Is imaging cells with a Dimension V possible?

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John Thornton replied on Sat, Feb 11 2012 8:50 AM

rated by 0 users

Suggested by Bede Pittenger

Hi Alex,

Yes, you can definitely image cells with the Dimension V.  It would be good to know a little more detail about the problems that the student is having.  However, I can provide some general guidelines below that may answer their question.

You can image the cells in contact or TappingMode in fluid with the Dimension V.  You would choose similar probes as with other tools with this application.  For contact, low spring constant silicon nitride cantilever - the 0.06 N/m on the DNP or the 0.01N/m cantilever on the MLCT.  For Tapping, the 0.32N/m cantilever on the DNP probes are good a good start,

When using the fluid cell, be sure to use the protective fluid skirt that goes around the base of the scanner guard and the sides of the fluid cell to protect the scanner when imaging in fluid.  We have had fluid cells with and without the piezo in the probe holder to oscillate the cantilever with Dimension design.  The current design has the piezo in the tip holder, and is called the Direct Drive Fluid Cell for Dimension.  If using the fluid cell without the piezo, the Z Modulation parameter will need to be set to Enabled before the cantilever can be tuned.  If the fluid cell has the piezo, then this step can be skipped.  

First, align the laser on the probe before placing the tip in solution.  The laser path will change slightly when the solution is added, so he will probably need to make a slight adjustment with the laser position knobs along the axis of the cantilever.  Then, adjust the PSD knobs so that you have a nice sum and vert defl signal near zero.

When placing the probe in solution, I like to use a pipette to place a drop of the fluid on the end of the probe before I place it in the solution with the sample (petri dish, glass slide, cover slip, etc.).  This will help prevent air bubbles and make it easier for the probe to go into the solution.  At this point, you may want to go into Navigate/Focus Surface to bring the probe into the solution a little bit more.  Then, focus on the probe (Locate Tip), then focus on the surface (Focus Surface/Navigate).  In general, I will recommend that operators do this step in air if the sample is dry at the beginning of the experiment, but this is not the case with cells.  The optical image will be reflected light, so you will not see the cells as well in the optics as with an inverted scope, but you should still be able to see them and focus on the surface.

At this point, if running Tapping, tune the cantilver in fluid.  You will usually find a peak between 8 to 12Hz.  Setup the RMS amplitude to around 300mV.  For AutoTune to work here, you will need to change the Start and End Frequencies to be slightly large than this range, and reduce the Target Ampltude from 500 to 300.  For that reason, I will often run the tune manually, but either way will work.

When you are running the Dimension in air, the tip and sample are 1mm apart when you go through focusing on the tip and sample.  In fluid, they will end up farther apart (~1.4mm) once you have gone through this routine.  So, it will take longer to go through the slow part of the engage routine (this is no longer an issue in the current Dimension products).  This is not really a problem for contact mode, but it may go slower in Tapping.  Watch for false engagements.  If the system engages, put in a slight decrease to the Amplitude Setpoint in Tapping, or slight increase to the Deflection Setpoint in Contact.  If the Z center has a visible jump toward the Extended part of the Z range, then you may be false engaged.  You can hit the Engage button again and see if it continues to step toward the surface.

Once engaged, adjust your parameters and see how it goes. If running Tapping, you can check the cantilever oscillation by bring up Cantilever Tune (when engaged, you may have to go through the pull down menus, not the tuning fork icon), and put in a 50nm tip offset and check the peak at your driving frequency.  If needed, you can use the Offset cursor to readjust the operating frequency. 

You might try this first in contact mode, then Tapping.  I like to get engaged at small scan sizes, and then zoom out once I have a stable engagement.  The fact that the cells are fixed should make them relatively straightforward to image, but you never know.  Sample prep is always important for this type of application.  If they are not held down well, you may not be able to image them.

I hope this helps.  Let me know if I can be of further assistance.

John

john.thornton@bruker-nano.com