[Neuroscience] Re: Fast drug application 'liquid filament' help!

jonesmat via neur-sci%40net.bio.net (by jonesmat from physiology.wisc.edu)
Wed Jul 18 17:18:01 EST 2007


On Jul 16, 5:31 pm, Bill <connelly.b... from gmail.com> wrote:
> Hi,
>
> Does anyone here use a liquid filament style, fast drug applicator?
> E.g. theta pipette with control and drug in each barrel, which is the
> rapidly moved via a piezoelectric actuator to squirt the drug solution
> onto the cell?
>
> Does anyone have any brands they could recommend?
>
> I'm also a big confused about how the whole setup would look.
> Obviously you'd need a course manipulator to get the pipette close to
> the cell, some kind of pipette holder, and the piezoelectric stepper.
> You'd presumably mount the manipulator to your isolation table, and
> then the actuator onto the manipulator?
>
> Thanks for any advice.


Geez Bill,

You seem to have one finger in every possible pie, from hippo to
thalamic slices to fast application to who knows what else.

As it happens, we routinely use piezo-driven fast application.

Assuming you want to make real fast, submillisecon solution exchanges,
suitable for studying, say, AMPA or GABAA receptor kinetocs, there are
two basic flavors. If you don't need to be that fast, then there are
probably more options. We do the former, and I have used both flavors.

Flavor A > Very fancy, very fast, very expensive:
This would be a large piezo-electric element, typically several
centimeters in length and 2 cm in width, either made of a "stack" of
smaller piezo disks or a single cantilevered block. They are made by
Physik Instrumente, and by Burleigh. In either case, from the outside
it'll just look like an aluminum bar or cylinder with a 3 mm diameter
cable hanging out the back, and someplace to screw in a small pipette
holding attachment. You can mount it approximately the same way you
would mount a headstage (but there are some additional vibration
issues to worry about, because the headstage doesn't recoil, whereas
this thing most definitely does). These are driven by very strong
voltage sources (> 1000 volts), they usually move extremely fast, but
they also usually have a quite limited degree of total movement (200
microns max?). One would mount the flowpipes or theta tube to the
front end, via a little screw-clip or something, which can be easily
homemade, or probably purchased at exhorbitant cost from the company.
The lateral movement of the pipette is executed via a piston-like
movement of the piezo, at least for the Physik stacks.

Flavor B > Not fancy at all, very fast (but not as fast), relatively
cheap:
This is what I prefer. I routinely get 200 microsecond 10-90% solution
rise and fall-times, so definitely fast enough for rigorous kinetic
work most of the time. However it's largely homemade, so if you're
looking for plug & play technology and have a large budget, go with
the Physik or Burleigh systems.
   Here the piezo element is a little tiny wafer-thin chip called a
"bimorph", about 10 mm x 20 mm x 1 mm. These are bought separately
from Morgan Electro Ceramics, and only cost $35 each. BUT, Morgan now
has a $3000 minimum limit, so you would probably want to split a batch
of 80 bimorphs between a few different people. You can use the same
bimorph for a year or more if you take good care of it (which means,
don't bash it with anything). Eventually they wear out after many tens
of thousands of steps.  You have to solder leads to this yourself to
connect to a voltage or current source. We use a linear amplifier from
WPI so we can give bidirectional steps, and control the degree of
motion in an analog manner. We need to do this because we are often
hopping between more than two pipes (usually 3-4, depending on the
specific expt, see J. Neurosci. 18:8590 for some examples of complex
step protocols). You usually can't do this easily with Flavor A,
because of the limited motion. Anyway, the bimorph only needs tens of
volts, so any old SIU can be used to drive it, especially if you only
need two barrels. The bimorph is mounted by clamping the back corner
with whatever clip is handy and sturdy. A black binder-clip would
actually work. You have to make your own piece for holding the
pipette. I use a tiny piece of perspex that I cut a groove in with a
hacksaw, then tapped some holes for small teflon screws to clamp the
pipette down with. This is then epoxied onto the front end of the
bimorph.
The bimorph bends when you apply a current or voltage, so the total
movement of the flowpipes basically just depends on how long they are.
You can get several hundred microns of movement easily, which is
useful for multi-barrel expts on patches, or expts when you want to
perfuse micro-islands with whole neurons and dendritic arbors growing
on them.

Both of these flavors have their own drawbacks, and regardless of what
I said about plug & play before, there WILL be significant fiddling
around required in order to a) get the solution exchange into the
submillisecond range while simultaneously b) avoiding the phenomenon
of "ringing" (wiggling of the pipette movement due to applying a very
rapid jerky step). Lots of different tricks may come into play here,
which will be different depending on what system you use.

Cheers,

Matt







More information about the Neur-sci mailing list