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Frequently Asked Questions

Questions: 
Question: Does the Photoconductive Stimulation Device (PSD) sense cellular activity?
Answer: No, the PSD does not sense any cellular activity. The device is used solely for depolarizing cells in a controlled manner. In some cases, sensing may be necessary. The PSD does operate alongside standard fluorescent imaging systems for this purpose.
 
Question: Do I need any "special" cells to use the PSD?
Answer: No, the PSD operates with any standard mammalian excitable cells, including neurons and cardiac myocytes.
 
Question: What kind of equipment do I need to prepare cells for use with the PSD?
Answer: The standard tools to culture cells are necessary. No special tools are required for this purpose. Neurosilicon does provide the silicon materials on which to culture the cells.
 
Question: How does the PSD stimulate cells without damaging or physically touching them?
Answer: The PSD uses a phenomenon called the Photoconductive Stimulation Effect. It is based in the principle that when visible light strikes silicon in combination with a specific current, a local field potential is generated. This LFP is what causes the cell to depolarize noninvasively without causing any damage to the cell.
 
Question: Do I need a microscope to use the PSD with?
Answer: Yes, the PSD does require a microscope for operation. The PSD only supports the use of a standard upright microscope with the light source coming from above.
 
Question: Do PSD silicon substrates need to be coated with laminin? Our lab’s protocol calls for use of just poly-L-lysine?
Answer: Laminin is presented as an example of what is used in our labs at Neurosilicon – whatever protocol you use for glass cover slips in your lab should work with the PSD silicon substrates.
 
Question: Is a separate incubator necessary for culturing on the PSD substrates?
Answer: No, culturing is exactly the same as for cover slips, so any incubator should suffice, needs no isolation or special treatment, and grow them in whatever culture media they usually use.
 
Question: Is a special medium necessary for culturing cells on the PSD substrates?
Answer: No, whatever you use for your own cell culture should work fine.
 
Question: In our experience some silicone grease is toxic to neurons. Can you confirm a specific brand that works for you?
Answer: We use Corning Vacuum grease; it has never given us problems.
 
Question: What should I use as my PSD firing scheme (frequency and intensity)?
Answer: Firing can be done at any frequency; it depends on your application.
If you just want to test things and see cells fire with a calcium dye for example, slow rates are fine (1-2 Hz).

The voltage and duration dose not really change much once established, 2-5 milliseconds and 4.5 volts is a good starting point.

The idea is to watch the cells as you stimulate them, increase the voltage and duration (from 3->5 volts and 2->5 ms) and when they start to fire, those are the settings you will want to use.
 
Question: When starting using the PSD what light wavelength should I use?
Answer: All wavelengths work, I would start with a GFP or YFP cube.
Intensity makes a big difference (both for cell viability and fluorescent reporter life) a guide would be to stay within a light level that is as high as your cells can be exposed to without significantly damaging them after ~5 minutes of continuous exposure.

When considering how you develop your experimental protocols, it may be fine for a short 5 second stimulation after which you are going to observe an event over the next few minutes, but if you are stimulating for 30 seconds every 5 minutes over several hours, we recommend to adjust the aperture slightly and turn up the voltage slightly (~5.5) to compensate so as to keep the light intensity levels to within reasonable levels over the course of the analysis.
 
Question: Using whole mount preparations that are up to a few hundred microns thick, can we target cells in X,Y, and Z as opposed to just X and Y?
Answer: Contact with the surface of the substrate and a clear path for light to reach the substrate surface are key for successful photoconductive stimulation. Provided that the sample allows sufficient light to pass, samples as thick as ~100 microns, should be excitable using the PSD in the X and Y.

If the preparation (or slice) remains electric connected at the cellular level then excitation of the cells closest to the surface of the substrate could have an excitation response in the cells in the Z but there is no way to directly (specifically) excite cells in the Z while circumventing those closest to the substrate surface.
 
Question: How/where does PSD Experimental Chamber mount and/or connect onto the microscope?
Answer: Page (19) of the PSD user's manual outlines the procedure for mounting the experimental on the microscope stage. Please request the manual from Neurosilicon for more information.
 
Using the guide rails on the experimental chamber, move the chamber into place beneath the objective on the existing microscope stage.

Then using standard laboratory tape fix the chamber in pace beneath the objective for easy “on” and “off” of the stage.
 
Question: Are there plans for being able to use this with an inverted scope any time in the future?
Answer: Currently, we are working to develop a solution for using the PSD with inverted microscopes. Through the use of an “objective inverter” users are able to utilize the PSD with their inverted microscopes. Please contact us for more information.
 
Question: Is it possible to lay a slice on the wafer without growing it on and have the PSD work effectively?
Answer: Yes, “growing” the sample on the surface of the substrate is not necessary for effective photooducitve stimulation. Contact with the surface of the substrate and a clear path for light to reach the substrate surface are key for successful photoconductive stimulation as well as an optimal thickness.
 
Question: Can you image a large field of cells and stimulate only a small portion of the area you are imaging?
Answer: Yes, but this will require a secondary light source to deliver the excitation light.
 
Briefly, one can set the power level on the control below the level for excitation at the light intensity required for imaging.

The secondary light source (say a fiber optic light wand) can then be set to an intensity that will yield excitation at the PSD controller power setting that does not excite under imaging conditions.

In this way the increased light intensity of the secondary light source can be used to specifically cellular excitation while the light used for image collection will not result in activation.
 

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