MedWorm provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest medical blog items that have been tagged with 'fluorescent'. Sat, 03 Sep 2011 02:38:21 +0100 http://www.medworm.com/index.php?rid=3362486&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fwww.molecularbrain.com%2Fcontent%2Fdownload%2Fsupplementary%2F1756-6606-3-5-s3.mov Atsushi Miyawaki’s lab has developed a series of neat tools for visualizing cell cycle progress. For zebrafish, the zFucci system consists of two fluorescent proteins, mKO2 and mAG, that are fused to Cdt1 and geminin genes.  Cell cycle- regulated proteolysis of these fusion proteins causes each cell to display orange fluorescence in G1 phase nuclei and green fluorescence in both the nucleus and cytoplasm of S/G2/M phase cells. Video of cell cycle transitions in culture. Click for the video. The last time I saw Atsushi give a talk, he showed an incredible time lapse video from the zebrafish cleavage stage that I haven’t been able to find online.  However, here is a video from later in development of the zebrafish that is still pretty remarkable. Development of a zebrafish visu... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=3362486 Sat, 13 Mar 2010 16:08:39 +0100 3362486 http://www.medworm.com/index.php?rid=3354459&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2010%2F03%2F10%2Fcnifers-of-acetylcholine%2F “If you find yourself needing to reread this paragraph, perhaps it’s not that well written. Or it may be that you are low on acetylcholine.” Acetylcholine (ACh) is a major modulator of brain activity in vivo and its release strongly influences attention. If we could visualize when and where ACh is released, we could more fully understand the large trial to trial variance found in many in vivo recordings of spike activity, and perhaps correlate that to attentional and behavioral states mediated by ACh transmission. Back in grad school, when I was desperately trying to figure out what biological question to answer with my GluSnFR glutamate sensor, I ended up in a meeting with Kleinfeld, his grad student Lee Schroder and Palmer Taylor. We plotted a strategy to make a FRET... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=3354459 Thu, 11 Mar 2010 05:53:24 +0100 3354459 http://www.medworm.com/index.php?rid=3201695&cid=t_140550_139_f&fid=38879&url=http%3A%2F%2Fmedia.rawvoice.com%2Fpmn_twiv%2Fwww.twiv.tv%2FTWiV067.mp3 Hosts: Vincent Racaniello, Alan Dove, and Marc Pelletier Vincent, Alan, and Marc talk about chronic wasting disease of deer caused by prions, blocking the semen-derived enhancer of HIV infection with surfen, and making green transgenic rabbits using a lentiviral vector. This episode is sponsored by Data Robotics Inc. Use the promotion code VINCENT to receive $50 off a Drobo or $100 off a Drobo S. Download TWiV #67 (64 MB .mp3, 88 minutes) Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email. Links for this episode: Chronic wasting disease in West Virginia Chronic wasting disease alliance and CDC webpage Semen-derived inhibitor of viral infection (SEVI) Surfen inhibits the action of SEVI Making green transgenic rabbits using simian immunodeficiency vir... virology blog blogs http://www.medworm.com/rss/comments.php?id=3201695 Sun, 24 Jan 2010 14:44:22 +0100 3201695 http://www.medworm.com/index.php?rid=2977425&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2009%2F11%2F09%2Fthree-cheers-for-gcamp%2F Three papers are out online in Nature Methods that show big improvements in calcium imaging with genetically encoded sensors.  They are are based on the fluorescence intensity indicator, GCaMP.   GCaMP, first developed by Junichi Nakai, consists of a GFP that has been circularly permuted so that the N and C termini are fused and new termini are made in the middle of the protein.  Fused to one terminus is calmodulin and the other is a peptide, M13, that calmodulin (CaM) binds to in the presence of calcium. The name is supposed to look like GFP with a CaM inserted into it, G-CaM-P.  Normally the GFP is dim, as there is a hole from the outside of its barrel into the chromophore.  Upon binding calcium, this hole is plugged and fluorescence increases. The first paper, A genetically encod... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=2977425 Mon, 09 Nov 2009 23:54:11 +0100 2977425 http://www.medworm.com/index.php?rid=2809765&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2009%2F09%2F18%2Foptogenetic-induction-of-memory-recall%2F Speaking of reactivating specific memories, at the 2009 Society for Neuroscience meeting, Matteo Rizzi of Michael Häusser’s lab is presenting the realization of an idea that has been floating around in some research proposals I’ve read over the last year.  Express channelrhodopsin-2 under control of the immediate early gene c-fos, induce a strong memory formation via fear conditioning, and then drive the recall of that memory by stimulating the neurons that are expressing ChR2. Immediate early genes are activated shorty after high levels activity in neurons, though the precise patterns are different depending on which promoter (c-fos, Zif268, etc) you use, making precisely HOW they reflect recent neuronal activity patterns unclear.  Nevertheless, the activation of the c-fos ... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=2809765 Fri, 18 Sep 2009 19:40:52 +0100 2809765 http://www.medworm.com/index.php?rid=2800550&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2009%2F09%2F16%2Flight-switchable-protein-interactions%2F A fully genetically-encoded approach to light-activated transcription is getting closer now that a new, generalizable method of light-switchable protein interactions has been published.  In Nature’s advance online publication, Spatiotemporal control of cell signalling using a light-switchable protein interaction, Anselm Levskaya of the Voigt lab at UCSF and co-authors demonstrate inducible, reversible control of protein binding, localization and signalling in mammalian cells.   apo-PhyB covalently binds to the chromophore phycocyanobilin (PCB) to form a light-sensitive holoprotein. PhyB undergoes conformational changes between the Pr and Pfr states catalysed by red and infrared light, reversibly associating with the PIF domain only in the Pfr state. This heterodimerization interac... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=2800550 Wed, 16 Sep 2009 16:06:58 +0100 2800550 http://www.medworm.com/index.php?rid=2747913&cid=t_140550_87_f&fid=36438&url=http%3A%2F%2Ffeedproxy.google.com%2F%7Er%2FCato-at-liberty%2F%7E3%2FnB9H7_RbfUM%2F Unfortunately, there are many good (and sad) examples of Uncle Sam’s insatiable desire to regulate the smallest aspects of our lives.  Legislators can’t even let us decide which light bulbs to buy.  Government believes that it knows best, and is banning the venerable incandescent bulb. Lighting consultant Howard Brandston makes a plaintive plea for lighting that serves people rather than politics: The Energy Independence and Security Act of 2007 will effectively phase out incandescent light bulbs by 2012-2014 in favor of compact fluorescent lamps, or CFLs. Other countries around the world have passed similar legislation to ban most incandescents. Will some energy be saved? Probably. The problem is this benefit will be more than offset by rampant dissatisfaction with lighting.... Cato-at-liberty blogs http://www.medworm.com/rss/comments.php?id=2747913 Mon, 31 Aug 2009 12:53:17 +0100 2747913 http://www.medworm.com/index.php?rid=2645408&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2009%2F07%2F27%2Fvoltage-imaging-with-sub-millisecond-single-action-potential-resolution%2F I have been itching to post about this work since David DiGregorio presented it at a meeting at Janelia last year. His group’s results, Submillisecond Optical Reporting of Membrane Potentials In Situ Using a Neuronal Trace Dye, were published in the Journal of Neuroscience last week.  Their method of optical voltage sensing is the first one that looks like its ready for “prime-time” action outside of the labs of developers of these sorts of techniques.  It has sufficient speed (<1 ms resolution), sensitivity (25% dF/F per 100mV), and limited membrane perturbation to see single action potentials, without dramatically altering the shape of these currents.     Membrane depolarization causes DPA to rapidly partition to the inner membrane leaflet, quenching DiO.   Like... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=2645408 Mon, 27 Jul 2009 15:28:54 +0100 2645408 http://www.medworm.com/index.php?rid=2398985&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2009%2F05%2F08%2Finfrared-fluorescent-proteins%2F Hunting for new fluorescent proteins in the coral reefs of the Caribbean and Australia is a task that a lucky few researchers have managed to get funding for. Scuba diving in some of the world’s most beautiful places; it’s not a bad gig, if you can get it.  Most fluorescent protein scientists are confined to a lab, mutating existing fluorescent proteins from jellyfish and coral. Shifting their excitation and emission spectra has allowed multiple fluorescent proteins to be used as molecular highlighters at the same time, since their colors are distinct from each other. Some members of this palette are shown in Brain Windows top image bar.  After over a decade of research, the spectrum is pretty well covered.  Except for one area…  The infrared. The near-infrared band i... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=2398985 Fri, 08 May 2009 19:33:12 +0100 2398985 http://www.medworm.com/index.php?rid=2259405&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2009%2F03%2F12%2Fbackground-perceval-the-atpadp-sensor%2F Recently, Brain Windows mentioned the report A genetically encoded fluorescent reporter of ATP:ADP ratio. We invited Dr. Jim Berg, the lead author of the study to provide additional background to our readers. Below, Jim provides a fascinating look at rationale behind sensor development.  I really like that they came at this problem with a biological question in mind, something I would recommend before anyone start the development of a genetically encoded indicator.   A pixel-by-pixel ratio of the 490 nm excitation image by the 430 nm excitation image from two cultured HEK293 cells expressing Perceval during control conditions (left) and after 40 min of metabolic inhibition with 5 mM 2-deoxyglucose (right)   Here’s a little insight into why we decided to develop a fluorescent sens... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=2259405 Thu, 12 Mar 2009 19:52:55 +0100 2259405 http://www.medworm.com/index.php?rid=2182654&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2009%2F02%2F11%2Fsymposium-a-revolution-in-fluorescence-imaging%2F This coming Tuesday and Wednesday (Feb 17th & 18th) at UCSD, there will be a symposium honoring Roger Tsien, featuring presentations from 32 former and current members of the Tsien Lab. The topics are quite diverse, concentrated in genetically-encoded indicators, but also featuring fluorescent cell penetrating peptides for cancer therapy, photophore ligases for imaging synaptic development, and even a radical new design for the internal combustion engine. The quality of speakers and subjects looks to be outstanding.  Here is a complete schedule.  You may notice that at 11:15 AM on Tuesday in Price Center East Ballroom, I will be presenting recent progress we have made in the development of genetically-encoded calcium indicators and their application to in vivo imaging.  Don’t... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=2182654 Thu, 12 Feb 2009 04:45:42 +0100 2182654 http://www.medworm.com/index.php?rid=2092629&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2009%2F01%2F09%2Fbrainstorm-1-the-calcium-memory-sensor%2F As mentioned in the previous post, this is the first installment of BrainStorm, a section of ideas I have under development, but don’t have the time to physically work on.  This section will contain organically developed ideas, organized by project.  Reader feedback is encouraged. How can we identify the group of neurons that encode a particular thought?   I don’t want to simply see correlations of in activity of a few scattered neurons with a given thought, but identify the entire neuronal ensemble.  Which neurons are active at a precise moment in a task?  How are they wired together? Which are the drivers of activity? Existing technology is inadequate to identify the entire neural ensemble that encodes a thought. Immediate early gene expression  patterns have not been s... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=2092629 Fri, 09 Jan 2009 19:06:39 +0100 2092629 http://www.medworm.com/index.php?rid=2033252&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2008%2F12%2F12%2Ffluorescent-proteins-in-scholarpedia%2F This article is an excellent review of all fluorescent proteins that contains both classic and very current references.  I recommend it as the first place to go to learn about fluorescent proteins in detail. It makes my contributions to the GFP page of wikipedia look quite primitive. The author, Rob Campbell, is relatively famous in the field of fluorescent proteins for his monomerization of dsRed, the brilliant red fluorescent protein from coral. Many of the fluorescent proteins in Brain Windows title bar were derived from mutations of this monomeric red fluorescent protein. Less well known is that he taught me how change restriction sites on plasmid DNA via overlap extension PCR during my first year of grad school. Great work Rob! Red Discosoma Coral Posted in Fluorescent Protein&... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=2033252 Fri, 12 Dec 2008 18:23:59 +0100 2033252 http://www.medworm.com/index.php?rid=2027180&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2008%2F12%2F10%2Fpreview-structure-of-g-camp2%2F A high-resolution crystal structure of the genetically-encoded calcium indicator G-CaMP2 would aid in rational design of improved calcium indicators. Crystallization of G-CaMP2 was first reported here : Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2 M. M. Rodríguez Guilbe, E. C. Alfaro Malavé, J. Akerboom, J. S. Marvin, L. L. Looger and E. R. Schreiter Fluorescent proteins and their engineered variants have played an important role in the study of biology. The genetically encoded calcium-indicator protein GCaMP2 comprises a circularly permuted fluorescent protein coupled to the calcium-binding protein calmodulin and a calmodulin target peptide, M13, derived from the intracellular calmodulin target myosin ligh... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=2027180 Thu, 11 Dec 2008 04:07:29 +0100 2027180 http://www.medworm.com/index.php?rid=1975206&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2008%2F11%2F20%2Fsome-interesting-posters-sfn%2F Here’s a few posters that caught my eye at SfN.  Click the titles for the full abstract Optimizing two-photon activation of channelrhodopsin-2 for stimulation at cellular resolution J. P. RICKGAUER1,2, D. W. TANK1,2;  Spiral pattern of 2-photon excitation can drive neurons to spike.  A low NA objective helps. Need to do piezo-based Z-scanning if you use high NA, don’t with low NA. In vivo two-photon imaging 1 mm deep into cortical brain tissue with novel microprism probe  *T. H. CHIA, M. J. LEVENE;  A cute method to image 1mm into cortex with 2-photon imaging. They used 2-6 month old mice. The just took a triangular prism whose hypotenuse was silvered and stuck it in the cortex. Then they internally reflected the beam off the prism and fired it sideways into cortex. Got g... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=1975206 Fri, 21 Nov 2008 03:27:11 +0100 1975206 http://www.medworm.com/index.php?rid=1964123&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2008%2F11%2F15%2Fraw-data-vesicular-release-from-astrocytes%2F When I was working on my Ph.D. thesis, I was trying to find some biological question to definitively answer with GluSnFR, my glutamate sensitive fluorescent reporter. One possibility was the study of glutamate release from astrocytes.  Around that time, 2003/2004, there was increasing evidence that glutamate was not just scavenged by astrocytes, but was also released from astrocytic vesicles. It released in response to calcium elevations within the cell.  Existing methods for measuring this release were somewhat crude, so it seemed a great test system for GluSnFR.   Unfortunately, since there seemed to be no specialized areas on the astrocyte where the vesicles fused, and the release rate was relatively slow, we were unable to detect glutamate release with GluSnFR. I thought this might ... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=1964123 Sat, 15 Nov 2008 23:21:38 +0100 1964123 http://www.medworm.com/index.php?rid=1642713&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2008%2F07%2F21%2Fthe-great-geci-shootout%2F Dierk Reiff’s lab has done another head-to-head in vivo showdown between various GECIs and a synthetic dye. Their paper, Fluorescence changes of genetic calcium indicators and OGB-1 correlated with neural activity and calcium in vivo and in vitro, is very interesting and deserves a full write-up. I will present a detailed analysis of the paper in a future update.  For now, check the abstract. Recent advance in the design of genetically encoded calcium indicators (GECIs) has further increased their potential fordirect measurements of activity in intact neural circuits. However, a quantitative analysis of their fluorescence changes (F) in vivo and the relationship to the underlying neural activity and changes in intracellular calcium concentration ([Ca2+]i) has not been given... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=1642713 Mon, 21 Jul 2008 19:25:52 +0100 1642713 http://www.medworm.com/index.php?rid=1631146&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2008%2F07%2F16%2Fsensing-salty-currents-with-mermaids%2F A new genetically-encoded voltage sensor paper is out from a friend and former mentor of mine, Atsushi Miyawaki. One memorable moment when working in his lab during the RIKEN summer program of 2002 was when Atsushi took me into his office and whipped out a custom green laser pointer. These had been banned in Japan, as fans would shine their powerful light into the eyes of pitchers and batters at baseball games. Atsushi was really proud of his. He smiled and then started sweeping the light point over the rocks in his fishtank. Each ‘rock’ was actually coral his lab had collected from fluorescent protein hunting trips, and each glowed a different color when the green light hit it. He has been putting these novel discoveries to good use. In Improving membrane voltage measurements ... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=1631146 Thu, 17 Jul 2008 02:58:25 +0100 1631146 http://www.medworm.com/index.php?rid=1596486&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2008%2F07%2F08%2Fvoltage-sensitive-imaging-powering-up%2F I’m starting to come around on voltage imaging. I haven’t been a fan of it for a number of reasons. The response sizes suck.  Classic dyes and genetically encoded systems get a few percent fluorescence change at best.  The response speeds suck. Measuring continuous current injections from -100mV to +150mV is not very interesting.  Action potentials are interesting.  But they are fast. Toxicity. The dyes kill neurons, or strongly perturb their electrical properties. OK, voltage-sensitive imaging isn’t totally useless, for example see Carl Petersen’s recent paper on Spatiotemporal Dynamics of Cortical Sensorimotor Integration in Behaving Mice (2007). But if the above problems could be solved, then voltage sensitive imaging would be a strong competitor to calciu... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=1596486 Tue, 08 Jul 2008 22:29:44 +0100 1596486 http://www.medworm.com/index.php?rid=1560925&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2008%2F07%2F01%2Fslick-labeling-and-new-fps%2F There is a nice writeup of the single-neuron labeling with inducible Cre-mediated knockout (SLICK) paper from Guoping Feng’s lab over at the Alzheimer’s Research forum. The method simultaneously knocks out a gene in a small number of cells, while highlighting the knocked-out cells with a cytosolic fluorescent protein. In a comment to the Schizophrenia Research Forum, Joseph Gogos points out a similar technique his lab published last year in Current Biology. Also in the writeup is coverage of the new fluorescent protein variants from the Tsien Lab.  These include mOrange2 made by Nathan Shaner, which is a much more photostable version of mOrange. This should immediately replace mOrange in most constructs.  Also of note is TagRFP-T from Michael Lin and his trusty undergraduat... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=1560925 Tue, 01 Jul 2008 19:40:08 +0100 1560925 http://www.medworm.com/index.php?rid=1556392&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2008%2F06%2F30%2Fgiving-synapses-a-born-on-labelinthe%2F Memories are thought to be encoded by the patterns of synaptic connections in the brain. Learning can either delete or change the strength of existing synapses, or add new synapses. Following a learning process, how can we tell which synapses were added to encode this new memory?   One strategy is to make a timelapse movie of the synapses.  In mice, this can be accomplished by installing a cortical window on the skull, and imaging the changes in structure of GFP labelled neurons. However, this is technically demanding, only works with sparsely labeled neurons, and accesses only a small subset of the neurons which may be involved in the learning process.   Ideally, one could have a tag which can discriminate between synapses existing before learning takes place, and new ones generated ... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=1556392 Mon, 30 Jun 2008 17:25:43 +0100 1556392 http://www.medworm.com/index.php?rid=1325144&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2008%2F03%2F25%2Fjournal-club-gfp-reconstitution-across-synaptic-partners-grasp%2F This week, I’m guest presenting a recent Neuron paper over at the Nature Network’s neuroscience journal club. The authors propose a clever new technique, Genetic Reconstitution Across Synaptic Partners, to track the locations of selected synaptic connections backed by an impressive set of in vivo proof-of-principal experiments in C. elegans. The gist of the strategy is to split a fluorescent marker into two non-functional components and then distribute each half on different sides of circuit’s connection. Only at synaptic connections would the two components be close enough to undergo trans-complementation and reconstitute a functional marker. Check out the full presentation and join the discussion. (Source: Brain Windows) Brain Windows blogs http://www.medworm.com/rss/comments.php?id=1325144 Tue, 25 Mar 2008 16:51:14 +0100 1325144 http://www.medworm.com/index.php?rid=1298766&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2008%2F03%2F12%2Foptical-imaging-of-neuronal-glutamate-release-and-spillover-with-glusnfr%2F This post is difficult to craft. I’ve been struggling with whether to write an epic post describing the history of glutamate imaging, the major advances and players in the field and where I fit into it, or a simple post focused on my new paper. Since glutamate imaging is my field, I’ve got tons to say about it, but also there is probably no way to avoid significant personal bias in my account. So, I’ll go with the short form. For those interested in further reading, please check out these earlier reports, including our brief mention of neuronal glutamate measurements with GluSnFR prototypes, neuronal glutamate measurement with FLIPE and the optimization of FLIPE constructs from Wolf Frommer’s group, and the use of FLIPE’s in brain slice to look at broad patter... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=1298766 Wed, 12 Mar 2008 22:11:05 +0100 1298766 http://www.medworm.com/index.php?rid=1243476&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2008%2F02%2F19%2F3d-and-multicolor-superresolution-imaging%2F Progress in superresolution imaging is still moving very quickly. Here are two more great papers in the field. First, Huang et al. from Xiaowei Zhuang’s group published a Science paper that moves superresolution imaging into three dimensions. Previously, STORM and PALM techniques were most useful for thin sections where the z-axis depth is well-constrained. Breaking the diffraction limit in the z-dimension was thought to possibly require recording from multiple angles, standing wave TIRF or optical lattice microscopy. Instead, the authors simply inserted a weak cylindrical mirror in between the imaging lens and the objective. This distorted the shape of the point spread function in the x- and y-dimensions, dependent on the z-axis distance from the focal plane. By examining the shape ... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=1243476 Tue, 19 Feb 2008 21:48:34 +0100 1243476 http://www.medworm.com/index.php?rid=1240219&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2008%2F02%2F18%2Fpulse-shaping-for-2-photon-signal-enhancement%2F Gains in signal to noise ratios of organic dyes and genetically encoded indicators often come in modest steps following screening of large numbers of compounds or clones. Improvements are usually specific to individual chromophores, leading to the pigeonholing of development efforts on a small handful of indicators that have already undergone systemic optimization (i.e. cameleons, G-CaMP and troponin-based GECIs). Indicator photobleaching imposes strict limits on the amount of information which can be extracted by optical indicators. Improvement of specific indicators and their constituents is a worthy and necessary goal, but more generalizable improvements can be made by changing the nature of the illumination source. A series of papers from a variety of groups has shown that careful mani... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=1240219 Tue, 19 Feb 2008 06:11:59 +0100 1240219 http://www.medworm.com/index.php?rid=1001041&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2007%2F11%2F02%2Fbrainbow-mice-are-out%2F Jeff Lichtman’s Brainbow mouse paper is out! Not that I really need to report that news, as it is, of course, on the cover of Nature. Jean Livet comes up with some really clever genetic strategies involving incompatible, overlapping Lox sites to generate random, combinatorial patterns of multiple fluorescent proteins inside the cell. Around 90 different shades can be discerned by spectral deconvolution. Besides making pretty covers, why is this so cool? Well, this technique provides a method for generating high resolution maps of the brain. With a single fluorescent tag, the processes of neighboring cells blur together and became impossible to trace unambiguously. With brainbow, many neighboring axons are clearly resolvable. This is the perfect genetic tool to use for a large-scale,... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=1001041 Fri, 02 Nov 2007 19:46:05 +0100 1001041 http://www.medworm.com/index.php?rid=999436&cid=t_140550_132_f&fid=35006&url=http%3A%2F%2Fnsaunders.wordpress.com%2F2007%2F11%2F02%2Fover-the-brainbow%2F I’m a big fan of cell imaging - pretty pictures of fluorescent cells always enhance a conference presentation. A Harvard team have taken fluorescent labelling to a new level, using combinatorial fluorescent proteins to image brain tissue in 90 distinct colours. What’s more, they’ve come up with a memorable word in “brainbow”. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system (Nature abstract) Colours light up brain structure (Nature News) Cell stains create a ‘brainbow’ (BBC Science News) Search: brainbow at del.icio.us (Source: What You're Doing Is Rather Desperate) What You're Doing Is Rather Desperate blogs http://www.medworm.com/rss/comments.php?id=999436 Fri, 02 Nov 2007 01:05:55 +0100 999436 http://www.medworm.com/index.php?rid=961700&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2007%2F10%2F18%2Fthree-quick-paper-picks%2F Here are three papers that are worth reading over. No time for full reviews. New Single-FP GECIs The Russian fluorescent protein team has come out with some new single fluorescent protein G-CaMP/pericam-like sensors. They fiddled with the linker sites at the 145 and 148AA insertion points and found a great deal of fluorescence sensitivity to the amino acid composition at those sites. They note two new sensor constructs Case12 and Case16 that have 12-16.5x maximal changes in fluorescence upon calcium binding, a significant improvement over G-CaMP2. The tradeoff appears to be that they are dimmer. They show calcium responses in HeLa, PC-12 and cortical neuron cells, but no direct head-to-head with other sensors in cells. Multipoint multiphoton microscopy In this technical paper, an MIT gro... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=961700 Thu, 18 Oct 2007 19:55:09 +0100 961700 http://www.medworm.com/index.php?rid=935304&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2007%2F10%2F08%2Fupdate-on-far-field-superresolution-optics%2F Its thesis crunch time for me, so I have had limited time to do ‘extracurricular’ reading and reporting for Brainwindows. However, there have been some very exciting developments in the field of superresolution fluorescence imaging that deserve a mention. First, let’s take a look at this excellent review of far-field superresolution imaging techniques by Stefan Hell. I was almost able to understand the basics of the current techniques after reading it. Hopefully my summary doesn’t contain too many errors ☺. Axial resolution is particularly bad in conventional superresolution techniques. Confocal imaging and 2 photon imaging provides ~450 nm resolution at best, while 4Pi microscopy with immersion lenses above and below the sample has delivered ~100nm axial resolution images of fix... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=935304 Mon, 08 Oct 2007 23:02:55 +0100 935304 http://www.medworm.com/index.php?rid=710312&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2007%2F07%2F02%2Fan-update-and-new-fret-biosensor-software%2F Apologies for the long delay between updates. I’ve been writing up and editing my work, also spent the last 3 weeks at the CSHL Ion Channel Physiology course. Jeff Diamond, Mark Farrant, Kenton Swartz and Michael Hausser ran a very informative and entertaining program. So, what’s new in FRET sensor land? Producing useful FRET sensors requires some structural insight, theoretical knowledge, patience to screen many variants, and luck. Making a new sensor that works marginally well is often not that difficult. However, making a reversible sensor with high speed and S/N takes a lot of time and effort. How does a researcher select the protein substrate, linker sequences and fluorescent proteins from the vast space available? A new paper and software from Kevin Truong’s group ... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=710312 Mon, 02 Jul 2007 20:36:25 +0100 710312 http://www.medworm.com/index.php?rid=500882&cid=t_140550_122_f&fid=35068&url=http%3A%2F%2Fbrainwindows.wordpress.com%2F2007%2F03%2F25%2Fcshl-meeting-session-vii-super-resolution-optical-techniques%2F Jean-Louis Bessereau – Ultrastructural mapping of functional domains of synapse at the synapse using high pressure imaging High pressure freezing instantaneously converts up to 0.3mm thick water into amorphous ice. C Elegans only .1mm thick at maximum. HPF entire worm to obtain EM of ‘living’ synapses. Vesicle priming occurs within 100nm of presynaptic density, directly across from post receptors. However, vesicle recycling occurs only at sites >150nm lateral from presynaptic release sites. Mark Ellisman – Multiscale light and electron microscopic imaging of the nervous system Two-color correlated light and EM microscopy using FlAsH and ReAsH. Quantum dot immunohistochemistry for multicolor correlated light and EM. QDs of different wavelength are differently sized and can be dis... Brain Windows blogs http://www.medworm.com/rss/comments.php?id=500882 Mon, 26 Mar 2007 07:22:36 +0100 500882