Here’s a quick description/’review’ of the method for making pipettes fluorescent by quantum dot coating described in Andrásfalvy et al 2014. We used the protocol for some in-vivo 2p guided sharps recordings a while ago and really liked how cheap, simple and versatile the method was.
We previously used a flourescent dye (alexa) in our pipette solution, and while giving great contrast, this resulted in fluorescent dye accumulation after repeated recordings. Qdots just leave a visible but not overly bright ring where the pipette enters the tissue, which turns out to be pretty helpful in guiding subsequent recordings.
Another potential upside of qdots might be being able to use different channels for a fluophore on the inside of the pipette and for the qdots.
Finally, we found that qdots work better than alexa for thin pipettes, where the tiny inside diameter makes it very challenging to locate the tip of the pipette with a 2p.
We used the protocol from:
Andrásfalvy et al.: Quantum dot–based multiphoton fluorescent pipettes for targeted neuronal electrophysiology, Nature Methods 11, 1237–1241 (2014)
from Andrásfalvy et al. 2014
Pipette bubble number
This method makes use of the ‘bubble number’ test, which is a quick and easy way for measuring the tip opening diameter of micropipettes, and for making sure that pipettes dont get clogged with the qdots:
Bowman & Ruknudin: Quantifying the geometry of micropipets, 1999 Cell Biochemistry and Biophysics
The method estimates the tip opening by measuring the pressure required to expel bubbles from the pipette in methanol. It’s possible to precisely measure tip diameters with this, but here we just used it as a ballpark estimate and to very quickly check for clogging.
Attach the pipette to a syringe that allows you to apply pressure, dip the pipette in methanol while holding light pressure, and steadily increase the pressure until small bubbles rise from the tip. Fine adjustments of the pressure should now reveal a precise point at which bubbles either appear, or not. With some calibration, this point should allow a good prediction of the tip resistance. I usually get down to ~5ml compressed air volume from 10ml, for ~15MOhm pipettes, but this value depends on a few factors, and should be calibrated.
Preparing the quantum dots
In order to ‘paint’ the glass pipettes with the qdots, they need to be suspended in hexane.
The qdots usually come in tuolene, we’ve tested this protocol with red (630nm) ones from Sigma.
To get the qdots out of the tuolane and into hexane, we need:
A small 2000g bench top centrifuge, a pipette, some centrifuge compatible tubes, acetone, methanol, and hexane.
- Wash the qdots in a 50/50 mixture of acetone and methanol. We’ve used up to 2 parts of the aceton&methanol mix to 1 part of the qdots (1 mg/mL in toluene).
- Spin down until either a pellet forms, or sometimes the qdots just adhere to the wall of the tube – as long as the solvent can be removed cleanly with a pipette, and replaced with new clean solvent, it’s alright. This step can be repeated to further wash the qdots. We haven’t noticed any differences after the 1st wash, so we stuck with just doing 2.
- Once the qdots are suspended in clean acetone/methanol, remove the acetone/methanol and add as much hexane to the qdots as desired. A less diluted solution will require fewer (possibly only one) dip of the pipette to coat. We found that dilution to the original volume (of the acentone methanol toluene qdot mix) worked well and gave us good control over the pipette coating.
Coating the recording pipettes
Now, just dip the tips of the pipettes in the qdot hexane solution, and dry off. It is very important to keep enough pressure on the pipette to keep the dots from clogging the tip – you always want enough pressure to keep bubbling the tip. A quick bubble test in methanol after the qdot dipping is an easy way to check if the tip was clogged. If needed, multiple dips can be used to accumulate more qdots. The coating can be checked by eye with a UV light, just from a little LED flashlight.
Keeping the qdot solution around for more than a few days might require adding new hexane every now and then, most small plastic containers that we tried failed to stop evaporation. Once the solution dries out completely it can be pretty tricky to get the qdots back into a nice solution.
