<html><head><meta http-equiv="Content-Type" content="text/html charset=utf-8"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class="">Hi Tim<div class=""><br class=""></div><div class="">we get our mirrors from EOPC. I think it is the SC-10 with 1kHz.</div><div class=""><br class=""></div><div class="">Yes, the zebrafish embedding is far from perfect. We are thinking about several ways of improving it and we are keeping the tricaine level as low as possible.</div><div class=""><br class=""></div><div class="">Best</div><div class="">Jan</div><div class=""><br class=""></div><div class=""><br class=""><div><blockquote type="cite" class=""><div class="">On May 3, 2016, at 3:22 PM, Feinstein, Timothy N <<a href="mailto:tnf8@PITT.EDU" class="">tnf8@PITT.EDU</a>> wrote:</div><br class="Apple-interchange-newline"><div class=""><div class="WordSection1" style="page: WordSection1; font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px;"><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">Thank you Jan, of course your 2007 paper is the more appropriate reference. Do you know where to find a resonant SPIM mirror? Is there a supplier for those? <o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class=""> </span></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">Also I recall that we had a discussion on the mailing list about embryos suffocating in FEP tubes within a few hours. Is there a reliable way to prevent that? We are using 0.5% tricaine. I keep the fish head down and position it as close to the end of the tube as I can, but there is always a balance between not close enough to the end and so close that the embryo falls out if it wriggles a bit. <o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class=""> </span></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">Best ,<o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class=""> </span></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class=""> </span></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">Tim<o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class=""> </span></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">Timothy Feinstein, Ph.D.<o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">Research Scientist<o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class="">University of Pittsburgh Department of Developmental Biology<o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class=""> </span></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125);" class=""> </span></div><div class=""><div style="border-style: solid none none; border-top-color: rgb(181, 196, 223); border-top-width: 1pt; padding: 3pt 0in 0in;" class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><b class=""><span style="font-size: 10pt; font-family: Tahoma, sans-serif;" class="">From:</span></b><span style="font-size: 10pt; font-family: Tahoma, sans-serif;" class=""><span class="Apple-converted-space"> </span>Jan Huisken [<a href="mailto:huisken@mpi-cbg.de" style="color: purple; text-decoration: underline;" class="">mailto:huisken@mpi-cbg.de</a>]<span class="Apple-converted-space"> </span><br class=""><b class="">Sent:</b><span class="Apple-converted-space"> </span>Friday, April 29, 2016 3:01 AM<br class=""><b class="">To:</b><span class="Apple-converted-space"> </span>Feinstein, Timothy N<br class=""><b class="">Cc:</b><span class="Apple-converted-space"> </span><a href="mailto:openspim@openspim.org" style="color: purple; text-decoration: underline;" class="">openspim@openspim.org</a><br class=""><b class="">Subject:</b><span class="Apple-converted-space"> </span>Re: [OpenSPIM] Is anyone using digital scanning to reduce banding?<o:p class=""></o:p></span></div></div></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">Dear Tim,<o:p class=""></o:p></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">I think you are mixing up two issue.<o:p class=""></o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><b class="">1. Getting rid of stripes.</b><o:p class=""></o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">Yes, you can do a little bit in post-processing but the stripes can be quite complex and are not necessarily straight. Oftentimes you see a lot of lensing and I do not think you can easily remove it with image processing.<o:p class=""></o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">a) Double-sided illumination helps. Depending on the sample you may want to do simultaneous or sequential double-sided illumination. There are various ways of merging the two images.<o:p class=""></o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">b) A resonant mSPIM mirror is cheap and easy to integrate. You will get rid of most stripes, but again this depends on the sample. The details for a) and b) are in our Opt. Lett. paper (Huisken & Stainier, 2007). Basically, you are pivoting the light sheet around the center of your field of view, e.g. with 1kHz. The stripes are “washed out” during the exposure time of your camera. We use this on most of our systems and it helps a lot to reduce stripes.<o:p class=""></o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">c) Multi-view fusion can also help to some extent.<o:p class=""></o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><b class="">2. DSLM vs. SPIM</b><o:p class=""></o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">Yes, DSLM should also help reducing stripes but I have no experience with that. Basically, your light sheet is not coherent anymore and the sheet does not “interfere with itself”. However, the setup is more expensive and requires you to power and control another element: the galvo scanner to sweep the beam up and down. You need a proper scan mirror and not just the cheap mSPIM mirror. The additional benefit is that the resulting light sheet is more uniform than the cropped Gaussian light sheet. However, you need to illuminate each line with higher intensity which may result in saturation and worse dynamic range. Obviously you need to wait for the scan and need to synchronize your readout. Depending whether you have a global shutter or rolling shutter camera you need to take some precaution.<o:p class=""></o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">Best<o:p class=""></o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">Jan<o:p class=""></o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div><div class=""><div class=""><div class=""><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="" class="">—<o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><b class="">Jan Huisken</b><span style="" class=""><o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><i class="">Head of Max Planck Research Group</i><span style="" class=""><o:p class=""></o:p></span></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="" class=""> </span></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><span style="" class="">Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG)<o:p class=""></o:p></span></div></div></div></div></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div><div class=""><blockquote style="margin-top: 5pt; margin-bottom: 5pt;" class=""><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">On Apr 27, 2016, at 4:13 PM, Feinstein, Timothy N <<a href="mailto:tnf8@PITT.EDU" style="color: purple; text-decoration: underline;" class="">tnf8@PITT.EDU</a>> wrote:<o:p class=""></o:p></div></div><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class=""><o:p class=""> </o:p></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 12pt; font-family: 'Times New Roman', serif;" class="">Hello all,<span class="Apple-converted-space"> </span><br class=""><br class="">I found the Selzer group's solution (Keller et al. 2013) quite interesting<br class="">and would love to know if anyone had a positive experience doing that.<br class="">The banding from my basic openSPIM is definitely a problem for<br class="">segmentation and analysis.<br class=""><br class="">As far as I can tell fixing the banding will mostly involve buying a scan<br class="">mirror. I don't have a quote yet for the Cambridge VM500 scanning mirror<br class="">that the Selzer group used but its ThorLabs equivalent (#GVS101?) costs<br class="">about $1k. That seems reasonable and not that hard for regular folks to<br class="">implement. <br class=""><br class="">In related news I just noticed that the TeraStitcher plugin for Vaa3D now<br class="">has an option to fight banding in light sheet data. I have not tested it<br class="">yet but potentially that feature could be useful. I will just post my<br class="">usual warning for folks trying TeraStitcher for the first time: it<br class="">stitches really well but it could be a hair more user-friendly.<br class=""><br class="">Best,<span class="Apple-converted-space"> </span><br class=""><br class=""><br class="">Tim<br class=""><br class="">Timothy Feinstein, Ph.D.<br class="">Research Scientist<br class="">University of Pittsburgh Department of Developmental Biology<br class=""><br class=""><br class="">_______________________________________________<br class="">OpenSPIM mailing list<br class=""><a href="mailto:OpenSPIM@openspim.org" style="color: purple; text-decoration: underline;" class="">OpenSPIM@openspim.org</a><br class=""><a href="http://openspim.org/mailman/listinfo/openspim" style="color: purple; text-decoration: underline;" class="">http://openspim.org/mailman/listinfo/openspim</a></div></div></blockquote></div></div></div></div></blockquote></div><br class=""></div></body></html>