The Analytical Imaging Facility has access to four Eppendorf microinjection systems that all essentially the same but differ slightly in vintage and functions.

One system is typically installed on CCD#4.

The others float from station to station.  They may be used for microinjection or translocation pipette assays.


Microinjection instructions

Setup Checklist:
  2. Set up microscope as it should be set up for normal imaging.
  3. Set up microscope for phase contrast with proper Kohler illumination.
    phase.gif (6726 bytes)
  4. Load the needle.  Mount it on the machine. Guide the needle to the center of the field.  The rest is an art...

Any time you walk away from the station, please
   o  leave a note on it that work is in progress
   o  switch the injection mode from I1 or I2 to X for the older system or to Standby for the newer systems.

needle-in-dish.jpg (22573 bytes)


dish_of_cells.gif (1490 bytes) If cells are growing on coverslips in culture dishes, there are three surfaces that the cells may be living on.  They may adhere to the bottom of the plastic dish, to the bottom of the coverslip, or to the top of the coverslip.


dish_of_cells_needle.gif (2186 bytes)You must be focused on the cells on the top of the coverslip because the needle has access only to the top layer of cells.


dish_z_limit.gif (2130 bytes)The simplest way to set a Z limit is to lightly touch the side of one of the cells.  Then type the magic key sequence:   set; z-limit; enter.

set_button.gif (259 bytes)zlimit_button.gif (216 bytes)enter_button.gif (210 bytes)

or on the newer systems, just by tapping the "Limit" button.

If this Z limit is slightly too high or slightly too low, you can modify the Z limit in fractions of a micrometer.
z_limit.gif (14072 bytes)

To cancel a Z limit,cancel_button.gif (218 bytes)zlimit_button.gif (216 bytes).


If you want to label the cells with a fluorescent dextran so you can see immediately the amount injected.

Standard 0.5-1 mg/ml Dextran 10,000 MW lysine fixable (Molecular Probes; stays after fixation, handy for IF). You can go much lower with the dextran if your solution is extra thick (we have used about 10 times less when he was injecting DNA in the nucleus).  Standard is 1:30 from 2 mg/ml stock.


Checklist!! You may need to provide the following things for microinjection:

  1. You need to purchase Femptotips II from Eppendorf or you need microneedles from another source.  If you pull your own needles, you need a container to store needles in. Here are two sample containers:
    The container at the left holds more than 30 needles and is available commercially. The container at the right is a simple culture dish with plasticine (non-drying clay) to hold the needles so that the tips cannot be damaged.
    Needles may be coated to make them more slippery.
  2. Microloaders. When in stock, we sell microloaders, which are specially thin pipette tips, for $5.00 each. We recommend you buy a box of Eppendorf order number 5242 956.003.

  3. Bring your own pipetteman to each session. Each needle will be filled with 1 to 3 ul. We do not supply the pipetteman because they walk off with users.
  4. Your cells must be on coverslips. These may be in just about any culture dish of your choice. But the bottoms of plastic culture dishes themselves are not flat enough for microinjection. Only glass coverslips are smooth and flat enough. 
  5. The solution to be injected must be filtered with 0.1 or 0.2 um filters. An alternate which may work is a high spin of the liquid to be injected. Two examples of filters are Corning disposable sterile syringe filters 21012-3 and Fisher centrifuge filters 09-719-31.    Here's the official Eppendorf method:  brinkman/prep.htm.
The Dept. Anatomy incubator in the common equipment room is available for AIF use.   All dishes in this incubator must be free clearly labeled with your lab and the date.  The culturing hoods may be available by special request; do not use them without specific permission from the lab designated to the hood.


Need to locate injected cells later?

EMS sells Cell-Vu gridded coverslips(cat. no. 72264-23). 1-800-523-5874.

We use Bellco's 22x22mm etched coverslips #1916-92525. I think we got them
through Fisher.  Their coverslips have photoetched grids with 520 alphanumerically
coded squares.

There are coveslips from eppendorf. The name is CELLocate coverslips.


Microinjection needle coating instructions

If you pull your own needles, you may find that they stick less to the cells if you treat them as follows:

You will need

  1. Take HMDZ out of container by sticking needle through rubber stopper on top and cover rubber stopper with parafilm to keep sealed.
  2. Squirt 100ul of Hexamethyldisilazane into the bottom of the container.
  3. Label the container with date & time & HMDS added.
  4. Leave sealed overnight or longer.
  5. First time opening container of needles, do it in a hood to pull off unbound HMDZ.


If you supply your own needles...

grip-head.jpg (22219 bytes) needle-in-dish.jpg (22573 bytes)
The AIF has lost a number of these grip heads for microcapillaries.  They just simply disappear.  This is a problem because 1.) when people need to do microinjection, there aren't any and 2.) they cost $50 each.

Therefore, all microinjection users who use their own needles instead of the commercially available Eppendorf ones need to purchase their own grip head.  We sell them at cost.

Ask Michael in advance and he will give you a grip head in a labeled baggie (so you may clip it to your notebook).


Selected Bibliography of papers using microinjection apparatus in AIF:


Other Links

return to index next page