The L-ETC, is an inexpensive and reliable CLARITY ETC chamber that can be easily assembled by a novice user. It provides several key advantages over previous ETC chamber fabrication methods including secure tissue containment, more consistent and efficient tissue cooling, fast lipid removal, easier chamber fabrication, and very low initial costs. The L-ETC chamber aligns the Electric Field with Clearing Solution flow and is the main focus of this proposal. The L-ETC system as a whole (pump, refrigerator, and power supply) will also be discussed in terms of settings.
The L-ETC chamber is easily fabricated from ‘off the shelf’ parts, and are therefore subject to industry standards, thereby minimizing the risk of leaks. The tissue to be cleared is securely contained in a fine mesh cylindrical tissue strainer. The tissue strainer is fit into off the shelf plumbing components between platinum mesh plate electrodes, creating the L-ETC. The detergent buffer clearing solution (as described in (Structural and molecular interrogation of intact biological systems, Deisseroth 2013)[https://www.nature.com/nature/journal/v497/n7449/full/nature12107.html]) is pumped through the L-ETC, forcing the solution over and through the tissue, expelling generated bubbles from around the tissue. A DC power supply applies an electrical field over the tissue and a refrigerator removes the heat from the solution generated at the platinum-solution interface. The L-ETC applies a uniform electric field, allowing for rapid tissue clearing. The temperature and pressure of the solution can be monitored at the inlet or outlet near the tissue. If necessary, the use of a simple refrigerator allows for stable, uniform cooling without the need for non-uniform ice baths or expensive cooling equipment.
The L-ETC system has shown success with mouse brains and spinal cords and rat lungs. Half mouse brains (n=20) have been successfully cleared in 2-3 days with the L-ETC and whole mouse brains (n=4) have also been cleared in 3-5 days. Smaller, 128 μm slices of mouse brain can be cleared in under a day (n=3). Rat lung slices (5 mm) were cleared in 2 days.
A pdf describing this project in detail can be found here. Instructions on how to assemble and key parameters are discussed. Note that you can build your own chamber, have the ONE Core build your own, or you can bring your tissue to the ONE Core for clearing. Please contact us for more information. Assistance with deep tissue imaging can be found at the Advanced Light Microscopy Core Site.
\[/ Item Description PN Supplier Qty need Qty in package Price per Package Total $ Total Cost Per Chamber 1 Solution Inlet - - - - - - - 2 Thermometer 6182K51 McMaster 1 1 17.91 17.91 17.91 3 3/8" ID Barbed Tube Plug 2974K401 McMaster 4 10 8 8 3.2 4 3/8" ID Clear PVC Tube 5231K355 McMaster 12" 300 11.25 11.25 - 5 1 1/4" Hex Threaded Nipple 46825K51 McMaster 1 1 1.94 1.94 1.94 6 1" PVC Pipe (spacers) 48925K93 McMaster .178" 60" 5.27 5.27 0.02 7 1" PVC Pipe (spacers) 48925K93 McMaster .681" - - - 0.06 8 Sample Holder 21008-949 VWR 2 50 72.3 72.3 2.892 9 1" PVC Pipe (spacers) 48925K93 McMaster .927" - - - 0.08 10 1 1/4" Coupling 46885k185 McMaster 2 1 6.12 12.24 12.24 11 Platinum 2" x 3" with 4" stem PLATINUM The Amateur Chemist 1/5 1 25 25 5 12 1 1/4" to 3/4" Reducer Hex Nipple 46885K305 McMaster 2 1 2.91 5.82 5.82 13 3/8" Tube to 3/4" Female Threaded Pipe 5372K222 McMaster 2 5 7.89 7.89 3.156 14 3/8" Cross Connectors 5463K97 McMaster 2 5 8.51 8.51 3.404 15 Pressure Gage Not Nec - - - - - - 16 Solution Outlet - - - - - - - 17 Positive Wire (NP) Lab - - - - - - 18 Negative Wire (NP) Lab - - - - - - 19 High Strength Sealant (NP) 7475A67 McMaster 1 1 9.41 9.41 9.41 20 Dorm Refridgerator (NP) Lab - - - - - - 21 Pump (NP) (~.4 L/min) Lab - - - - - - 22 Power Supply (NP) (28V 0.05A) Lab - - - - - - 23 TaegaSeal PTFE Tape 4591K12 McMaster 1 1 2.31 2.31 2.31 Totals - - - - - - - - -- - - - - - - - - 176.13 65.13\]
Many of the 3D files shown are from McMaster.
Please see the files linked above for more information about this project.
ONE Core acknowledgement
Please acknowledge the ONE Core facility in your publications. An appropriate wording would be:
“The Optogenetics and Neural Engineering (ONE) Core at the University of Colorado School of Medicine provided engineering support for this research. The ONE Core is part of the NeuroTechnology Center, funded in part by the School of Medicine and by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under award number P30NS048154.”