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Find Surface settings need to be tuned for the specific sample and probe combination. For many samples,
the standard settings of Find-
Surface will be suitable and, thus, tuning won’t be needed. However, for
very soft or adhesive samples tuning will be required. When tuning Find Surface settings follow these
steps:
1.
Submerge the probe in the medium;
2.
Press “Find Surface”;
3.
Set up “Config exp” with a single indentation in D
-mode (explained in the next chapter);
4.
“Run Experiment”;
5.
Look at the load-indentation curve, adjust Find Surface settings (
increase ‘
Z above surf
ace’
,
activate adhesion mode) and indentation profile accordingly (increase displacement) and repeat
the procedure until out-of-contact measurements are obtained.
Caution:
If the same probe is used to measure the range of samples e.g. from very soft
to stiff or very adhesive to non-adhesive in a high throughput manner, tune the
settings for the softest and most adhesive sample, and then the same settings will
automatically work for the stiff and least adhesive samples.
3.2
Setting transit height and focal plane
Next, one can set up the transit height of the probe stage and focal plane of the objective stage to be able
to fasten up high-throughput measurements when moving from well to well. Follow these steps:
1.
Pres “
Find Surface
”
.
2.
Note the position of the probe stage Z(µm), e.g. 23 640 µm.
3.
If all wells are filled with the same volume of material and, thus, has approximately the same
surface position, fill in ~1000 µm lower value in Transit (µm), e.g. 22 000 µm or move up by 1000
µm and press
“
Set transit height
”
.
4.
If samples in different wells have different positions of the surface, then you will need to find the
one with the highest surface position and set transit height based on that well. Otherwise, you
can also set transit height much higher e.g. just below the level of the medium, however, this
going to make the measurements longer.
5.
Next, bring back the probe close to the surface of the sample by pressing Find-Surface again.
6.
Move up the objective stage in steps of 500 µm until you start seeing the shadow of the probe.
Then, decrease the step size and move up until you find the probe in focus. Probe tip position in
the medium will differ from its position in the air, thus, adjust the
“Find probe” position as
described in Section 2.5.
7.
Now move down the objective stage until you see the surface of the sample in focus and press
“Set focal plane”. Keep in mind that the probe tip is only in focus when touching the sample during
indentation.
Now, when moving from well to well, you can quickly go to transit height and focal plane, and use these
positions to set up high-throughput experiments (see
).
Caution:
keep in mind the working distance of your objective. If the sample
’
s surface
is high (thick sample), you might not be able to reach it with the objective due to the
working distance of the objective. Use a long working distance objective or lower
magnification objective. Compatible options from Nikon are x4, x10, x20, x40.
