| Low biological noise probe
for high-speed neural imaging of electrical signals |
OI announces its new
"blue" line of high-speed voltage sensitive dyes for cortical
imaging, originally synthesized by Rina Hildesheim, and available
exclusively from OI (patent pending; blue dye series includes RH-1691,
and RH-1692). Voltage sensitive dyes (VSDs) are optical transducers
of membrane potential changes. Applied to the brain, they bind to
the external surface of the membranes of living cells without interrupting
their normal function. Once introduced into a preparation, VSDs rapidly
(within a microsecond) alter the intensity and/or wavelengths of fluorescent
light they emit as a function of changes in neuronal membrane potential.
 Priority
in supply of dyes will be given to Optical Imaging's Brain Imager customers (in case of supply shortage)
Photodynamic Damage and Bleaching
Photodynamic damage depends on illumination
intensity. At 4-10 msec time resolution, saturating a camera (10,000,000
electrons/pixel) with the dye fluorescence, using a 1:1 tandem lens,
allowed averaging of about 256 trials, each of 1 sec, without significant
photodamage. Bleaching proved very variable in cat visual cortex
| RH
1691:
mol. weight
∈ (EtOH)
∈ (H2O) |
589
~57000
~37000 |
RH 1692:
mol. weight
∈ (EtOH)
∈ (H2O) |
555
~57000
~37000 |
RH 1838:
mol. weight
|
539
|
|
 |
The similarity
between the cortical dye signal from a small population of neurons
and an intracellular recording.
Two traces showing simultaneous intracellular and optical recording
for 6 seconds, performed in deeply anesthetized cat: a condition in
which spontaneous changes in membrane potential are highly synchronized
in a large population of neurons. The intracellular recording is depicted
by the green traces. The action potentials were truncated. The optical
signal from the population next to the electrode is depicted by the
red traces. Modified from Sterkin et al., 1998
NOTE:
The following instructions are recommendations only; you may wish
to adjust concentrations and procedures as appropriate for your own
laboratory.
Because of difference in staining, different
VSDs may be optimal for different preparations. Blue dyes have been
designed and tested for in vivo use in mammalian cortex, particularly
monkey and cat.
- Store dry
- Store below 5°
- Prepare fresh dye solution for each experiment
- Dye is supplied by Optical Imaging as
dry crystals in 10 mg vials.
- Typically, between 0.5 mg (cat) and 2.0
mg (monkey) are used per hemisphere for cortical applications.
The amount required depends on the volume of dye required to perfuse
your preparation.
- Stock solutions are not stable when stored.
Before beginning an experiment, it is necessary to make fresh
solution from dry dye.
| Making Fresh Stock Solution |
- Using a clean, dry spatula, pick out the
approximate amount of dye you need for the day's experiment. It
is difficult to weigh small amounts of dye, so a final concentration
adjustment should be performed using a spectrometer (see below).
- Dissolve the dye into the buffer of your
choice, using about 7 ml of buffer per mg of dye. Buffer solution
protocols differ among laboratories; check the literature to find
a buffer appropriate for your particular preparation.
- Pass the dye solution through a filter
of .45 or .25 micron pore size.
- Dilute a 50 µl sample of the dye
solution to 1 ml in buffer, saline, or water.
- Place the diluted sample in a cuvette,
and measure the optical density at 580 nm.
- By adding dye or buffer, adjust the dye
concentration of the stock solution to an OD of 4-7 (OD 0.2 to
0.35 in the 20-fold diluted measurement sample).
- Keep the solution protected from strong
light until it is used.
Staining proceeds by allowing dye held in
an elevated pistonless syringe to flow slowly into the cranial chamber's
inlet port via connecting tubing. The chamber itself is closed,
except for a second outlet port, which allows free flow to another
pistonless syringe, also attached by tubing. Normal staining time
is three hours.
This continuous flow procedure is required
to avoid a stagnant layer of dye-free CSF forming above the pia.
The set up:
- One of the chamber's port tubes is connected
to an open-topped syringe containing the dye solution, held about
10 cm above the cortex. This forms the inlet side.
- The other port is connected to a second
open-topped syringe, held at slightly lower level, so as to allow
very slow flow (~ 5 ml/hour). This forms the outlet side. Continuous
adjustment to maintain even flow is not required.
- To recirculate dye, adjust the outlet
syringe's height upward and the inlet syringe's downward, reversing
the direction of flow. Reversing every hour is typical.
- At no time should additional pressure
be applied to the cortex.
| Catalog
number |
Type |
Minimum
order quantity |
| RH
1691 |
Blue
dye |
10mg |
| RH 1692 |
Blue dye |
10mg |
| RH 1838 |
Blue dye |
10mg |
To
order dyes please contact info@opt-imaging.com
Selected references - Dyes
|
