<html><head><meta http-equiv="Content-Type" content="text/html; charset=UTF-8"></head><body>A little correction... the velocity of the cells is 15 μm/min, not /sec, meaning that the desired imaging speed would be on the order of 2-3 volumes/min, which should be a bit more manageable!<div><br></div><div>Mel</div><div><br></div><div style="font-size:100%;color:#000000"><!-- originalMessage --><div>-------- Original message --------</div><div>From: Menelaos Symeonides <msymeoni@uvm.edu> </div><div>Date: 6/2/16 7:48 AM (GMT-05:00) </div><div>To: openspim@openspim.org </div><div>Subject: [OpenSPIM] Planning an appropriate SPIM build for my experiments </div><div><br></div></div>Hello OpenSPIM,<br><br>I've been following this list and reading papers in the field for about <br>a year. In our lab, we've come to a point where we need to carry out <br>long term fast nontoxic imaging of cell cultures that is only possible <br>with SPIM, and have just obtained funding to build our own microscope to <br>do these experiments. I was hoping to pick the brains of this mailing <br>list to hopefully design an appropriate setup, perhaps based on the <br>OpenSPIM platform, so that we can carry out these experiments.<br><br>In general, the experiment involves imaging HIV-infected T cells <br>embedded in a 3D collagen hydrogel as they migrate through the ECM and <br>spread the infection to other cells and do various other things related <br>to the infection, e.g. bystander cell killing. Here are some of the <br>considerations:<br><br>1. We have fluorescently-tagged viruses in the FITC channel, a live cell <br>apoptosis sensor in the iRFP channel, and will also need to somehow <br>label nuclei and perhaps also membranes. That would be a maximum of 4 <br>channels, with 3 channels being "acceptable".<br><br>2. The cells will need to be imaged for 2 to 3 days, with a time <br>resolution on the order of 2 Hz (for the full volume). T cells migrate <br>at about 15 μm/sec, and we would like sufficient time resolution to <br>capture each cell at least twice during each body length movement (body <br>length is about 10 μm, hence needing around 2 Hz).<br><br>3. The entire volume (or close to that) of the hydrogel will need to be <br>imaged at each timepoint. The volume of the hydrogel is up to our <br>discretion, but generally it is on the order of 150 to 300 μL.<br><br>4. Spatial resolution is not of particular concern, as we are more <br>interested in the cells as whole entities rather than e.g. the details <br>of virus assembly in each cell. However, we would like comparable <br>resolution in all three dimensions, as there is no real "plane" we are <br>interested in and the cells can go in any direction.<br><br>5. The hydrogel can be fairly optically dense, and we experience <br>prohibitive distortion at anything deeper than ~100 μm with widefield <br>single photon imaging.<br><br>6. Of course, temperature control and media perfusion will be required. <br>Given the nature of the pathogen, we will need to be able to fully <br>decontaminate the sample chamber and anything that has come into contact <br>with the media. We've been looking at Ernst Stelzer's TC-LSFM to get <br>ideas for the tissue culture chamber. We have access to a fabrication <br>service at our institute that can help us design and build the chamber.<br><br><br>Just to give you a general idea, we have about $100K available to us to <br>purchase parts for this, not including the fabrication fee for the <br>culture chamber for which we have other money set aside.<br><br>Other than general ideas of what direction we should be heading in, or <br>links to papers for setups we could emulate, I also have some more <br>specific questions:<br><br>A. Would two-photon imaging be an inevitable requirement given the <br>density of the sample?<br><br>B. If the sort of speed of acquisition we are looking for is beyond <br>reach for any system we could feasibly build ourselves (especially given <br>the need for exposing in multiple fluorescence channels at each <br>position), what should we sacrifice in our experimental plan to make it <br>more within reach?<br><br>C. Does anyone know of any labs in the Northeast US/Quebec that have <br>implemented a similar setup that we could go see in person? We are based <br>in Vermont.<br><br><br>I appreciate the work everyone on this list puts in for newcomers, so <br>thank you!<br><br>Best regards,<br>Mel<br><br><br>-- <br>Menelaos Symeonides<br>Post-Doctoral Associate, Thali Lab<br>Department of Microbiology and Molecular Genetics<br>University of Vermont<br>318 Stafford Hall<br>95 Carrigan Dr<br>Burlington, VT 05405<br>msymeoni@uvm.edu<br>Phone: 802-656-1161<br><br>_______________________________________________<br>OpenSPIM mailing list<br>OpenSPIM@openspim.org<br>http://openspim.org/mailman/listinfo/openspim<br></body></html>