{"id":5700,"date":"2026-06-13T12:13:20","date_gmt":"2026-06-13T16:13:20","guid":{"rendered":"https:\/\/labrigger.com\/blog\/?p=5700"},"modified":"2026-06-13T12:13:20","modified_gmt":"2026-06-13T16:13:20","slug":"the-labrigger-2p-calcium-imaging-catechism","status":"publish","type":"post","link":"http:\/\/labrigger.com\/blog\/2026\/06\/13\/the-labrigger-2p-calcium-imaging-catechism\/","title":{"rendered":"The Labrigger 2p Calcium Imaging Catechism"},"content":{"rendered":"\n<figure class=\"wp-block-image size-large\"><a href=\"https:\/\/labrigger.com\/blog\/wp-content\/uploads\/2026\/06\/image.png\"><img loading=\"lazy\" decoding=\"async\" width=\"774\" height=\"1024\" src=\"https:\/\/labrigger.com\/blog\/wp-content\/uploads\/2026\/06\/image-774x1024.png\" alt=\"\" class=\"wp-image-5701\" srcset=\"http:\/\/labrigger.com\/blog\/wp-content\/uploads\/2026\/06\/image-774x1024.png 774w, http:\/\/labrigger.com\/blog\/wp-content\/uploads\/2026\/06\/image-227x300.png 227w, http:\/\/labrigger.com\/blog\/wp-content\/uploads\/2026\/06\/image-768x1017.png 768w, http:\/\/labrigger.com\/blog\/wp-content\/uploads\/2026\/06\/image.png 1100w\" sizes=\"auto, (max-width: 774px) 100vw, 774px\" \/><\/a><\/figure>\n\n\n\n<p class=\"has-medium-font-size\"><strong><em>Step 1: Ensure that you\u2019re getting enough photons\/cell\/s<\/em><\/strong><\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li>Select an indicator.\u00a0<\/li>\n\n\n\n<li>Determine what you want to measure: Do you need to see every spike? Or is detecting bursts enough?, and what\u00a0<em><a href=\"https:\/\/doi.org\/10.1038\/nature12354\">minimum deltaF\/F<\/a><\/em>\u00a0that corresponds to for your indicator.<\/li>\n\n\n\n<li>Find the\u00a0<em><a href=\"https:\/\/doi.org\/10.1038\/nature12354\">decay time<\/a><\/em>\u00a0for your indicator.<\/li>\n\n\n\n<li>Pick <a href=\"https:\/\/doi.org\/10.1016\/j.bpj.2012.07.058\">the\u00a0<em>d-prime<\/em>\u00a0level<\/a> at which you want to detect those events.<\/li>\n<\/ul>\n\n\n\n<p><strong>Then set your imaging parameters according to the baseline\u00a0<\/strong><strong><em>F<\/em><\/strong><strong>\u00a0you need to have.<\/strong>\u00a0<\/p>\n\n\n\n<p>That will be a number of photons per second per cell. Typically on the order of 1000 to 10,000 photons\/s\/cell. You can estimate how many photons you&#8217;re getting by <a href=\"https:\/\/labrigger.com\/blog\/2010\/07\/30\/measuring-the-gain-of-your-imaging-system\/\">estimating the gain of your system<\/a>.<\/p>\n\n\n\n<p class=\"has-medium-font-size\"><strong><em>Step 2: Ensure that your photons\/cell\/s in an ROI are dominated by an individual neuron and not swamped by neuropil.<\/em><\/strong><br>\u2022 Take data and <a href=\"https:\/\/doi.org\/10.1038\/nn.4365\">compare neuron ROI traces with nearby neuropil ROI traces<\/a>.\u00a0<em>Lower correlations indicate lower neuropil contamination in the neuron ROI.<\/em><br>\u2022 Use neuropil subtraction and <a href=\"https:\/\/doi.org\/10.1038\/nn.4365\">examine neuronal correlations in the population<\/a>.<br><em>Population level average correlations are lower when the neuropil contamination is effectively removed.<\/em><\/p>\n\n\n\n<p class=\"has-medium-font-size\"><br>One can use small PSFs and\/or sparse labeling at first. These conditions can be relaxed if the data quality indicators above are not degraded.<\/p>\n\n\n\n<p class=\"has-medium-font-size\"><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/7869748\/\">Source for header image.<\/a><\/p>\n\n\n\n<figure class=\"wp-block-image size-large\"><a href=\"https:\/\/labrigger.com\/blog\/wp-content\/uploads\/2026\/06\/image-1.png\"><img loading=\"lazy\" decoding=\"async\" width=\"1024\" height=\"214\" src=\"https:\/\/labrigger.com\/blog\/wp-content\/uploads\/2026\/06\/image-1-1024x214.png\" alt=\"\" class=\"wp-image-5702\" srcset=\"http:\/\/labrigger.com\/blog\/wp-content\/uploads\/2026\/06\/image-1-1024x214.png 1024w, http:\/\/labrigger.com\/blog\/wp-content\/uploads\/2026\/06\/image-1-300x63.png 300w, http:\/\/labrigger.com\/blog\/wp-content\/uploads\/2026\/06\/image-1-768x160.png 768w, http:\/\/labrigger.com\/blog\/wp-content\/uploads\/2026\/06\/image-1.png 1380w\" sizes=\"auto, (max-width: 1024px) 100vw, 1024px\" \/><\/a><\/figure>\n\n\n\n<p class=\"has-medium-font-size\"><\/p>\n","protected":false},"excerpt":{"rendered":"<p><a href=\"https:\/\/labrigger.com\/blog\/wp-content\/uploads\/2026\/06\/image.png\"><\/a><\/p>\n<p class=\"has-medium-font-size\"><strong><em>Step 1: Ensure that you\u2019re getting enough photons\/cell\/s<\/em><\/strong><\/p>\n<p>Select an indicator.\u00a0<\/p>\n<p>Determine what you want to measure: Do you need to see every spike? Or is detecting bursts enough?, and what\u00a0<em>&#8230;<\/p>\n<div class=\"read-more\"><a href=\"http:\/\/labrigger.com\/blog\/2026\/06\/13\/the-labrigger-2p-calcium-imaging-catechism\/\">Read More<\/a><\/div>\n<p><\/em><\/p>\n","protected":false},"author":1,"featured_media":5701,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[3],"tags":[],"class_list":["post-5700","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-tips"],"_links":{"self":[{"href":"http:\/\/labrigger.com\/blog\/wp-json\/wp\/v2\/posts\/5700","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/labrigger.com\/blog\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/labrigger.com\/blog\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/labrigger.com\/blog\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/labrigger.com\/blog\/wp-json\/wp\/v2\/comments?post=5700"}],"version-history":[{"count":1,"href":"http:\/\/labrigger.com\/blog\/wp-json\/wp\/v2\/posts\/5700\/revisions"}],"predecessor-version":[{"id":5703,"href":"http:\/\/labrigger.com\/blog\/wp-json\/wp\/v2\/posts\/5700\/revisions\/5703"}],"wp:featuredmedia":[{"embeddable":true,"href":"http:\/\/labrigger.com\/blog\/wp-json\/wp\/v2\/media\/5701"}],"wp:attachment":[{"href":"http:\/\/labrigger.com\/blog\/wp-json\/wp\/v2\/media?parent=5700"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/labrigger.com\/blog\/wp-json\/wp\/v2\/categories?post=5700"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/labrigger.com\/blog\/wp-json\/wp\/v2\/tags?post=5700"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}