup&#8221; page. (When aligning brightfield sections, this page is skipped.) On this page, you have the option to edit marker names, colors, and order. Here, we deselected all but the first cycle&#8217;s DAPI channel, ensuring that only this channel will be included in the generated output OME-TIF high-plex file.<\/p>\n\n\n\n<figure class=\"wp-block-image size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"1002\" height=\"667\" src=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-CycIF-ChannelConfig-1.png\" alt=\"Screenshot of Slide Align Wizard\" class=\"wp-image-4886\" srcset=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-CycIF-ChannelConfig-1.png 1002w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-CycIF-ChannelConfig-1-300x200.png 300w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-CycIF-ChannelConfig-1-768x511.png 768w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-CycIF-ChannelConfig-1-370x246.png 370w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-CycIF-ChannelConfig-1-270x180.png 270w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-CycIF-ChannelConfig-1-570x379.png 570w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-CycIF-ChannelConfig-1-740x493.png 740w\" sizes=\"auto, (max-width: 1002px) 100vw, 1002px\" \/><figcaption class=\"wp-element-caption\">MIKAIA<strong><sup>\u00ae<\/sup><\/strong> Slide Align Wizard &#8212; Page 3: Multiplex Fusion Channel Setup (<a href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-CycIF-ChannelConfig-1.png\" target=\"_blank\" rel=\"noreferrer noopener\">full res.<\/a>)<\/figcaption><\/figure>\n\n\n\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"Export_configuration\"><\/span>Export configuration<span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">Next comes the general configuration page, where the output folder, naming scheme, and resolution can be configured. In the case of CycIF, the &#8220;&lt;Stain&gt;&#8221; placeholder is ignored in the output naming scheme, since all channels result in the same output file.<\/p>\n\n\n\n<figure class=\"wp-block-image\"><img loading=\"lazy\" decoding=\"async\" width=\"1002\" height=\"667\" src=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Config.png\" alt=\"\" class=\"wp-image-4887\" srcset=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Config.png 1002w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Config-300x200.png 300w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Config-768x511.png 768w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Config-370x246.png 370w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Config-270x180.png 270w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Config-570x379.png 570w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Config-740x493.png 740w\" sizes=\"auto, (max-width: 1002px) 100vw, 1002px\" \/><figcaption class=\"wp-element-caption\">MIKAIA<strong>\u00ae<\/strong> Slide Align Wizard &#8212; Page 4: Export config (<a href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Config.png\" target=\"_blank\" rel=\"noreferrer noopener\">full res.<\/a>)<\/figcaption><\/figure>\n\n\n\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"Reviewing_alignment_result_before_exporting\"><\/span>Reviewing alignment result before exporting<span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<div class=\"wp-block-media-text has-media-on-the-right is-stacked-on-mobile\"><div class=\"wp-block-media-text__content\">\n<p class=\"wp-block-paragraph\">The last page shows one row per case (here only 1 case = 1 row). The alignment starts automatically, starting with the topmost case. This only takes a few seconds. <\/p>\n\n\n\n<p class=\"has-gridlove-txt-color has-text-color has-link-color has-small-font-size wp-elements-a5243ada6937b50feb6acf5ac74990cd wp-block-paragraph\">MIKAIA<strong><sup>\u00ae<\/sup><\/strong> Slide Align Wizard &#8212; Page 5: Batch alignment &amp; export (<a href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-1.png\" target=\"_blank\" rel=\"noreferrer noopener\">full resolution<\/a>)<\/p>\n<\/div><figure class=\"wp-block-media-text__media\"><img loading=\"lazy\" decoding=\"async\" width=\"1002\" height=\"667\" src=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-1.png\" alt=\"\" class=\"wp-image-4889 size-full\" srcset=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-1.png 1002w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-1-300x200.png 300w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-1-768x511.png 768w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-1-370x246.png 370w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-1-270x180.png 270w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-1-570x379.png 570w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-1-740x493.png 740w\" sizes=\"auto, (max-width: 1002px) 100vw, 1002px\" \/><\/figure><\/div>\n\n\n\n<p class=\"wp-block-paragraph\">Once the alignment has been computed, BEFORE (&#8220;original&#8221;) and AFTER (&#8220;aligned&#8221;) thumbnails are generated and can be inspected by clicking on the &#8220;Open&#8221; button with the lens-icon. This will bring up the &#8220;Inspect alignment results&#8221; dialog . On the left half, it shows the channels\/stains in carousel mode (one at a time), whereas on the right half, all images are displayed in a gallery, side by side. When hovering an image with the mouse pointer, press the SPACE key to zoom in to the maximum available preview resolution. Press SPACE again to zoom out again.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">If the automatic alignment has failed,you can use this dialog to manually place three landmarks in both the reference image and in the channel that did not auto-align. After both sets of three landmarks have been placed, click the &#8220;Re-align&#8221; button to repeat the alignment. If the alignment still does not yield satisfactory results, uncheck the &#8220;Include this stain&#8221; checkbox to skip this alignment pair during the following alignment.<\/p>\n\n\n\n<figure class=\"wp-block-image size-large\"><img loading=\"lazy\" decoding=\"async\" width=\"1024\" height=\"562\" src=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Inspector-1-1024x562.png\" alt=\"Screenshot of Slide Align Wizard\" class=\"wp-image-4892\" srcset=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Inspector-1-1024x562.png 1024w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Inspector-1-300x165.png 300w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Inspector-1-768x422.png 768w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Inspector-1-370x203.png 370w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Inspector-1-270x148.png 270w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Inspector-1-570x313.png 570w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Inspector-1-740x406.png 740w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Inspector-1.png 1202w\" sizes=\"auto, (max-width: 1024px) 100vw, 1024px\" \/><figcaption class=\"wp-element-caption\">MIKAIA<strong><sup>\u00ae<\/sup><\/strong> Slide Align Wizard &#8212; Page 5: Batch Alignment, Inspect Alignment Results Dialog (<a href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Inspector-1.png\" target=\"_blank\" rel=\"noreferrer noopener\">full res.<\/a>)<\/figcaption><\/figure>\n\n\n\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"Exporting_aligned_fused_OME-TIF\"><\/span>Exporting aligned &amp; fused OME-TIF<span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<div class=\"wp-block-media-text is-stacked-on-mobile\"><figure class=\"wp-block-media-text__media\"><img loading=\"lazy\" decoding=\"async\" width=\"1002\" height=\"667\" src=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Aligning-in-progress-1.png\" alt=\"\" class=\"wp-image-4893 size-full\" srcset=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Aligning-in-progress-1.png 1002w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Aligning-in-progress-1-300x200.png 300w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Aligning-in-progress-1-768x511.png 768w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Aligning-in-progress-1-370x246.png 370w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Aligning-in-progress-1-270x180.png 270w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Aligning-in-progress-1-570x379.png 570w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Aligning-in-progress-1-740x493.png 740w\" sizes=\"auto, (max-width: 1002px) 100vw, 1002px\" \/><\/figure><div class=\"wp-block-media-text__content\">\n<p class=\"wp-block-paragraph\">Now, press &#8220;Export all&#8221; to start exporting all cases to disk. This generates a deformed copy of each input slide (except of the reference slides). Once a job has finished, the resulting *.multifile.csv file will be created.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><\/p>\n\n\n\n<p class=\"has-gridlove-txt-color has-text-color has-link-color has-small-font-size wp-elements-ab4877926048bb787d59d700e4ddf7ae wp-block-paragraph\">MIKAIA<strong><sup>\u00ae<\/sup><\/strong> Slide Align Wizard &#8212; Page 5: Export in progress (<a href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-Page-Alignment-Aligning-in-progress-1.png\">full res.<\/a>)<\/p>\n<\/div><\/div>\n\n\n\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"Viewing_and_analyzing_generated_High-plex_OME-TIF_in_MIKAIA%C2%AE\"><\/span>Viewing and analyzing generated High-plex OME-TIF in MIKAIA<strong><sup>\u00ae<\/sup><\/strong><span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">High-plex OME-TIF generated in the previous steps can now be viewed and analyzed.<\/p>\n\n\n\n<figure class=\"wp-block-image size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"2000\" height=\"1116\" src=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-CycIF-result.png\" alt=\"Screenshot of High-plex OME-TIF in MIKAIA\" class=\"wp-image-4891\" srcset=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-CycIF-result.png 2000w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-CycIF-result-300x167.png 300w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-CycIF-result-1024x571.png 1024w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-CycIF-result-768x429.png 768w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-CycIF-result-1536x857.png 1536w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-CycIF-result-370x206.png 370w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-CycIF-result-270x151.png 270w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-CycIF-result-570x318.png 570w, https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-CycIF-result-740x413.png 740w\" sizes=\"auto, (max-width: 2000px) 100vw, 2000px\" \/><figcaption class=\"wp-element-caption\">MIKAIA<strong><sup>\u00ae<\/sup><\/strong> : High-plex OME-TIF generated in the previous steps can now be viewed and analyzed (<a href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/wp-content\/uploads\/2026\/01\/MIKAIA-Slide-Align-CycIF-result.png\" target=\"_blank\" rel=\"noreferrer noopener\">full res.<\/a>)<\/figcaption><\/figure>\n\n\n\n<p class=\"has-vivid-cyan-blue-to-vivid-purple-gradient-background has-background wp-block-paragraph\">For analysis options, please visit <a href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/overview-of-spatial-analysis-apps-for-mif-slides\/\"><strong>Overview of MIKAIA\u2019s Spatial Analysis Apps for Multiplexed  Immunofluorescence Slides<\/strong><\/a>.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"MIKAIA%C2%AE_Slide_Align_is_powered_by_award-winning_science_HistokatFusion_by_Fraunhofer_MEVIS\"><\/span><strong>MIKAIA<sup>\u00ae<\/sup>&nbsp;Slide Align<\/strong>&nbsp;is powered by award-winning science: HistokatFusion by Fraunhofer MEVIS<span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<div class=\"wp-block-columns is-layout-flex wp-container-core-columns-is-layout-8f761849 wp-block-columns-is-layout-flex\">\n<div class=\"wp-block-column is-layout-flow wp-block-column-is-layout-flow\">\n<p class=\"wp-block-paragraph\"><strong>Top precision<\/strong><\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li>1<sup>st<\/sup>&nbsp;at the ANHIR challenge 2019<\/li>\n\n\n\n<li>2<sup>nd<\/sup>&nbsp;at ACROBAT challenge 2023<\/li>\n<\/ul>\n<\/div>\n\n\n\n<div class=\"wp-block-column is-layout-flow wp-block-column-is-layout-flow\">\n<p class=\"wp-block-paragraph\"><strong>Top performance<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\">CPU &lt; 10s, GPU &lt; 1s for accurate alignment of two consecutive sections<\/p>\n<\/div>\n<\/div>\n\n\n\n<p class=\"wp-block-paragraph\" style=\"font-size:16px\"><strong>Fore more details, <\/strong>visit the&nbsp;<strong><a href=\"https:\/\/www.mevis.fraunhofer.de\/en\/portfolio\/solutions\/histokatfusion.html\">Fraunhofer MEVIS&#8217;s HistokatFusion website<\/a>.<\/strong><\/p>\n\n\n\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"Trying_out_MIKAIA_Slide_Align\"><\/span>Trying out MIKAIA Slide Align<span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">In the free MIKAIA<sup>\u00ae<\/sup> lite edition, the Slide Align can be tried out, but the maximum output resolution is limited to 5 microns\/px. Get in touch (mikaia@iis.fraunhofer.de) if you want to try out an eval version of MIKAIA studio, incl. Slide Align export at full resolution.<\/p>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"Data_source\"><\/span>Data source<span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">The scans are kindly provided by <a href=\"https:\/\/www.dakewemedical.com\/digital-pathology.html\"><strong>DAKEWE Medical<\/strong><\/a> with permission to use them for this article.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"Funding\"><\/span>Funding<span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">This MIKAIA<sup>\u00ae<\/sup>&nbsp;extension has been kindly made possible thanks to venture capital provided by the&nbsp;<strong><a href=\"https:\/\/www.fraunhofer-zukunftsstiftung.de\/\">Fraunhofer Future Foundation&nbsp;<\/a><\/strong>(Fraunhofer Zukunfsstiftung). Project: &#8220;Histology AI Author&#8221;, consortium: Fraunhofer IIS &amp; Fraunhofer MEVIS<\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><\/p>\n","protected":false},"excerpt":{"rendered":"<p>Spatial Proteomics was named the Nature Method of the Year 2024. One predominant method is Cyclic Immunofluorescence (CycIF). Here, a tissue sample is stained and scanned during a first cycle. Next, the stain is bleached away, and the sample is restained for other antigens before being scanned again. This process can be repeated in multiple [&hellip;]<\/p>\n","protected":false},"author":2,"featured_media":4892,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[35,28,24],"tags":[7,29,111],"coauthors":[56],"class_list":["post-4879","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-life-science","category-mikaia-university","category-spatial-biology","tag-mikaia","tag-mikaia-app-note","tag-workflow"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v27.7 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Manual Spatial Proteomics: Fusing Cycles into OME-TIF was Never Easier<\/title>\n<meta name=\"description\" content=\"Manual cyclic spatial proteomics with your own low-plex fluorescence scanner is now easier than never before: use MIKAIA&#039;s new Slide Align module to align and fuse cycles into a high-plex OME-TIFF.\" \/>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/cycif-alignment\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Manual Spatial Proteomics: Fusing Cycles into OME-TIF was Never Easier\" \/>\n<meta property=\"og:description\" content=\"Manual cyclic spatial proteomics with your own low-plex fluorescence scanner is now easier than never before: use MIKAIA&#039;s new Slide Align module to align and fuse cycles into a high-plex OME-TIFF.\" \/>\n<meta property=\"og:url\" content=\"https:\/\/websites.fraunhofer.de\/smart-sensing-insights\/cycif-alignment\/\" \/>\n<meta property=\"og:site_name\" content=\"SMART SENSING insights\" \/>\n<meta property=\"article:publisher\" content=\"https:\/\/www.facebook.com\/FraunhoferIIS\" 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