[OpenSPIM] Planning an appropriate SPIM build for my experiments

[Michael Weber]

Hi Mel,

that’s a long list of requirements. Here are some thoughts:

- test your samples on existing light sheet setups
- also test/consider a diSPIM-like configuration
- 2-Photon is beyond reach for $100k - and hard to do for 3-4 channels
- a fiber-coupled laser launch with 4 lines will be expensive - you might want to start with less lines and upgrade later
- a feasible detection combination would be a single sCMOS camera plus a filter wheel

Best,
Michael

> On Jun 4, 2016, at 7:45 PM, Menelaos Symeonides <msymeoni at UVM.EDU> wrote:
> 
> 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!
> 
> Mel
> 
> -------- Original message --------
> From: Menelaos Symeonides <msymeoni at uvm.edu>
> Date: 6/2/16 7:48 AM (GMT-05:00)
> To: openspim at openspim.org
> Subject: [OpenSPIM] Planning an appropriate SPIM build for my experiments
> 
> Hello OpenSPIM,
> 
> I've been following this list and reading papers in the field for about 
> a year. In our lab, we've come to a point where we need to carry out 
> long term fast nontoxic imaging of cell cultures that is only possible 
> with SPIM, and have just obtained funding to build our own microscope to 
> do these experiments. I was hoping to pick the brains of this mailing 
> list to hopefully design an appropriate setup, perhaps based on the 
> OpenSPIM platform, so that we can carry out these experiments.
> 
> In general, the experiment involves imaging HIV-infected T cells 
> embedded in a 3D collagen hydrogel as they migrate through the ECM and 
> spread the infection to other cells and do various other things related 
> to the infection, e.g. bystander cell killing. Here are some of the 
> considerations:
> 
> 1. We have fluorescently-tagged viruses in the FITC channel, a live cell 
> apoptosis sensor in the iRFP channel, and will also need to somehow 
> label nuclei and perhaps also membranes. That would be a maximum of 4 
> channels, with 3 channels being "acceptable".
> 
> 2. The cells will need to be imaged for 2 to 3 days, with a time 
> resolution on the order of 2 Hz (for the full volume). T cells migrate 
> at about 15 μm/sec, and we would like sufficient time resolution to 
> capture each cell at least twice during each body length movement (body 
> length is about 10 μm, hence needing around 2 Hz).
> 
> 3. The entire volume (or close to that) of the hydrogel will need to be 
> imaged at each timepoint. The volume of the hydrogel is up to our 
> discretion, but generally it is on the order of 150 to 300 μL.
> 
> 4. Spatial resolution is not of particular concern, as we are more 
> interested in the cells as whole entities rather than e.g. the details 
> of virus assembly in each cell. However, we would like comparable 
> resolution in all three dimensions, as there is no real "plane" we are 
> interested in and the cells can go in any direction.
> 
> 5. The hydrogel can be fairly optically dense, and we experience 
> prohibitive distortion at anything deeper than ~100 μm with widefield 
> single photon imaging.
> 
> 6. Of course, temperature control and media perfusion will be required. 
> Given the nature of the pathogen, we will need to be able to fully 
> decontaminate the sample chamber and anything that has come into contact 
> with the media. We've been looking at Ernst Stelzer's TC-LSFM to get 
> ideas for the tissue culture chamber. We have access to a fabrication 
> service at our institute that can help us design and build the chamber.
> 
> 
> Just to give you a general idea, we have about $100K available to us to 
> purchase parts for this, not including the fabrication fee for the 
> culture chamber for which we have other money set aside.
> 
> Other than general ideas of what direction we should be heading in, or 
> links to papers for setups we could emulate, I also have some more 
> specific questions:
> 
> A. Would two-photon imaging be an inevitable requirement given the 
> density of the sample?
> 
> B. If the sort of speed of acquisition we are looking for is beyond 
> reach for any system we could feasibly build ourselves (especially given 
> the need for exposing in multiple fluorescence channels at each 
> position), what should we sacrifice in our experimental plan to make it 
> more within reach?
> 
> C. Does anyone know of any labs in the Northeast US/Quebec that have 
> implemented a similar setup that we could go see in person? We are based 
> in Vermont.
> 
> 
> I appreciate the work everyone on this list puts in for newcomers, so 
> thank you!
> 
> Best regards,
> Mel
> 
> 
> -- 
> Menelaos Symeonides
> Post-Doctoral Associate, Thali Lab
> Department of Microbiology and Molecular Genetics
> University of Vermont
> 318 Stafford Hall
> 95 Carrigan Dr
> Burlington, VT 05405
> msymeoni at uvm.edu
> Phone: 802-656-1161
> 
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