{"id":219185,"date":"2024-10-18T00:00:08","date_gmt":"2024-10-18T04:00:08","guid":{"rendered":"https:\/\/www.thetransmitter.org\/?p=219185"},"modified":"2024-10-18T14:47:27","modified_gmt":"2024-10-18T18:47:27","slug":"new-tissue-clearing-techniques-let-microscopes-peer-deeper-into-living-brains","status":"publish","type":"post","link":"https:\/\/www.thetransmitter.org\/methods\/new-tissue-clearing-techniques-let-microscopes-peer-deeper-into-living-brains\/","title":{"rendered":"New tissue-clearing techniques let microscopes peer deeper into living brains"},"content":{"rendered":"","protected":false},"excerpt":{"rendered":"

Washing mouse brain tissue with a blood protein or complex sugar can illuminate cells 550 micrometers into the cortex without compromising its normal physiology.<\/p>\n","protected":false},"author":14,"featured_media":219188,"comment_status":"open","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_relevanssi_hide_post":"","_relevanssi_hide_content":"","_relevanssi_pin_for_all":"","_relevanssi_pin_keywords":"","_relevanssi_unpin_keywords":"","_relevanssi_related_keywords":"","_relevanssi_related_include_ids":"","_relevanssi_related_exclude_ids":"","_relevanssi_related_no_append":"","_relevanssi_related_not_related":"","_relevanssi_related_posts":"","_relevanssi_noindex_reason":""},"categories":[152],"tags":[21,318,148,74,561],"acf":{"primary_tag":148,"doi_url":"https:\/\/doi.org\/10.53053\/EIMQ8827","custom_js_library":"","hero_type":"video","hero_alt_image":null,"hero_youtube":"","hero_video":219189,"hero_layout":"landscape","hero_caption":"Crystal clear:<\/b> Tweaks to tissue clearing, a technique that previously worked only in fixed tissue, improve the imaging depth and resolution of in vivo two-photon microscopy, shown here in the mouse hippocampus.","hero_by":"Inagaki et al<\/em>, bioRxiv 2024 ","hero_credit":"","hero_bg_color":"tan","authors":[107167],"other_authors":"","related_title":"Explore more from The Transmitter<\/em>","related_hide":false,"related_filter":"latest","related_tag":null,"related_category":null,"related_custom":{"articles":null},"recommended_title":"Recommended reading","recommended_hide":false,"recommended_filter":"latest","recommended_tag":null,"recommended_category":null,"recommended_custom":{"articles":null},"comps":[{"acf_fc_layout":"copy_comp","copy":"About 10 years ago, whenever Takeshi Imai presented on his group\u2019s tissue-clearing technique at scientific conferences, audience members asked the same question over and over: When will this work on live tissues?\r\n\r\n\u201cThat was a long-standing dream in this field, and at that time, I always answered that that would be impossible,\u201d says Imai<\/a>, professor in the graduate school of medical sciences at Kyushu University.\r\n\r\nTissues are opaque because the substances in them scatter light to varying degrees. Tissue clearing<\/a> aligns these different refractive indices by removing lipids or adding in chemicals. These processes make it easier to visualize cells marked with a fluorescent tag, but both are toxic to living cells, Imai says. And even nontoxic clearing agents have to be used at such high concentrations that they become toxic in practice. So, tissue clearing has only worked for studying cell anatomy in fixed tissue<\/a>.\r\n\r\nBut now, Imai\u2019s team and an independent group led by Ed Boyden<\/a> have each identified refractive-index-altering compounds that work in living brains, without compromising cellular health and function. As a result, they can be used in conjunction with functional imaging. Both groups posted their methods<\/a> as preprints<\/a> to bioRxiv last month.\r\n\r\n[tt_sidebar_quote author='Hans-Ulrich Dodt']To clear something in vivo and still have meaningful physiology is new.[\/tt_sidebar_quote]\r\n\r\nThe new techniques do not produce completely transparent brains, as the methods for fixed tissue do, but instead increase the signal-to-noise ratio for any added fluorescent tags and the depth of cells visible using one- and two-photon imaging. Previous efforts to improve imaging resolution focused on the microscopes and not the tissue, says Boyden, professor of neurotechnology at the Massachusetts Institute of Technology.\r\n\r\n\u201cWe were wondering, \u2018What\u2019s the most opposite thing we could do?\u2019 And we realized that, to our knowledge, nobody had made the living brain itself more transparent,\u201d Boyden says. \u201cAnd so we thought we\u2019d give it a go.\u201d\r\n\r\n[tt_text class='']I[\/tt_text]n both methods, the researchers add a compound with a high refractive index to artificial cerebrospinal fluid (aCSF) and wash the mixture across the exposed brains of anesthetized mice. This raises the refractive index of the brain tissue, which in turn reduces light scattering and increases the visibility of cells deeper in the cortex.\r\n\r\nTweaking the refractive index of tissue by adding compounds with different refractive indices is an old idea, but \u201cto clear something in vivo and still have meaningful physiology is new,\u201d says Hans-Ulrich Dodt, professor at the Vienna University of Technology, who wasn\u2019t involved in either study but co-developed one of the first tissue-clearing techniques.\r\n\r\nImai\u2019s group conducted a year-long screening process of compounds with a high molecular weight and a refractive index around 1.37 at a solute concentration in a healthy range for cells, Imai says. They settled on a solution containing bovine serum albumin, calcium and magnesium. Adding the formula, dubbed SeeDB-Live, to aCSF essentially makes a one-photon microscope as powerful as a two-photon microscope, Imai says.\r\n\r\nBoyden\u2019s group also paid attention to osmolarity and molecular weight, but wanted to find an \u201coff-the-shelf chemical,\u201d Boyden says, because \u201cwe always want our techniques to be widely usable immediately.\u201d The group identified the complex sugar dextran, which enhanced the brightness and increased the number of visible cells at a depth of up to 550 micrometers for two-photon calcium imaging.\r\n\r\nThis improvement will not be immediately helpful for researchers who study subcortical structures, such as Dayu Lin<\/a>, professor of psychiatry, neuroscience and physiology at New York University. \u201cIt is a very cool, clever and simple technique,\u201d Lin told The Transmitter<\/em> in an email. \u201cHowever, it will not make a big difference to our structures of interest,\u201d including the amygdala and hypothalamus, \u201cwhich typically sit more than 4 millimeters below the brain surface, much deeper than this method can reach (around 0.5 millimeters).\u201d\r\n\r\nBut those deeper structures might become accessible if the clearing techniques are paired with a three-photon or other more powerful microscope, says Francesco Saverio Pavone<\/a>, professor of physics at the University of Florence, who was not involved in either work. \u201cThat would be extremely cool.\u201d\r\n\r\nImai\u2019s clearing approach did not harm the health of the tissue, based on the firing activity of control versus cleared brain slices\u2014a comparison that was \u201creally tricky,\u201d Imai says, because clearing the tissue makes it \u201cvery difficult to find\u201d cells for patch clamping to assess that. And the function of neurons in the visual cortex remained intact, according to tests of the cell\u2019s responses to visual stimuli through whole-brain calcium imaging.\r\n\r\nBoyden also tested cell health, by measuring the orientation tuning of neurons in the visual cortex in awake mice. \u201cWe picked that because we thought it\u2019d be a pretty demanding validation,\u201d Boyden says. \u201cLots of stuff has to all go correctly for that to work.\u201d There was no significant difference between control mice and mice with cleared brain tissue, the team reported.\r\n\r\nDodt says he would like to see even more validation that the normal functioning of the brain tissue is not affected by clearing before the technique is used in new studies. Boyden agrees: \u201cEven with mature tools, I think people should be careful and do good controls. I think that that applies to all technologies, but definitely for a new technology.\u201d\r\n\r\n[tt_text class='']B[\/tt_text]oth preprints followed an established tissue-clearing strategy, but a highly publicized paper published last month in Science<\/em>\u2014which used the dye from Doritos tortilla chips\u2014took an entirely new approach, says Hiroki Ueda<\/a>, team leader at the RIKEN Center for\u00a0Biosystems Dynamics Research, who was not involved in the study.\r\n\r\nThat team screened for compounds based on the frequencies of light they absorb, rather than their refractive indices. It\u2019s \u201ccounterintuitive,\u201d says study investigator Zihao Ou<\/a>, assistant professor of physics and materials science at the University of Texas at Dallas, but absorbing light at a particular frequency reduces scattering and increases transparency at other frequencies. Tissue clearing has generally relied on the same \u201ccocktail of chemicals,\u201d and disregarded those that absorb light, Ou says, \u201cbut now we are providing more ingredients, so potentially we can provide easier and more effective options.\u201d\r\n\r\nOu and his colleagues identified tartrazine, the yellow food dye in Doritos, as a potential clearing agent based on its absorption profile. When they applied it to the belly of a live mouse, the tartrazine cleared the skin and made the internal organs visible.\r\n\r\nThose results are not particularly impressive or novel, Dodt says, but the approach is: Screening for substances based on their chemical and physical properties, such as absorption, is more rigorous than the typical trial-and-error used in the field\u2014and he says he hopes the paper will attract more material chemists to work on the problem.\r\n\r\nUeda says he is curious to see if this technique will work in live brain tissue\u2014maybe not using tartrazine, but with another light-absorbing compound. The study investigators did not test if the dye altered cell physiology, Imai says, and the concentration needed is likely toxic.\r\n\r\n\u201cI don\u2019t think our current chemical will work for the brain at this concentration, but there are potentially future options,\u201d Ou says. \u201cIf we are not able to make it completely transparent, even a slight improvement can be useful.\u201d"},{"acf_fc_layout":"newsletter","title":"Sign up for our weekly newsletter.","subtitle":"Catch up on what you may have missed from our recent coverage.","bg_image":200913,"groups":[{"group":"4","name":"","hide_checkbox":true}],"linktext":"","linkurl":""}]},"_links":{"self":[{"href":"https:\/\/www.thetransmitter.org\/wp-json\/wp\/v2\/posts\/219185"}],"collection":[{"href":"https:\/\/www.thetransmitter.org\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.thetransmitter.org\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.thetransmitter.org\/wp-json\/wp\/v2\/users\/14"}],"replies":[{"embeddable":true,"href":"https:\/\/www.thetransmitter.org\/wp-json\/wp\/v2\/comments?post=219185"}],"version-history":[{"count":10,"href":"https:\/\/www.thetransmitter.org\/wp-json\/wp\/v2\/posts\/219185\/revisions"}],"predecessor-version":[{"id":219231,"href":"https:\/\/www.thetransmitter.org\/wp-json\/wp\/v2\/posts\/219185\/revisions\/219231"}],"acf:post":[{"embeddable":true,"href":"https:\/\/www.thetransmitter.org\/wp-json\/wp\/v2\/contributor\/107167"}],"acf:term":[{"embeddable":true,"taxonomy":"post_tag","href":"https:\/\/www.thetransmitter.org\/wp-json\/wp\/v2\/tags\/148"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.thetransmitter.org\/wp-json\/wp\/v2\/media\/219188"}],"wp:attachment":[{"href":"https:\/\/www.thetransmitter.org\/wp-json\/wp\/v2\/media?parent=219185"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.thetransmitter.org\/wp-json\/wp\/v2\/categories?post=219185"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.thetransmitter.org\/wp-json\/wp\/v2\/tags?post=219185"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}