I want to trigger the injection via a hand control (computer mouse). Is this still possible?
Yes, it is still possible; you can order this accessory separately (order number: 5252 070.011).
Can I use the hand control for the previous FemtoJet model also for the FemtoJet 4i/4x?
Yes, the hand control is compatible with the new FemtoJet 4 generation.
Can I transfer my old protocols to the new device (e.g. same parameter settings)?
Yes, in general the protocols are transferable.
Which grip head is included in the delivery package of the FemtoJet 4i/4x?
It is the Grip head set 4 size 0. It contains one grip head which is suitable for capillaries with an outer diameter of 1.0-1.1 mm. The set contains also o-rings and distance sleeve as spare parts.
How do I convert from hPa to psi?
1 hPa = 0.0145 psi; 1 psi = 68.95 hPa. Using our FemtoJets you can choose in the ‘Menu’ the required unit (hPa/ psi).
What parameters are decisive for the injection volume, and how can I determine these?
The injection volume depends on the set injection parameters (injection pressure, time) and the type and the exact opening diameter of the capillary, as well as the residence time of the capillary in the cell. Furthermore, the injected volume depends on the type of cell, the targeted compartment/position within the cell, and the solution to be injected. Due to the above described factors, it is not possible to give exact settings for a specific injection volume. The user always has the option of determining the injection volume for the present experiment by making a type of ""calibration curve"".
Possible methods of determining the volume:
1. Injection of an enzyme that is normally not present in the cell (e.g. luciferase) in 50-100 cells and determination of the injection volume by subsequent enzyme assay:
– Add pure luciferase (Sigma, final conc. 2 mg/mL) to the injection solution.
– Inject an exact number of cells with the FemtoJet 4i/x with a known setting.
– Lyse cells and determine the luciferase activity from the extract with a luminometer (as described in the literature).
– Prepare a dilution series from the injection solution (2 mg/mL) and determine the luciferase activity in this series.
– Plot a curve of the measured activity versus volume.
– Read off the injected volume from this curve and the measured activity of the cells.
– Calculate the volume per cell from this read volume.
2. Injection of a defined radioactivity into one water drop (20-100 times), followed by measurement of the radioactivity (Geiger counter). The approximate injection volume can be calculated from the radioactivity of the solution.
3. Injection of fluorescence (generally coupled with a carrier protein), quantification of the injection volume by means of the detection system.
4. Injection of the sample of interest into oil droplets: measurement of the vesicle sizes using microscope/camera software and calculating the volume of the vesicle.
When using our Femtotips, the determined settings can be retained from capillary to capillary. Typical volumes for adherent cell injections are 0.1-0.3 pL for injection into the cytoplasm and 0.01-0.05 pL for injection into the nucleus. Typical volumes for pronuclear injection into mouse zygotes are approx. 1-2 pL.
G. Minaschek, J. Bereiter-Hahn, and G. Bertholdt (1989): “Quantification of the Volume of Liquid Injected into Cells by Means of Pressure”; Experimental Cell Research 183, 434-442 (Explanation of the calculation of the injection volume depending upon pressure and time.)
What can you recommend with regard to sample preparation of the injection solution for microinjection?
Samples should always be centrifuged (for 15 min at maximum speed in a microcentrifuge) immediately before the capillaries are loaded.
Use the Eppendorf Microloader for filling Femtotips (from the rear). Use it only once. The liquid should be extracted from the top of the tube. Fill the liquid as close as possible to the capillary tip end. Make sure that no gas bubbles are in the liquid phase within the glass capillary.
If your solution contains proteins, you should work as quickly as possible after the capillary has been loaded. If the injection solution is not introduced into the medium immediately, there is a risk of dry out of the injection solution within the capillary and thus blocking the Femtotips.
Please find more detailed information in our Application Note No. 8 "Sample preparation for microinjection", which we are happy to send you upon request.
What should be taken into account when injecting proteins?
It is sometimes difficult to inject protein solutions, as any contamination can block the capillary. Therefore, the solutions must be prepared very carefully (e.g. cleaned by centrifugation columns or ultracentrifugation). Before loading the Femtotip, the solution should always be centrifuged for at least 10 min at the highest speed.
What are recommended concentrations for microinjection of protein or DNA?
As a guideline for microinjection into adherent cells, 20-200 ng/µL DNA and 1-5 mg/mL protein are recommended, depending of the promoter or the activity of the protein. Using antibody solution for injection, the concentration should be 10 to 50 times higher than for in vitro tests (e.g. Southern blot).
Do you know a publication which explains how to determine the diameter of microinjection capillaries and also indicates the influence of the diameter on the injection volume?
Yes, there is a publication:
Experimental Cell Research 210, 260-267 (1994): "Microinjection Technique: Routine System for Characterization of Microcapillaries by Bubble Pressure Measurement"
Can you recommend any parameters for the injection of DNA into adherent cells?
Typical parameters for the injection of DNA into mammalian cells using Femtotips or Femtotip II:
– Compensation pressure (pc): 25-50 hPa
– Injection pressure (pi): 80-200 hPa
– Injection time (ti): 0.3-0.5 sec
The parameters need to be optimized according to each experiment. The optimal way for penetration into adherent cells is to perform the injection movement at an angle of 45°.
What is the typical injection volume?
For adherent cells the optimal injection volume would be ~10 (-20) % of cell volume which corresponds to approx. 0.1-0.3 pL for injection into the cytoplasm and approx. 0.01-0.05 pL for injection into the nucleus. Under the microscope you will hardly observe a short faint swelling of the cell or nucleus.
Typical volumes for pronuclear injection into mouse zygotes are approx. 1-2 pL. A swelling of approx. 10% in diameter can be observed.
Which dextran is most suitable for microinjection?
All dextrans can be used irrespective of the molecular weight. For a special application, namely compartment-specific injections, we use dextran with a molecular weight in excess of 60 kDA, as it will not pass intracellular membranes.
Which Eppendorf devices do you recommend for the injection into insect embryos?
We recommend the micromanipulator TransferMan 4r or InjectMan 4 in combination with the electronic microinjector FemtoJet 4x, requiring an external pressure source. For penetration of objects with a strong shell (chitin) our PiezoXpert is advantageous transmitting additional mechanical forces to the tip of the capillary as short and precise impulses.
Which injection pressure do I have to select for microinjection into fish embryos?
Microinjections into embryos are normally performed manually with an injection pressure of 300-500 hPa and a very short injection time of 0.1-0.2 sec. The high injection pressure prevents the injection needle from clogging.
What is the typical injection volume when injecting into drosophila embryos?
The typical injection volume ranges between 60-100 pL.
Can you recommend any literature about microinjection into plant cells?
1. Schnorf, M. et al., An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis, Transgenic Research 1, 23-30 (1991)
2. Brücker et al., Microinjection of heme oxygenase genes rescues phytochrome-chromophore-deficient mutants of the moss Ceratodon purpureus, Planta 210, 529-535 (2000)
What is important for the microinjection into plant cells?
For penetration of the rigid cell wall of a plant cell the use of piezo-assisted micromanipulation would be a solution: The Eppendorf system composed of the manipulator InjectMan 4, electronically connected to the PiezoXpert allows to synchronize the capillary injection movement with piezo impulses, facilitating the penetration of rigid structures. Injection pressures up to 6000 hPa can be applied by the electronic injector FemtoJet 4i.
In case injection pressures higher than 6000 hPa are needed, the manual injector CellTram 4r Oil can be used (max. pressure 20.000 hPa).
What equipment is required to inject fluorescent dyes into plant cells and to then aspirate the entire content of the cell?
We recommend using the FemtoJet 4 (max 6000 hPa) or the manual CellTram 4r Oil microinjector (max 20,000 hPa) in combination with the micromanipulator InjectMan 4 or TransferMan 4r, which enables material to be injected into cells with high turgor pressure and to be extracted out of the cell. In case of free floating cells (>40 µm) a second micromanipulator and microinjector are reuierd for holding the cells.
Is it possible to control the manipulators or FemtoJet 4 via PC?
Yes, it is possible. Please ask our Sales Specialist.
Is it possible to perform automated injection steps with the FemtoJet 4?
Yes, semi-automated injection steps into cells can be performed when the FemtoJet® 4i/x is connected to the Eppendorf Micromanipulator InjectMan® 4. The electronic connection of both units (Eppendorf microinjector and manipulator system) allows performing to synchronize the injection movement of the micromanipulator and the injection pressure of the FemtoJet 4i/x. This semi-automated step is easily triggered by one click onto the joystick key of the manipulator.
How to connect the InjectMan 4 with the FemtoJet 4i/x?
The interface cable Order No 5192 082.007 is required which is included in the package of the InjectMan 4.
What is the compensation pressure pc that you can set on the FemtoJet 4?
The compensation pressure pc ensures that no culture medium flows into the capillary during microinjection experiments. Insertion of the capillary into the medium of the cell culture dish would induce capillary forces causing liquid flow into the injection capillary diluting the injection material. To prevent this, a permanent low compensation pressure pc is set which should lead to a permanent slight flow-out of liquid from the injection capillary. The individual pressure level can be determined in a preliminary test.
Which compressor do you recommend for the FemtoJet 4x pressure supply?
We recommend the Jun Air 3-4. If a non-oil system is required, we recommend the compressor Jun Air OF 301-4B. Since this non-oil compressor generates noise it should be placed separate from the workstation: Homepage Jun air
How do you remove pieces of broken glass capillaries from the capillary holder?
For this purpose we included a cleaning stylet with the capillary holder. In case it is lost, we offer a kit, the so-called O-ring-set 4 for grip head sets 4 (Order no. 5196 086.007) that contains this tool. Alternatively, you can also use a thin metal wire.
The injection tube connecting the microinjector device FemtoJet 4i/x with the Capillary Holder is missing. What do I have to order?
The 2 m long Injection tube (with bayonet mount) for connecting the FemtoJets to the Capillary holder can be ordered using following Order no. 5252 070.054
What is the outer diameter of the capillary holder 4?
The outer diameter of the capillary holder 4 is 4 mm, as it is of our predecessor, the so-called 'universal capillary holder'.
I want to use my own capillaries with Eppendorf microinjectors. Which capillary grip head would fit my tips?
All Eppendorf microinjectors (FemtoJet 4 and CellTram 4) are equipped with the same model of capillary holder 4. Different capillary grip heads are available for the use of capillary tubings with different outer diameters. Find below to determine which grip head suits your needs.
Grip head set 4 size 0: for microcapillaries with 1.0-1.1 mm outer diameter, Order no. 5176 210.003
Grip head set 4 size 1: for microcapillaries with 1.2-1.3 mm outer diameter, Order no. 5176 212.006
Grip head set 4 size 2: for microcapillaries with 1.4-1.5 mm outer diameter, Order no. 5176 214.009
Grip head set 4 size 3: for microcapillaries with 0.7-0.9 mm outer diameter, Order no. 5176 207.002
How can the different grip head sizes be distinguished?
The grip heads have different numbers of notches. The grip head 4, size 0 has no notch at all; the grip head 4, size 3 has, for example, three notches.
I read in the manual that the piston position of the CellTram 4 Air has an influence on the responsiveness in sample control. What are the recommended working positions / piston settings with the CellTram 4 Air?
The lower the selected piston position is, the more direct the responsiveness of your sample in the capillary is. This means, at piston positions of 1-3 the rotation of the drive wheel causes a faster or stronger movement of the sample within the capillary. In case you choose a piston position of 7-9, the sample in the capillary is moved less or more slowly per rotation of the drive wheel. Since many users have individual preferences in their injector responsiveness, we recommend to identify your optimal piston position for your particular application (e.g., holding of oocyte, transfer of sperms or cells, etc.) by testing different piston positions.