[OpenSPIM] Planning an appropriate SPIM build for my experiments

[Menelaos Symeonides]

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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