VOLTAGE SENSITIVE DYES

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.

Voltage sensitive dyes

Priority in supply of dyes will be given to Optical Imaging’s Brain Imager customers (in case of supply shortage)

Photodynamic Damage and Bleaching

Dye Characteristics

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

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

TBD
RH 1691
mol. weight
ε (EtOH)
ε (H2O)
589
~57000
~37000
RH 1692
mol. weight
ε (EtOH)
ε (H2O)
555
~57000
~37000
RH 1838
mol. weight 589

Dye Characteristics

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.
  • The dye should be stored dry and in the dark.
  • The dye should be stored at +4°C (-4°C is better but there is a larger risk of absorbing humidity).
  • Beware! Allow the vial to reach the room temperature before opening it.
  • Desiccations (storage under vacuum) is a good precaution for maximizing the dye lifetime (but not many groups use it).
  • Staining solution should be kept in the dark. It can be maintained in the refrigerator for up to a month. Check the OD before repeated use. If there is a drop one should use a fresh solution.
  • 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.
  • Weighting the dye for each imaging session is not recommended.
  • Since it is difficult to get the dye weight for a single staining the concentration shall be determined by spectrophotometry.
  • One usually uses an optical density of 2-4 (1 cm path length cuvette) at 630 nm. Since most spectrophotometer can measure optical density only up to a value of 3OD (transmission of 0.001) a cuvette of 2-4 mm path length is needed. (cheap plastic cuvettes are available).
  • A few grains of a solid dye shall be added to 2cc of ACSF (artificial cerebral spinal fluid) with the aid of a delicate small spatula or glass pipette (make sure the pH is in the range of 7.4 ; lower pH destroy the dye).
  • Before the measurement the cuvette shall be closed with parafilm and shaked strongly so that the dye will be thoroughly mixed.
  • After the initial measurement either the first solution is diluted or more grains shall be added.
  • After gaining experience one can estimate the OD by eye.
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 setup:
  • A minimal amount of staining solution is topically applied to the cortex after dura removal (never let the cortex dry during or after the surgery).
  • The dye shall be mixed over the cortex thoroughly.
  • The staining solution shall be replaced every 15 minutes.
  • Staining duration 1.5-2 hours.
  • The brain shall be covered with aluminum sheath to keep it nearly in the dark. After starting the dye with the ACSF shall be replaced several times
  • One should let the dye equilibrate across the cortical surface for another 30 minutes or so before starting the measurement.
Catalog Number Type Minimum order Quantity
RH 1691 Blue Dye 10mg
RH 1692 Blue Dye 10mg
RH 1838 Blue Dye 10mg
RH 2080 Blue Dye 5mg