We often have to pull long, tapered pipettes for anatomical tracer injections, virus injections, or for injections for bulk loading of calcium dyes.
Our preferred geometry for these pipettes differs from those of patch pipettes (which are short and stubby but must have a very smooth opening of 1.5-3µm), and the requirements aren't as stringent as for electrodes that need to impale cells (sharp pipettes). The ideal geometry involves a long, gradual taper that ends in a tip of the desired size (3-7µm for bulk loading pipettes, 10µm for biocytin / most anatomical tracers, ~30µm for virus injection).
There are 3 major steps:
- Pulling the pipettes
- Breaking the pipette tips
- Examining the tip under the microscope to ensure it is broken to the correct size
There are 3 specific hazards:
- Electricity and heat: When pulling pipettes, there is a tremendous amount of electrical current flowing through the filament; there is both an electrical hazard (shock) and a heat hazard, so do not touch the filament or adjust the filament when the device is on.
- Visual hazard: When the filament is hot, the filament gives off UV light, so do not look directly at the filament when pulling pipettes.
- Broken/sharp glass: The pipettes are made of glass; these pipettes can break, and pulled pipettes have very sharp points. Take care not to cut yourself, and dispose of all pipettes in sharps waste.
1. Pulling the pipettes
Pipettes that exhibit a gradual taper are best pulled on a vertical puller that can be set to use gravity as the only pulling force (that is, no electric solenoid assistance). In the VH lab, we have a Kopf 700C puller. The glass pipette of appropriate size is loaded onto the puller; the heat is set to the appropriate amount, and the pipette is pulled. DON'T LOOK AT THE FILAMENT DURING PULLING; it gives off UV light.
- Bulk loading pipettes: we use 1mm glass; we use a 3.5 turn filament with wide spacing; the heat should be set so that the current flowing through the loop is 16A (usually the heat setting is 55-60). The pipette end that is on the bottom will generally have the best geometry.
2. Breaking the pipette tips
Next, simply break the pipette on a KimWipe. This is basically all motor memory and takes some practice. Watch someone who knows how to do it.
- Bulk loading pipettes: fold a KimWipe so that a bit of it billows up from the surface; gently touch the pipette into the billowed KimWipe straight on.
3. Examining the tip under a microscope to ensure it is broken to the correct size
Next, look at the pipette tip under a calibrated microscope with at least a 40X objective. In the VH lab, we have an old Zeiss microscope with a 40X lens, and a reticle that we have calibrated to be 0.95µm per fine division at 40X.
- Place the pipette on a slide that has a little clay on it to support the shaft of the pipette.
- Prepare to focus on the shaft about 1cm up from the pipette tip. Move the stage left and right so this part of the shaft is approximately underneath the objective. The working distance of the 40X lens on our scope is about 1-2mm, so, with the naked eye, move the pipette so it is about 1-2mm away from the objective.
- To "find" the pipette, one will have to move the pipette under the microscope to see the shadow of shaft moving around. To do this, look at a side view of the pipette in the scope, to see if you will need to move the stage forward or backward to bring it into focus. Then, look through the microscope while you move the stage forward or backward; the pipette's shadow should come into view.
- Then adjust the focus knob to bring the shaft into focus. Move the stage so that you focus on the tip of the pipette.
- Verify the tip is the correct size, or try again if it is not.
- It is important that you have a clear view of the tip; be sure to bring in the condenser so that it is almost touching the slide. In the VH lab, Steve has found that polarized viewing increases the resolution a little bit (use the PH setting on the viewing path and the corresponding filter on the condenser path). You should be able to clearly see tips as small as 1µm. If you cannot see tips this small, then there is something wrong with the microscope setup.
Step 1: Pulling a bulk loading pipette. The current is about 16A (heat 57, solenoid off).
Step 3.3: Look at the side view to see if you need to move the stage forward or backward:
Step 3.2/3.6: There is a 1-2mm working distance for the 40X lens, and the condenser should be very close to the glass slide.
Step 3.6: Make sure the polarized glass filter (bottom) in the condenser path is IN:
Step 3.6: Use the PH setting on the observer light path: