Wet SEM Analysis of Quantum dot emulsions - Rice University

Wet SEM Analysis of Quantum dot emulsions
performed by Ove Thompson (FEI, Senior Field Applications Engineer),
Dr. Angelo Benedetto (SEM facility Manager, Rice University),
Roy Meszlenyi (Research lab manager, Dr. Vicki Colvin’s lab)
November, 3, 2006
Chemistry Department, Rice University, Houston Texas
Abstract
Aggregation study of Quantum dot particles in Oil in Water emulsions.
1. Introduction
Standard SEM (Scanning Electron Microscopy) method applies high Vacuum in the
sample chamber for Analysis. Under Vacuum conditions the emulsion evaporates and
changes the morphology of the sample. The Wet SEM Instrument system set up uses
Temperature, Pressure and Water injection to control humidity conditions in the
chamber to preserve the emulsion in the liquid physical state allowing optimal Analysis
conditions. Feasibility tests were performed to reconfirm if Wet SEM Instrument set up
and method is useful for the visual study of Qdot aggregation in Oil in Water emulsion.
2. Experiment section:
2.1. Materials and Preparation
Test sample emulsions were prepared by Neera Gopee based on experimental protocol
Appendix B, (Scope of Work/ Synthesize and Characterize Quantum dots, Dr. Dean’s
protocol). For feasibility Test of the Wet SEM method and Instrument set up: control/ blank oil
in water emulsion and sample oil in water emulsion containing Amino (PEG) Qdot 655
(cat#BX36-0012, lot# 0705-0050, Qdot corporation) was used.
If feasibility test analysis are successful the Qdot sample emulsion shipment of September 20,
2006 containing Wyu621, lot#061506 will be used as requested.
2.2. Characterization
A drop of sample emulsion applied by a Micropipette was placed in the Microscope chamber
in an open conical shaped stainless steel specimen holder. Images and a video of the emulsion
were obtained using a FEI, Quanta Field emission Scanning Electron Microscope, Model
Q400F operating at 20 kV. The image mode was set on secondary electron mode. The average
Magnification range was 400x to 3000x. Minimum of 200x, Maximum of 9800x
Test run 1: Blank control emulsion. The electron beam of the SEM system was pointed in the
center of emulsion. Sample chamber condition was Room temperature & Humidity (exact
Temp. and Humidity not recorded)

Test run 2: Qdot emulsion. The electron beam of the SEM system was pointed in the center of
emulsion. Sample chamber condition was Room temperature & Humidity (exact Temp. and
Humidity not recorded)
Test run 3: Blank control emulsion: The electron beam of the SEM system was pointed close
to edge of the sample. Sample chamber condition was:
Pre-conditioning: This step is necessary to ensure optimal humidity conditions Starting
Condition Temperature 275 K= 2º C, 100% Humidity, 8 Torr Pressure
Room temp.: Temperature increase to 295k= 22º C, ~30% Humidity, 8.1 Torr Pressure. Hold
chamber conditions. Characterization Analysis phase of emulsion.
Drying: Temperature decrease to 273 K= 0º C, ~ 100% Humidity, 2.44 Torr Pressure. Apply
Vacuum to dry sample. Morphology Analysis of emulsion.
Room temp.: Temperature increase to 293 K= 22º C, ~30% Humidity???, 2.44 Torr Pressure.
Hold chamber conditions. Check Morphology.
Test run 4: Qdot emulsion: The electron beam of the SEM system was pointed close to edge
of the sample. Sample chamber condition was :
Preconditioning: Starting Condition Temperature 3º C, 100% Humidity, 7 Torr Pressure
Room temp.: Temperature increase to 22º C, ~30% Humidity, 5.7 Torr Pressure. Hold
chamber conditions. Characterization Analysis phase of emulsion: visual study of Qdot
aggregation.
Drying: Temperature decrease to 0º C, ~ 100% Humidity, 5.6 Torr Pressure. Apply Vacuum to
dry sample. Morphology Analysis of emulsion. Apply Humidity: Rehydration phase of
emulsion.
Room temp.: Temperature increase to 22º C, ~30% Humidity, 4.95 Torr Pressure. Hold
chamber conditions. .Check Morphology of rehydrated emulsion
40º C: Temperature increase to 40º C, low Humidity, 1.9 Torr Pressure. Hold chamber
conditions. .Check Morphology at higher Temperature condition
60º C: Temperature increase to 60º C, low Humidity (probably dried out), 1.9 Torr Pressure.
Hold chamber conditions. .Check Morphology at higher Temperature condition
At the end images were taken of the dried Qdot sample in secondary electron mode and Back
scattering mode. In secondary electron mode an Energy Dispersive X-Ray Spectroscopy (EDS)
was performed and X-ray energy emission spectrum was taken to obtain Elemental Analysis
data.
4. Results and discussion
Test run 1: If the electron beam is pointed in the center of Blank/ control emulsion the heat
of beam causes bubbles. The gray background is the surface of the Emulsion. Except for the
bubbles which appear on the surface of emulsion occasionally no detailed morphology of the
emulsion or can be obtained in this experimental set up. To download video (use Microsoft
Media player) please go to we blink:

Test run 2: The effect is the same as in Test run 1. If the electron beam is pointed in the
center of the Qdot emulsion the heat of beam causes bubbles. The gray background is the
surface of the Emulsion. Except for the bubbles which appeared on the surface of the emulsion
occasionally no detailed morphology of the emulsion or Aggregation of Qdots can be obtained
in this experimental set up. To download video (use Microsoft Media player) please go to we
blink:
Test run 3: To download video (use Microsoft Media player) please go to we blink:
No useful data could be obtained from Test run 1 & 2. In this experimental set up the electron
beam was pointed close to the edge of the stainless steel sample holder (see video). The edge
of the sample holder served as reference point to help visualize the emulsions Interface to the
holder. The following Pre-conditioning, Room temperature, Drying steps were applied to the
Blank/ control emulsion to develop optimal experimental conditions to visualize details of
Qdot Aggregation in the subsequent Test run 4.
Pre-conditioning: This step is necessary to ensure optimal humidity condition to preserve
In this phase the Interface between emulsion to the left of the video (gray background, bubbles)
and the sample Holder to the right (bright white area with ripples) can be clearly distinguished.
Room temp.: This is the desired Characterization condition (Room temp. and humidity) for
Analysis. In this phase violent bubbling (Popcorn effect) of the emulsion (left gray region) can
be observed probably caused by the heat of the electron beam as it scans along the Interface.
Zooming into the emulsion region revealed forming of the bubbles and the surface morphology
of the emulsion droplets turned from a smooth gray surface (observed in Test run 1 & 2) into a
“Strawberry” type shape. No details other than bubble forming could be obtained.
Drying: This step was performed to check if the dried form of the emulsion reveals more of
the emulsion details. Temperature decrease to 273 K= 0º C, ~ 100% Humidity, 2.44 Torr
Pressure. Apply Vacuum to dry sample. Morphology Analysis of emulsion. . In this phase
enhanced violent bubbling of the emulsion (left gray region) can be observed probably caused
by the vacuum and the heat of the electron beam as it scans along the Interface.
Room temp. The “dried emulsion” was put back on Room temperature to check emulsion
behavior. As the electron beam scans along the sample the “Strawberry” type of emulsion
droplets developed and additional micro bubbles on the surface of droplets. No details other
than additional bubble forming could be obtained.
Test run 4: To download video (use Microsoft Media player) please go to we blink:
The sample chamber conditions in Test run 3 were applied to the emulsion containing Qdots in
the hope that the different steps would visualize Qdots and Aggregation of Qdots in the
emulsion.
Pre-conditioning: This step is necessary to ensure optimal humidity condition to preserve
In this phase the Interface between emulsion to the left of the video (gray Background,
bubbles) and the sample Holder to the right (bright white area with ripples) can be clearly
distinguished.
Room temp.: This phase is the desired Characterization condition (Room temp. and humidity)
for Analysis. Much less bubbling (Popcorn effect) of the emulsion (left gray region). More like
a “Moon surface visual effect” can be observed probably caused by the heat of the electron
beam as it scans along the Interface. Zooming into the emulsion region revealed forming of

ubbles. The surface morphology of the emulsion droplets turns from a smooth gray surface
into bubbles with much smoother surface than observed in Test run 3. No formation of
“Strawberry” type shape droplets like observed in Test run 3. No details other than bubble
formation could be obtained.
Drying: This step was performed to check if the dried form of the emulsion reveals more of
the emulsion details. Apply Vacuum to dry sample. Morphology Analysis of emulsion.. In this
phase the emulsion seems to be stable and no enhanced violent bubbling of the emulsion like in
Test run 3 (left gray region) could be observed probably caused by the vacuum and the heat of
the electron beam as it scans along the Interface. No details of Qdot aggregation could be
obtained
Rehydration: This step was added in this Test run. The rehydration was performed after the
drying step.
Room temp.: The Rehydrated emulsion was put back on Room temperature to check emulsion
behavior. Emulsion seems to be stable: As the electron beam scans along the sample the no
building of “Strawberry” type of emulsion droplets or development of additional micro bubbles
on the surface of droplets could be observed. No details of Qdot aggregation could be obtained.
40º C: This step was added in this Test run to simulate the Temperature conditions in the
emulsion Preparation protocol (See Appendix B, Dr. Dean’s protocol). The Temperature was
increased to 40º C to investigate what effect on the emulsion it has. The morphology started to
reveal probably the “waxy” part 1 (Polyglyceryl-3 distearate + Cetearyla alcohol + mineral oil)
of the emulsion.
60º C: This step was added in this Test run to simulate the Temperature conditions in the
emulsion Preparation protocol (See Appendix B, Dr. Dean’s protocol). The Temperature was
increased to 60º C to investigate what effect on the emulsion it has. The morphology we
believe reconfirmed the “waxy” part 1 (Polyglyceryl-3 distearate + Cetearyla alcohol + mineral
oil) of the emulsion: We suspect that the emulsion breaks down to a fluid (mineral oil) and
white colored spheres started to appear which might be crystals of the buffer used to prepare
emulsion..
At the end images of the Qdot sample were taken in secondary electron mode (see Figure 1)
and Back scattering mode (see Figure 2 and 3). In secondary electron mode an Energy
Dispersive X-Ray Spectroscopy (EDS) was performed and X-ray energy emission spectrum
was taken to obtain Elemental Analysis data. (shown in Figure 4 and 5)

Figure 1. SEM micrograph of Qdot emulsion in secondary electron mode (20 kV, 1250x
magnification)
Figure 2. SEM micrograph of Qdot emulsion in back scattering Image mode (20 kV, 9822x
magnification)

Figure 3. SEM micrograph of Qdot emulsion in back scattering Image mode (20 kV, 1250x
magnification)
Figure 4. Energy Dispersive X-Ray Spectroscopy (EDS) of Blank/ control emulsion: X-Ray
Energy emission spectra, Elemental Analysis, SEM secondary electron mode.

The three last Peaks: Cr, Fe, Ni are probably from the stainless steel sample SEM holder. The
rest of the Peaks are from the emulsion and contamination traces of the Water (cleaning sample
Holder with Water).
Figure 5. Energy Dispersive X-Ray Spectroscopy (EDS) of Qdot emulsion: X-Ray Energy
emission spectra, Elemental Analysis, SEM secondary electron mode. The last peak Cr was
probably from the stainless steel sample SEM holder. The Detector picked up Cd and Se
Signals which reconfirmed that emulsion contained CdSe Qdots. The rest of the Peaks are from
the emulsion and contamination traces of the Water (cleaning sample Holder with Water).
3. Conclusion
Test run 1 and 2 showed that no useful data can be obtained with the electron beam pointed in
the middle of the emulsions. Test run 3 and 4 demonstrated that if the electron beam is
pointed at the edge of the sample holder that this area plays an important factor as a Interface
reference point in visualizing the emulsions surface. The different chamber conditions in Test
run 4 did not visualize the Qdot aggregation in the emulsion. However interesting visual
effects in the morphological behavior of the surface of the emulsion exposed to the different
sample chamber condition could be observed and were described in the Experimental section
of this report: The Qdot emulsion seemed to be much more stable regarding the bubble “Pop
corn” and “Strawberry” droplet visual effects. One hypotheses might explain the better
physical stability of the Qdot emulsion when exposed to the heat generated by the electron
beam: The CdSe might act as a kind of conductor conduit guiding the beam right through the
emulsion to the bottom like a lightning rod the lightning to the ground. Whereas the blank/
Control without the electrical conductor is longer exposed to the heat of the beam causing
violent bubbling. The second more likely and reasonable hypotheses is simply by comparing
the blank/ control emulsion to the Qdot emulsion by eye: The Qdot emulsion seemed to appear

in the delivered Micro centrifuge tube more viscose than the blank/ control which might
explain the more stable behavior when exposed to the electron beam. Video, secondary
electron and backscattering Image mode pictures have been possible to make but give no
useful visual data about Qdot Aggregation. The X ray energy emission spectra could be
obtained. Due to the nature of the emulsion mixture and trace contaminations (from cleaning
Water etc.) quantitative Analysis has to evaluated cautiously and the data shown should be
treated as preliminary Test results.
Qualitative Elemental Analysis: Cd and Se signals most probably from the Qdots have been
picked up by the detection system.
Wet SEM Method evaluation:
1. The Wet SEM Instrument system (FEI, Quanta Field emission Scanning Electron
Microscope, Model Q400F) used in this experiment with the standard set up with open conical
sample holder is not useful for the study of Quantum dot aggregations in Quantum dot oil in
Water emulsions.
2. A different, closed Quantomix sample holder with membrane system using the
backscattering Image mode for Analysis might give more useful data results. For a concept to
this Technology please visit Quantomix Web home
page:http://www.quantomix.com/tecnology02.html
For detailed description of the sample cell: Click on QX-Capsule movie Icon.
3. A new “cutting edge” Instrument system is under development by the FEI Microcoscope
Company and might be a breakthrough method for Analysis of oil in Water emulsions:
Wet STEM (Scanning Transmission Electron microscopy). This system uses the Transmission
Electron Microscopy (TEM) technique and will be able to scan the sample like SEM under
humidity controlled conditions. Instead of surface scanning the electron beam will scan
through the emulsion and might give useful Analysis data about Qdot aggregation. Scattering
effects of the emulsion and cross section diameter (certain drop sample size limitations) of the
emulsion might be factors which may interfere and limit the Analysis. Test Analysis can be
performed with a Microscope prototype system which will be available in the United States in
January 2007.
4. The next generation of Microscope system which might be useful for emulsion sample
Analysis will be the Wet Cryo STEM system. It is the same Technology like the Wet STEM
described above. The major advantage of this system will be 3 dimensional Images of cross
sections of the frozen sample. This system is still under development and will be available in
the near future. The disadvantage of this instrument will be the high purchasing cost.