| 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. |
| |
