Using WETSEM Capsules

Purpose:
!
Using the WETSEM capsules is required for biological samples and allows for
the imaging of samples that are in solution or wet.
Materials:
!
QX-102 capsule or QX-102 calibration capsule, QX imaging buffer, sample
(unless using the calibration capsule), Poly-L-Lysine (positively charged coating), Poly
[sodium-4-styrenesulfonate] (negatively charged coating), Distilled water, QX aspirator
tips, MP-10 multi-well plate, and SEM.
Quick Tips:
IMPORTANT: QX-102 capsules are much taller than regular sample holders, be sure
to lower the stage all the way down before using any capsules to avoid SEM damage.
QX-102 capsules are 10.6mm tall and this should be taken into account for imaging
The capsules are fully sealed when the wings of the liquid dish and sealing stub are
aligned
Take extreme care with the capsule membrane. Do not touch, set facing down unless
on multi-well plate, and use caution when filling even when using QX aspirator tips.
Unused capsules have a shelf life of 18 months from production date and should be
stored in a dry and dark environment at room temperature.

Preparing Samples:
NOTE: QX-102 imaging buffer is formulated to reduce damage to the sample and is
recommended for use with any imaging done with the QX-102 capsule for best result.
- Charged particles in solution require a membrane coating based on the charge of the
particle so the particles will be more attracted to the membrane giving better image
results
Poly-L-lysing to attract negatively charged particles
Poly [sodium-4-styrenesulfonate] to attract positively charged particles
IMPORTANT: Both coatings need to be diluted to 0.1% w/v before adding to capsules.
- Neutral Particles will be attracted to the window without any coating
NOTE: The QX aspirator tips are designed to not allow the pipet to touch the
membrane, however, if using a regular pipet tip us the utmost caution when filling.
I.

Place capsule in multi-well plate and remove the sealing stub.

II.

Coating the capsule membrane

1.

Apply 15 l of 0.1% Poly-L-lysine or Poly [sodium-4-styrenesulfonate] solution

to the liquid dish and incubate for one hour at room temperature. Longer
incubation times, up to overnight, also give good results. Keep the liquid dish
filled with water until applying the sample.

2.

Remove the solution and rinse the liquid dish twice with distilled water.

3.

Alternatively, remove the water and dry the liquid dishes for later use.

III. Apply sample to coated membrane

1.

Apply 15 l of the suspension in the the liquid dish and incubate for one hour a
room temperature.

2.

(Optional) Wash with distilled water several times and exchange with 15 l
QX-102 imaging buffer prior to imaging.

3.

Replace sealing stub onto the capsule and twist until the wings are aligned.

IV. Ready to image sample.

Imaging Samples:
NOTE: Acceleration Voltage is recommended to be between 15 - 30 kV with an
acceptable minimum of 10 kV. Where working distance is recommended for 8 - 12
mm with an acceptable distance of 6 - 15 mm.
1. Use the same protocol for SEM use when inserting the capsule and beginning
sequence as with any other sample, with the adjustment for the capsule height.
2. Focus in on the metal grid on the window first to get preliminary focus, then adjust
contrast until the grid is very bright. (As shown below)

3. Move stage to one of the windows in the grid, lower acceleration voltage to 13 - 20 kV
change the detector to backscatter while changing the settings to use Z-Contrast.
(Detectors > Detector Settings > Drop down to BSED > click Z-Contrast)
4. Find sample and use standard focusing methods to get clearest images. (Use of the
manual contrast adjustment provides the best images when using backscattering).
5. Adjusting the spot size can also drastically improve the image quality. A spot size of 5
produced very nice images using the calibration capsule, as seen below.
6. Every sample will require different settings, so adjusting the settings is crucial when
trying to achieve the best images when using backscatter and WETSEM capsules.

SEM Images:
1.

Calibration sample