U.S. Patent May 5, 2020 Sheet 17 of 63 US 10,639,634 B2

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constraints. Conventional experimental measurements and
calculations indicate that for thicknesses of a liquid layer
below~1 jum, resolution is determined by SNR and estimates
of broadening do not match the experimentally obtained
resolution for liquid cells or samples on thick substrates. The
STEM SNR is estimated by calculating the intensity reach-
ing the detector from elastic scattering in the liquid and
membrane, which contributes the background, and the signal
is the intensity reaching the detector via elastic scattering
from the particle of interest. This method of calculation is
applicable for estimating the resolution on the order of ~1
nm, but will only be a rough approximation for systems
which permit lattice resolution because the elastic scattering
calculations do not account for Bragg diffraction. Nonethe-
less, using this method we estimate an SNR sufficient to
obtain STEM resolution below 0.2 nm for the conditions
applicable to the liquid cell described here, assuming a
minimum SNR of 3.

Next, we consider the constraints that must be satisfied to
enable quantitative, high-resolution EELS to be performed.
First we focus on the design criteria for obtaining EELS of
the liquid (rather than a solid particle in the liquid). At liquid
layer thicknesses, t, much less than the inelastic scattering
length, 2, the liquid EELS signal will be small compared to
that from the membranes, while at large thicknesses (U3)
plural scattering obscures both the valence and core-loss
regions. A good EELS signal can be obtained if, while
minimizing multiple scattering, each electron experiences an
average of one inelastic scattering event, i.e., / should be

=1. To make a more detailed estimate of the optimal liquid 3

thickness we calculate the expected SNR of the O K
core-loss edge using simulated EEL spectra assuming the
atomic density of oxygen for water. The SNR is determined
by calculating the expected O K signal over a 30 eV window

using hydrogenic cross sections while the noise is calculated 3

from the square root of the intensity under the same window,
including the background contribution. The background
intensity is given by the sum of the energy losses due to the
Si L edge at 99 eV, the N K edge at 402 eV and the combined
multiple scattering from core loss and plasmon losses. To
approximate the background intensity including multiple
scattering, the core-loss spectrum from the SiN, membrane
is simulated and convoluted with a simulated low-loss
spectrum. The low-loss spectrum is simulated as a series of
Gaussian plasmon peaks where the total plasmon intensity is
determined by Poisson statistics for a given t/A. The value of
vA used is the total value for the membranes and liquid,
again calculated using an Iakoubovskii approximation. All
the calculations are performed using computer programs for
the core-loss edges are approximated by using the Sigmal3
and Sigmak3 hydrogenic cross section programs and the
low-loss is simulated by a SpecGen program. The simula-
tions indicate an optimal O K SNR for a liquid thickness of
100 nm which corresponds to a total t/A. value of 0.9 (FIG,
1). For low-loss EELS where energy-loss features from the
liquid and the membrane will overlap, the membrane thick-
ness should be minimized to prevent mechanical deforma-
tion or fracture and the limits of the fabrication approach.
To estimate the capabilities of this liquid cell for core-loss
spectroscopy of a nanoparticle in liquid, we calculated the
expected SNR for Fe,O, for a cell with a 250 nm thick liquid
layer and estimate that a 2 nm thick nanoparticle should
produce a detectable signal for 1 nA of beam current, a 5s
dwell time and a 20 eV window over the Fe L,,; edge.
The liquid layer thickness can be adjusted during the
fabrication over a wide range, depending on whether spec-
troscopic information is needed from the liquid itself, or

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from nanoparticles in solution; thinner liquid layers being
preferred for EELS analysis of nanoparticles and thicker
layers for EELS of the liquid. Reducing the liquid-layer
thickness below a certain value, however, will not be worth-
while if the membrane thickness cannot be reduced because
the scattering from the membrane will determine the SNR
for small liquid layer thicknesses

For EELS analysis of the low-loss region for liquids
where features from the membrane and the liquid will
overlap it will be advantageous for low-loss scattering from
the liquid to dominate, requiring that the liquid V/A. be greater
than that of the membrane, if ) is determined primarily by
the low-loss intensity. For 50 nm SiN, membranes and
water, the liquid layer thickness should be at least 130 nm.
Low-loss intensity from the membrane can be removed from
a spectrum of the liquid by deconvolving a membrane-only
reference spectrum though there will always be some loss in
SNR.

Ultimately, the precise values of the desired liquid thick-
ness will depend on the liquid or nanoparticles being studied
and the experiment to be conducted. Using calculations of
SNR for the O K edge from water gives an optimal liquid
thickness of ~100 nm and based on considerations for
low-loss spectroscopy the liquid thickness should be at least
130 nm. A good target liquid thickness for EELS is then in
the range of 100 nm to 200 nm. In this thickness range the
Fe L signal should be detectable for hematite nanoparticles
~2 nm in thickness.

FIG. 56 shows an estimated SNR normalized to the
maximum value for the O K edge as a function of liquid
thickness for a cell with two 50 nm SiN membranes.
Optimal liquid thickness for studying the O K edge of
liquids is ~100 nm.

The sample cell is designed to resist mechanical failure.
SiN membranes have high strength and exhibit a high level
of fracture toughness. In conventional cell designs, windows
are unsupported across the viewing area (supported only at
the edges of the window) and are subject to pressure-driven
bulging. The relationship between membrane deflection and
pressure, for a square membrane, is given by

L834E15* a

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a

where G, is the initial stress in the membrane, E the Young’s
modulus, v the Poisson’s ratio, t the thickness, a the half-
width of the membrane, and 6 the deflection (note that for a
given value of a, the deflection is approximately a factor of
2 larger for a long rectangular membrane). Equation 1 is
valid for the situation when the thickness t is much smaller

5 than the half-width, a; for smaller widths, finite-element

analysis (FEA) is necessary. The options for controlling the
deflection are limited: the size of membrane, a, can be
reduced, or the initial stress increased.

Reducing the width of the membrane below approxi-
mately 20 um is impractical for fabrication reasons (typical
wafer thickness variations cause membrane widths to vary
by as much as a factor of two at these sizes), while the initial
stress cannot be increased above 1 GPa without compro-
mising the strength of the membrane. Even for a long

rectangular SiN membrane of width 20 jum, thickness 50 nm,

and initial stress of 0.3 GPa, the center deflection for each
membrane will be approximately 140 nm under 1 atmo-


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US 10,639,634 B2

Sheet 61 of 63

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etch, or selective, or timed downstream plasma etch, or
timed or end-pointed ion milling.

Forming etchant trench 90 on second surface 32 to expose
a portion of oxide layer 84 (FIG. 49) can include the use of
aselective, or timed wet chemical etch, or selective, or timed
reactive ion etch, or selective, or timed downstream plasma
etch.

Etching a portion of substrate 2 from first surface 8 to
second surface 32 to expose a portion of first structural layer
62 in an area that corresponds to viewing reservoir 10 (to be
formed) and a fluid port (to be formed) (FIG. 50) can include
the use of a selective, or timed wet chemical etch, or
selective, or timed reactive ion etch, or selective, or timed
downstream plasma etch.

Selectively etching sacrificial member (68, 70, 72) to
remove sacrificial member (60, 70, 72) from between first
structural layer 62 and second structural layer 86 to form
sample cell 200 (FIG. 51) can include the use of a selective
wet, or vapor phase chemical etch.

Disposing third oxide layer 98 on second surface 32 to fill
etchant trench 90 (FIG. 52) can include physical vapor
deposition, chemical vapor deposition, low-pressure chemi-
cal vapor deposition, plasma-enhanced chemical vapor
deposition, atomic layer deposition, plasma-enhance atomic
layer deposition.

Disposing protective layer 310 on third oxide layer 98
(FIG. 53) can include spin or spray coating.

With reference to FIG. 54, patterning protective layer 310;
etching third oxide layer 98 to expose viewing reservoir 10;

and etching third oxide layer 98 to expose electrodes (e.g., 3

50, 52) can include optical, electron-beam, or ion beam
lithography, selective, or timed, wet chemical etching, or
selective or timed reactive ion, or downstream reactive
plasma etching, or timed or end-pointed ion milling.

In forming sacrificial member (68, 70, 72), as shown in 3

FIG. 55, tapered profiles (e.g., wall 40) a broad angular
distribution of atomic deposition that occurs in atomic
sputtering in combination with an undercut lift-off mask
produces a smooth transition from micro- to nanoscale
features to provide a flow transition from microfluidic flow
to nanofluidic flow.

Fluid sampler 100 has numerous beneficial uses, includ-
ing containing a fluid in sample cell 200 and performing
transmission electron microscopy on the fluid disposed in
sample cell 200.

Fluid sampler 100 has numerous advantageous and ben-
ficial properties. In an aspect, fluid sampler 100 provides a
substantially uniform and thin fluid layer to enable high-
resolution imaging and spectroscopy, a means of confining
the chemistry of interest, which may be corrosive, or oth-
erwise deleterious to a microscope or other measurement
apparatus, inside the sampler, thus protecting the micro-
scope or other measurement apparatus and enabling the
study of a wider range of chemistries, a means of achieving
higher pressures than can otherwise be attained, a means to
control fluid flows with substantially improved temporal
control

The articles and processes herein are illustrated further by
the following Example, which is non-limiting.

EXAMPLE

High-Resolution Imaging and Spectroscopy at High Pres-
sure ina TEM.

This Example describes quantitative core-loss electron
energy-loss spectroscopy of iron oxide nanoparticles and
imaging resolution of Ag nanoparticles in liquid down to

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0.24 nm, in both transmission and scanning-transmission
modes, in a novel, monolithic liquid cell developed for the
transmission electron microscope (TEM). At typical SiN
membrane thicknesses of 50 nm the liquid layer thickness
has a maximum change of only 30 nm for the entire TEM
viewing area of 200 umx200 jm.

Transmission electron microscope (TEM), with its ability
to deliver atomic-scale spatial, and <100 meV spectroscopic
resolution, has enabled countless breakthroughs in materials
science. Environmental TEMs (ETEMs) were developed to
study reactions in gaseous environments. ETEMs can be
limited in terms of the pressures and chemistries that can be
accessed. Study of materials and processes in liquid envi-
ronments is challenging and requires the use of special cells
to encapsulate the liquid and protect the microscope. Cre-
ating a thin, uniform liquid layer in a device that permits
high spatial and spectroscopic resolution was difficult.

Conventional liquid cells comprise a pair electron-trans-
parent windows between which a layer of liquid is sand-
wiched. Control over the membrane separation is achieved
by polystyrene microspheres, silicon dioxide or epoxies, or
wafer bonding. Scanning TEM (STEM) imaging in microm-
eter-thick layers allows atomic resolution in gases. These
conventional two-piece cells cannot accurately and repro-
ducibly define a thin liquid layer and maintain its uniformity
over a large observation area. The pressure difference
between the inside and outside of the cell can cause signifi-
cant membrane deformation (bulging) and large variations
in liquid layer thickness. Both the membrane bulging effect
and the difficulty of using spacers to reliably control the
liquid layer thickness pose a major challenge to atomic-
resolution TEM and quantitative electron energy-loss spec-
troscopy (EELS).

The sample cell in this Example overcomes these limits
and includes a pillar-supported, monolithic liquid cell that
eliminates spacers and limits bulging. This Example shows
a combination of atomic-scale imaging, in both TEM and
STEM modes, and quantitative EELS using this design. We
discuss the design of the sample cell and identify structural
and material parameters that affect its performance.

The effect of design parameters on the performance of the
device was considered. We examined membrane and liquid
thickness to achieve atomic-scale resolution in TEM and
STEM modes. Conventionally, the image resolution in TEM
mode for thick samples or a thick liquid cell has been
estimated by considering the effect of chromatic aberration
and assuming an energy spread estimated by the so-called
Landau energy distribution. This Landau distribution is
observed for thicknesses larger than t/A=3 (t is thickness and
2 is the inelastic mean free path) where individual plasmon
and core-loss features are obscured. Chromatic aberration
causes inelastically scattered electrons to be focused to a
different plane from the elastically scattered electrons, con-
tributing a background to the bright-field image; therefore,
there will be an increasing loss of image contrast as the
fraction of inelastically scattered electrons rises. For a total
SiN thickness of 100 nm, a 75 nm thick layer of water, and
an accelerating voltage of 300 kV, we find that the fraction
of unscattered electrons is close to 25%. The remaining 75%
of electrons, which are scattered by the membranes and
liquid, include both inelastically and elastically scattered
electrons. High-Resolution imaging is possible with a loss in
contrast and a diminished signal-to-noise ratio (SNR), and
resolution is weakly dependent on thickness in this range.

Image resolution in thick samples in STEM mode with an
annular dark-field detector is determined by either broaden-
ing of the incident probe by multiple scattering or SNR


US 10,639,634 B2

Sheet 2 of 63

May 5, 2020

U.S. Patent

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19

the pillar edge to between the pillars. No significant varia-
tion in these values was measurable across the entire cell
viewing area

FIG. 59 shows the HRTEM image and corresponding fast
Fourier transform (FFT) demonstrating lattice fringes out to
0.065 nm of a 40 nm diameter Fe,O, nanorod. The image
was acquired with a total SiN thickness of 100 nm, and a
liquid-layer thickness of ~80 nm (~40 nm over the nanorod)
in the beam path. 10 frames were aligned and summed for
the image to improve the SNR giving a total acquisition time
of 2.5 s. The membrane and liquid contribute a background
and noise, but do not prevent high-resolution imaging. We
note that the observation of a peak in the FFT corresponding
to 0.065 nm does not indicate a resolution of 0.065 nm, but
does demonstrate good stability of the system. For thick
liquid cells, the best resolution in TEM mode is achieved for
particles on the lower membrane, while the opposite is true
for STEM. For a particle on the bottom membrane, beam
broadening in the upper membrane and liquid degrades the
STEM resolution; in TEM mode, the image of a particle on
the upper membrane is degraded by broadening as the
electrons pass through the liquid and lower membrane
STEM images using an annular dark-field (ADE) detector
offer better signal-to-noise than TEM images for thick
samples and have therefore been preferred previously for
imaging in thick liquid layers. To explore the range of
applicability of the top-bottom criterion we have performed
lattice imaging in both STEM and TEM modes at various
locations across the cell membrane area and we find that
lattice resolution is attainable on the same object in both
TEM and STEM, repeatedly and reproducibly. FIG. 60
shows an example of a cluster of Ag nanoparticles exhibiting
0.24 nm lattice fringes ((111) planes of Ag) in both TEM and
STEM images. This demonstrates that the “top-bottom”
effect is negligible for an appropriately designed cell.

With reference to FIG. 61, panel A shows an HRTEM
image of an Ag nanoparticle cluster in liquid. Panel B shows
an HRSTEM images of the same cluster imaged in panel A.
Both images show lattice-fringe resolution of 0.24 nm. The
insets are the Fast Fourier Transforms of the images dem-
onstrating the resolution.

With reference to FIG. 62, panel A shows a core-loss
EELS spectrum of an iron oxide nanoparticle in liquid. The
ratio between the L, and L, peaks enables the identification
of the iron oxidation state as Fe**. Panel B shows core-loss
EELS spectrum from the liquid showing an N peak from the
SiN membrane and an O peak generated primarily by the
liquid.

FIG. 63 shows O K edge EELS data from a liquid-
containing region and a vapor-containing region of the same
liquid cell after deconvolution and intensity normalization.
Some of the O K signal (the vapor spectrum) comes from the
SiN, membranes.

Both core-loss and low-loss EELS on particles in liquid
and liquids alone are possible with the cell. Panel A of FIG.
61 shows EELS data obtained from a ~40 nm diameter
FeO, nanorod in water/isopropanol acquired with a total
exposure time of 20 s. Near-edge fine structure is observable
in both the O K edge at 532 eV and the Fe L, , edge at 708
eV despite the background from the liquid and membranes.
Quantification of the Fe oxidation state is possible by
measurement of the Fe L white-line ratio. A method of
white-line quantification was applied to a spectrum from a
hematite nanorod obtained with a 0.1 eV/channel dispersion;
after background subtraction and Richardson-Lucy decon-
volution with the low-loss spectrum, a double arctan func-
tion was used to subtract the continuum contribution under

30

40

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the white lines and the integrated intensity for the L, and L
peaks was obtained with 2 eV windows. The calculated
L:L, ratio is 5.67+0.1 (uncertainty is given as +one stan-
dard deviation). The uncertainty estimates are determined by
assuming the uncertainty in the intensity values for each
peak is given by the square root of the intensity (Poisson
statistics). Noise contributed from the increased background
intensity will increase the quantification uncertainty relative
to measurements in high-vacuum conditions. However, this
confirms that quantitative analysis of the near-edge structure
from particles within a liquid cell is possible. Panel B of
FIG. 62 shows core-loss EELS data from a liquid mixture of
water and ethanol. In the core-loss spectrum the N K edge
from the membrane is visible as well as a small O K edge
primarily from the liquid. The membrane also contributes to
the O K edge, but detailed comparison between spectra from
empty cells (membranes only) ad from cells filled with the
ethanol-water mixture indicates the O K intensity increases
by more than a factor of 2 when the cell is filled with liquid.

FIG. 63 shows two spectra from a cell filled with a water/
ethanol mixture, one from a liquid-containing region and
one from a nearby vapor-filled region; after deconvolution
with the low-loss spectrum and normalization of the inten-
sities, it is possible to determine the O K contribution from
the liquid itself. The thin vapor layer in parts of the cell
contributes a negligible O signal so the O signal from the
vapor region is from O in the membranes.

‘Vapor formation during imaging can create small regions
of vapor under the beam and this process can ultimately limit
the time available for data acquisition from a sample in
liquid. Although this was occasionally observed during
imaging with this cell, the data shown here were not
acquired from regions where bubble formation occurred.

While one or more embodiments have been shown and
described, modifications and substitutions may be made
thereto without departing from the spirit and scope of the
invention. Accordingly, it is to be understood that the present
invention has been described by way of illustrations and not
limitation. Embodiments herein can be used independently
or can be combined.

Reference throughout this specification to “one embodi-
ment,” “particular embodiment,” “certain embodiment,” “an
embodiment,” or the like means that a particular feature,
structure, or characteristic described in connection with the
embodiment is included in at least one embodiment. Thus,
appearances of these phrases (e.g., “in one embodiment” or
“in an embodiment”) throughout this specification are not
necessarily all referring to the same embodiment, but may.
Furthermore, particular features, structures, or characteris-
tics may be combined in any suitable manner, as would be
apparent to one of ordinary skill in the art from this
disclosure, in one or more embodiments.

All ranges disclosed herein are inclusive of the endpoints,
and the endpoints are independently combinable with each
other. The ranges are continuous and thus contain every
value and subset thereof in the range. Unless otherwise
stated or contextually inapplicable, all percentages, when
expressing a quantity, are weight percentages. The
suffix “(s)” as used herein is intended to include both the
singular and the plural of the term that it modifies, thereby
including at least one of that term (e.g., the colorant(s)
includes at least one colorants). “Optional” or “optionally”
means that the subsequently described event or circumstance
can or cannot occur, and that the description includes
instances where the event occurs and instances where it does
not. As used herein, “combination” is inclusive of blends,
mixtures, alloys, reaction products, and the like.



US 10,639,634 B2

17

sphere, translating to an undesirable liquid layer thickness
variation of more than 200% for an initial cavity thickness
of 100 nm.

Instead of reducing the total membrane width to the level
of a few micrometers, a more suitable alternative to achieve
the desired small membrane deflections is to introduce
regular support structures to periodically connect the upper
and lower membranes, equivalent to reducing the width, a,
in equation 1, while permitting the total membrane width
and viewing area to remain large (hundreds of micrometers).
FEA calculations for a pillar-supported, monolithic cell
show that membrane deflections can be reduced to accept-
able levels (s20 nm for ~10° Pa (1 atm) pressure) once the
pillar-to-pillar separation is reduced below 2 um to 3 um
(FIG. 57). This decouples the size of the viewing area from
membrane deflection and enables precise control over the
liquid layer thickness. FIG. 57 shows (panel A) membrane
deflection as a function of pillar spacing for different pres-
sures, and panel B shows membrane deflection as a function
of pressure for membranes of different thicknesses and a
constant pillar edge-to-edge spacing of 1 jum. In all cases the
initial membrane stress is 180 MPa. The inset shows a finite
element simulation of the deflections of a structure with a
membrane thickness of 50 nm and a support pillar pitch and
edge-to-edge spacing of 2 um and 1 jum respectively. The
vertical displacements in the image are exaggerated by 5x
for clarity.

With reference to FIG. 58, the sample cell was fabricated
by first depositing a layer of SiN, followed by a layer of

silicon oxide and a layer of polysilicon. Holes are then 3

etched through the poly-Si/SiO, bilayer to the underlying
SiN before the second SiN membrane layer is deposited.
Two etch ports are opened in the upper layer of SiN on either
side of the viewing area, and out of the electron beam path.

A hot KOH etch is then used to leach out the poly-Si/SiO, 3

bilayer, leaving a cavity with a precisely controlled height.
Because SiN etches very slowly as compared to Si in KOH
(3.3 nmv/h versus 2.2 jum/min), lateral etches over hundreds
of micrometers are possible in a relatively short time («2
hours), without compromising the SiN membranes, enabling
the creation of cells with large viewing areas. The etch ports
also serve as in/outlets for a microfluidic system.

Again, FIG. 58 shows fabrication of the sample cell for
fluids in which (panel A) LPCVD SIN is deposited on both
sides of a Si wafer; panel B shows deposition of sacrificial
bilayer of poly-Si/SiO,, wherein poly-Si etches laterally
rapidly in KOH, leaving the thin SiO, layer to be etched
vertically, and SiO, layer acts as a protective layer should
metal patterns be used on the lower SiN layer; panel C
shows bilayer patterning via photolithography and reactive
ion etching; panel D shows photoresist removal; panel E
shows second LPCVD SiN deposition; panel F shows etch
ports formed in upper SiN layer by photolithography and
reactive ion etch; panel G shows removal of sacrificial layer
by KOH etch; panel H shows an optical micrograph of
completed device (overall width=2 mm): inset shows higher
magnification view of pillar-supported membrane, wherein
pillar spacing is 1 um edge-to-edge, and panel I shows a
scanning electron microscope image of cross-section of
cavity between two pillars.

The small spacing between the membrane supports not
only helps to keep deflections at the few-nanometer level
(<10 nm) when the cell is in operation, it also serves to
prevent capillary forces from collapsing and sticking
together the membranes when the cell is dried after etching,
or when fluid is introduced during sample loading. The
Young-Laplace pressure for a cylindrical meniscus of radius

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40

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60

18
rand surface energy 7 is y/r. For water (y=0.073 J/m? at 20°
C.) the pressure difference across the meniscus in a 100 nm
tall cavity is approximately 1.5x10° Pa (15 atm). Using
Equation (1) and ignoring corrections for large deflections,
this constrains the maximum distance between supports to
be less than 3 um for 100 nm thick membranes. An attractive
feature of such a cell is its ability to support very high
pressures—up to 5x10° Pa (~50 atm) before there is any
danger of the membrane breaking. This may permit the
observation of catalytic processes that occur at high pres-
sure. Once completed, the cells can be loaded via a pulled-
glass micropipette and filled by capillary action through the
etch ports. The cells may then be sealed with UV-curing

resin, or, if fluid flow is desired, with a microfluidic system.

To demonstrate the capabilities of the cell, we have
performed high-resolution imaging in both TEM and STEM
modes using suspensions of Ag nanoparticles in water/
isopropanol mixtures, and collected images and EELS data
from FeO, nanorods in water/ethanol. The data were col-
lected at 300 keV using both an ETEM equipped with an
aberration corrector for the image-forming lens and a non-
corrected TEM, both with imaging filters, and with the liquid
cell in static (no-flow) mode. The HRTEM images were

collected with a parallel beam (convergence semi-angle a=0

mrad) on the aberration-corrected ETEM. The HR-STEM
images were collected using an annular dark-field detector
with collection semi-angles between 30 mrad and 75 mrad,
and a condenser aperture-spot size combination capable of a
nominal resolution of 0.136 nm. EELS data in STEM mode
(shown in panel A of FIG. 59) were collected with a
collection semi-angle B=21 mrad and convergence semi-
angle a=10 mrad. Additional EELS data were also acquired
in TEM mode with a=2.6 mrad and B=9.6 mrad (shown in
panel B of FIG. 59). EELS data acquired under these
conditions were also used to calculate the hematite nanorod
oxidation state.

With regard to FIG. 59, panel A shows energy-filtered
TEM w2. map of a cell partially-filled with a water/isopro-
panol mixture showing the liquid meniscus. The calibration
bar is in units of t/A. Panel B shows a thickness profile taken
along the line in panel A. (IMFP: Inelastic mean free path)
The maximum reduction in liquid layer thickness caused by
capillary forces in the liquid-filled region is approximately

5 25 nm. The deflection in the vapor-filled region at 10° Pa (~1

atm) is negligible.

With regard to FIG. 60, panel A shows an HRTEM image
of an Fe,O, nanorod. Panel B shows a Fourier transform of
the image in panel A demonstrating the presence of lattice
fringes out to a spacing of 0.065 nm.

Panel a of FIG. 59 isa thickness map of a liquid-filled cell
near a boundary with a vapor-containing region and a line
profile of the map is shown in panel B. The regular array of
support pillars is seen and the liquid region is on the

5 left-hand side of the image. The thickness profile indicates

that the membranes are bowed inward, rather than outward,
due to the capillary force of the liquid. The v/ value in the
liquid region decreases from ~0.9 near the pillar to ~0.8
between the pillars. Using a known value of the liquid
thickness near the pillar and accounting for the 100 nm of
SiN in the beam path indicates a} value of ~330 nm for the
liquid mixture of water and ethanol. This value is signifi-
cantly larger than values calculated for liquid water using
various approximations derived from measurements on sol-

5 ids, and the experimentally measured value for liquid water

is ~400 nm. Using this A value we can deduce a variation in
the liquid layer thickness from 90 nm to 66 nm going from


US 10,639,634 B2

23 24
14. A fluid sampler comprising: 16. The fluid sampler of claim 15, wherein the holder
a sample cell that comprises: comprises:

a substrate comprising: an armature receiver disposed on the lidded compartment
a first end; and that receives a transfer arm.
a second end opposing the first end: 5 17. The fluid sampler of claim 16, further comprising the
a first surface traversing a length of the substrate transfer arm. . ; ;
from the first end to the second end: and 18. The fluid sampler of claim 17, wherein the transfer

a second surface opposing the first surface and #1 comprises:

traversing the length of the substrate from the first an armature comprising:

10 a first armature end that is received by the armature
end to the second end; receiver of the holder,
a first port disposed in the substrate and that receives a > p
a second armature end that is arranged opposite of the
fluid; .
. d di d he sub: d in fluid first armature end and distal to the armature receiver;
a second port disposed in the substrate and in flui an electrical feedthrough disposed at the second arma-
communication with the first port; 1s ture end; and
a viewing reservoir disposed on the substrate in fluid a gasket receiver that receives a gasket for producing a
communication with the first port and the second vacuum seal in combination with a vacuum chamber.
port and that receives the fluid from the first port and 19. The fluid sampler of claim 17, wherein the transfer
communicates the fluid to the second port, the view- arm further comprises:
ing reservoir comprising: 20 a plurality of wires in electrical communication with the
a first view membrane disposed on the first surface of electrical feedthrough and comprising:
the substrate; and a first wire to communicate the first electric potential
a second view membrane disposed opposing the first from the electrical feedthrough to the first electrical
view membrane on the substrate, contactor; and
wherein the fluid is interposed between the first view 25 a second wire to communicate the second electric
membrane and the second view membrane; and potential from the electrical feedthrough to the sec-
a pillar interposed between the first view membrane ond electrical contactor.
and the second view membrane, the pillar separating 20. A fluid sampler comprising:
the first view membrane from the second view @ sample cell that comprises:
membrane at a substantially constant separation dis- °° asu fray ce
tance that is invariable with respect to a temperature a first end; .
a second end opposing the first end;
and invariable with respect to a pressure to which the first surf length of the subs
ie ceil is subject a first surface traversing a length of the substrate
fist clecirode in electrical ‘cation with th from the first end to the second end; and
a ist electro e in electrical communication with the ,, fa second surface opposing the first surface and
rst port; an oo. traversing the length of the substrate from the first
asecond electrode in electrical communication with the end to the second end;
second port, a first port disposed in the substrate and that receives a
° “ . Ps Ps
wherein the pillar, the first view membrane, and the fluid;
second view membrane are monolithic; 40 a second port disposed in the substrate and in fluid
a holder in which the sample cell is disposed and com- communication with the first port;
prising: a viewing reservoir disposed on the substrate in fluid
a transmission aperture arranged transmissively coin- communication with the first port and the second
cident with the viewing reservoir such that a probe port and that receives the fluid from the first port and
beam subjected to the fluid sampler is transmitted 45 communicates the fluid to the second port, the view-
tandemly through the transmission aperture and the ing reservoir comprising:
viewing reservoir an electrode member comprising: a first view membrane disposed on the first surface of
a first electrical contactor in electrical communication the substrate; and
with the first electrode through mechanical engage- a second view membrane disposed opposing the first
‘ment with the first electrode; and 0 ree ema on Ue SUSE nef
a second electrical contactor in electrical communica- wherein the fluid is interposed between the first view
P 5 membrane and the second view membrane; and
tion with the second electrode through mechanical . .
4 a pillar interposed between the first view membrane
engagement with the second electrode, ‘
: and the second view membrane, the pillar separating
wherein the first electrode and second electrode produce 5
1 field in respons 1 fa first °° the first view membrane from the second view
an electric field in response to application of a first membrane at a substantially constant separation dis-
electric potential to the first electrode from the first tance that is invariable with respect to a temperature
electrical contactor and application of a second electric and invariable with respect to a pressure to which the
potential to the second electrode from the second sample cell is subjected,
electrical contactor, and 60 wherein the pillar, the first view membrane, and the
the fluid electrokinetically flows from the first port and second view membrane are monolithic:
through the viewing reservoir to the second port in a fluid container disposed on the substrate and compris-
response to presence of the electric field. ing:
15. The fluid sampler of claim 14, wherein the holder a first fluid reservoir in fluid communication with the
comprises: 65 first port and that:

a lidded compartment in which the sample cell and the stores the fluid for communication to the first port:
electrode member are disposed. and


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US 10,639,634 B2

21

As used herein, “a combination thereof” refers to a
combination comprising at least one of the named constitu-
ents, components, compounds, or elements, optionally
together with one or more of the same class of constituents,
components, compounds, or elements

All references are incorporated herein by reference.

The use of the terms “a” and “an” and “the” and similar
referents in the context of describing the invention (espe-
cially in the context of the following claims) are to be
construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by con-
text. “Or” means “and/or.” Further, the conjunction “or” is
used to link objects of a list or alternatives and is not
disjunctive; rather the elements can be used separately or
can be combined together under appropriate circumstances.
It should further be noted that the terms “first,” “second,”
“primary,” “secondary,” and the like herein do not denote
any order, quantity, or importance, but rather are used to
distinguish one element from another. The modifier “about”
used in connection with a quantity is inclusive of the stated
value and has the meaning dictated by the context (e.g., it
includes the degree of error associated with measurement of
the particular quantity).

What is claimed is:
1. A fluid sampler comprising:
a sample cell that comprises:
a substrate comprising:
a first end;
a second end opposing the first end;
a first surface traversing a length of the substrate
from the first end to the second end; and
a second surface opposing the first surface and
traversing the length of the substrate from the first
end to the second end;

a first port disposed in the substrate and that receives a
fluid;

a second port disposed in the substrate and in fluid
communication with the first port;

a viewing reservoir disposed on the substrate in fluid
communication with the first port and the second
port and that receives the fluid from the first port and
communicates the fluid to the second port, the view-
ing reservoir comprising:

a first view membrane disposed on the first surface of
the substrate; and

a second view membrane disposed opposing the first
view membrane on the substrate,

wherein the fluid is interposed between the first view
membrane and the second view membrane; and

a pillar interposed between the first view membrane
and the second view membrane, the pillar separating
the first view membrane from the second view
membrane at a substantially constant separation dis-
tance that is invariable with respect to a temperature
and invariable with respect to a pressure to which the
sample cell is subjected,

wherein the pillar, the first view membrane, and the
second view membrane are monolithic, without the
presence of any spacer material.

2. The fluid sampler of claim 1, further comprising:

a first electrode in electrical communication with the first
port; and

a second electrode in electrical communication with the
second port,

wherein the first electrode and second electrode produce
an electric field in response to application of a first

0

40

60

65

22

electric potential to the first electrode and application of

a second electric potential to the second electrode, and

the fluid electrokinetically flows from the first port and

through the viewing reservoir to the second port in
response to the electric field.

3. The fluid sampler of claim 1, further comprising:

a first conduit in fluid communication with the first port
and the viewing reservoir and interposed between the
first port and the viewing reservoir, the first conduit
communicating the fluid from the first port to the
viewing reservoir; and

a second conduit in fluid communication with the second
port and the viewing reservoir and interposed between
the second port and the viewing reservoir, the second
conduit communicating the fluid from the viewing
reservoir to the second port.

4. The fluid sampler of claim 3, wherein a physical

geometry of the first conduit:

provides microfluidic flow of the fluid from the first port;
and

transitions the microfluidic flow to nanofluidic flow into
the viewing reservoir.

5. The fluid sampler of claim 3, wherein a physical
geometry of the second conduit: provides nanofluidic flow
of the fluid from the viewing reservoir; and transitions the
nanofluidic flow to microfluidic flow into the second port.

6. The fluid sampler of claim 1, further comprising:

a fluid container disposed on the substrate and compris-
ing:

a first fluid reservoir in fluid communication with the
first port and that:
stores the fluid for communication to the first port;
and
communicates the fluid with the first port.

7. The fluid sampler of claim 6, wherein the fluid con-
tainer further comprises:

a second fluid reservoir in fluid communication with the

second port and that:

stores the fluid for communication with the second
port; and

communicates the fluid with the second port.

8. The fluid sampler of claim 1, wherein the separation
distance between the first view membrane and the second
view membrane is from 10 nanometers (nm) to 1000 nm.

9. The fluid sampler of claim 1, wherein the sample cell
transmits electrons across viewing reservoir through the first
view membrane, the fluid in the viewing reservoir, and the
second view membrane response to being subjected by
electrons from an external electron source.

10. The fluid sampler of claim 1, wherein the first view
membrane and the second view membrane independently
comprise silicon nitride, silicon, silicon dioxide, silicon
carbide, boron nitride, graphene, diamond, or a combination
comprising at least one of the foregoing materials.

11. The fluid sampler of claim 1, wherein the fluid
comprises a gas, a liquid, or a combination comprising at
least one of foregoing fluids.

12. The fluid sampler of claim 1, further comprising a
holder in which the sample cell is disposed.

13. The fluid sampler of claim 12, wherein the holder
comprises:

a transmission aperture arranged transmissively coinci-
dent with the viewing reservoir such that a probe beam
subjected to the fluid sampler is transmitted tandemly
through the transmission aperture and the viewing
reservoir.


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US 10,639,634 B2

5

reservoir 10 respectively have thicknesses (D3, D4) that can
be the same or different from each other.

In an embodiment, with reference to FIG. 7 (an exemplary
cross-section through line B-B of FIG. 2), a physical geom-
etry (e.g., a contour of a bounding wall 40) of first conduit
20 provides microfluidic flow of fluid from first port 16 and
transitions microfluidic flow to nanofluidic flow into view-
ing reservoir 10. Similarly, a physical geometry of second
conduit 28 can provide nanofluidic flow of fluid from
viewing reservoir 10 and can transition the nanofluidic flow
to microfluidic flow to second port 24. As a result, conduit
(20, 24) provide microfluidic flow of the fluid, and viewing
reservoir 10 provides nanofluidic flow of the fluid.

According to an embodiment, with reference to FIG. 8,
FIG. 9, FIG. 10, FIG. 11, and FIG. 12, sample cell 200
includes first electrode 52 disposed on substrate 2 and in
electrical communication with first port 16; and second
electrode 50 disposed on substrate 2 and in electrical com-
munication with second port 24. First electrode 52 and
second electrode 50 produce an electric field through the
fluid disposed in viewing reservoir 10 in response to appli-
cation of a first electric potential to first electrode 52 and
application of a second electric potential to second electrode
50. In this manner, the fluid electrokinetically flows from
first port 16 and through viewing reservoir 10 to second port
24 in response to a presence of the electric field.

In an embodiment, with reference to FIG. 13, FIG. 14,
FIG. 15, FIG. 16, and FIG. 17, fluid sampler 100 includes
fluid container 3 disposed on substrate 2 of sample cell 200.

Fluid container 3 can include first fluid reservoir 5 bounded 3

by wall 7 in fluid communication with first port 16 and that
stores fluid for communication to first port 16 and commu-
nicates fluid with first port 16. Fluid container 3 also can
include second fluid reservoir 9 bounded by wall 11 and in

fluid communication with second port 24. Here, second fluid 3

reservoir 9 stores fluid for communication with second port
24; and communicates fluid with second port 24. In an
embodiment, lid 21 is disposed on fluid reservoir (5, 7, and
the like) to contain the fluid on sample cell 200. In an
embodiment, lid 21 seals the fluid in fluid container 3 and
ports (16, 24) conduits (20, 28) and viewing reservoir 10.
Fluid can be introduced in to fluid reservoirs (e.g., 5, 7) such
that sample cell 200 can be disposed in a vacuum chamber
in an absence of further introduction of fluid into fluid
container 3 from an external source. In an embodiment, fluid
container 3 can be interfaced to an external fluid source
while being disposed, e.g., in a vacuum chamber for fluid
communication of the fluid between fluid container 3 and the
external fluid source (e.g., see FIG. 23). A number of fluid
reservoirs can be selected based on a number of fluid ports
(e.g,, 16, 24, and the like) disposed on substrate 2. The fluid
disposed in the different fluid reservoirs (e.g., 5, 9, and the
like) can be the same or can be different.

Electrodes (e.g., 50, 52, and the like) provide electrical
connections for heating, temperature sensing, moving com-
ponents, electrical biasing, and the like of sample cell 200.

In an embodiment, with reference to FIG. 18, FIG. 19,
FIG. 20, FIG. 21, FIG. 22, sample cell 200 can include a
plurality of fluid ports (16, 24, 21, 23) in fluid communica-
tion with viewing reservoir 10; a plurality of pillars 14
disposed in viewing reservoir 10 and interposed between
first view membrane 34 and second view membrane 36;
plurality of conduits (20, 28) that communicates fluid from
the plurality of fluid ports (16, 24, 21, 23) and viewing
reservoir 10; a plurality of electrodes (50, 52) in electrical
communication with and exposed to fluid ports (16, 24, 21,
23); and flow container 3 disposed on substrate 2 and

2

40

&

3

6

including a plurality of fluid reservoirs (5, 9, 13, 17) that are
in fluid communication with fluid ports (16, 24, 23, 21),
wherein led 21 is disposed on fluid reservoirs (5, 9, 13, 17)
to seal the fluid in sample cell 200. Although flow container
3 is shown as disposed on first surface 8, and electrodes (50,
52) are disposed on second surface 32, it is contemplated
that any permutation of the locations and arrangements of
fluid container 3 and electrodes (50, 52) can be provided on
substrate 2.

In an embodiment, with reference to FIG. 23, fluid
sampler 100 can include fluid line 300 connected to fluid
container 3. Fluid line 300 communicates fluid with fluid
reservoir (e.g. 5, 9, and the like). A number of fluid lines 300
can be selected based on a number of fluid reservoirs
disposed in fluid container 3. In an embodiment, fluid
sampler 100 includes: first gas line 300A in fluid commu-
nication with first fluid reservoir 5 and that supplies the fluid
to first reservoir 5; and second gas line 300B in fluid
communication with second fluid reservoir 9 and that
receives the fluid from second reservoir 9, such that the fluid
flows from first fluid line 300A to first fluid reservoir 5, from
first fluid reservoir 5 to viewing reservoir 10, from viewing
reservoir 10 to second port 24, and from second port 24 to
second fluid line 300B.

In an embodiment, with reference to FIG. 24, sample cell
200 includes electrical lines 400 in communication with
electrodes (50, 52, and the like) and heater 402 disposed at
second end 6. A sample, e.g., a solid sample can be disposed
on heater 402 and probed by a probe beam (e.g,, electron
beam, laser beam, and the like. Heater 402 can heat sample
cell 200 and the sample disposed thereon.

In an embodiment, with reference to FIG. 25, FIG. 26,
FIG. 27, FIG. 28, FIG. 29, fluid sampler 100 includes holder
202 in which sample cell 200 is disposed. Holder 202
includes transmission aperture 208 bounded by wall 220 and
arranged to be transmissively coincident with viewing res-
ervoir 10 of sample cell 200 such that a probe beam (e.g., an
electron beam, laser beam, neutron beam, X-ray beam, and
the like) subjected to fluid sampler 100 is transmitted
tandemly through transmission aperture 208 and viewing
reservoir 10. Additionally, holder 202 includes a plurality of
electrical contactors 226 disposed in electrode member 224.
In an embodiment, sample cell 200 includes first electrode
52 in electrical communication with first port 16; and second
electrode 50 in electrical communication with second port
24, and holder 200 to includes electrode member 224 that
includes: first electrical contactor 226A (e.g., a pogo pin) in
electrical communication with first electrode 52 through
mechanical engagement with first electrode 52; and second
electrical contactor 226B (e.g., a pogo pin) in electrical
communication with second electrode 50 through mechani-
cal engagement with second electrode 50. Here. first elec
trode 52 and second electrode 50 can produce an electric
field in response to application of a first electric potential to

5 first electrode 52 from first electrical contactor 226A and

application of a second electric potential to second electrode
50 from second electrical contactor 226B. In this manner,
the fluid electrokinetically flows from first port 16 and
through viewing reservoir 10 to second port 24 in response
to presence of the electric field.

Holder 202 can include lidded compartment 214 in which
sample cell 200 and electrode member 224 are disposed.
Lidded compartment 214 includes lid 210; lid 206 disposed
on and in mechanical contact with lid 210; and armature

5 receiver 212 disposed on lidded compartment 214 and that

receives transfer arm 250 (see, e.g., FIG. 30). Lid 206 and
lid 210 bound internal compartment 222 in which sample


US 10,639,634 B2

9

membrane 34 and second view membrane 36, separation
distance D1 is conserved at pillar 14. Moreover, under a
tensive force (due to a tensile stress) subjected to pillar 14,
pillar 14 provides substantially constant separation distance
D1 between view membranes (34, 36) and remains in
physical contact with view membranes (34, 36). The com-
pression force or tensive force can be a result of a tempera-
ture or pressure under which sample cell 200 is subjected. It
is contemplated that the temperature or pressure subjected to
sample cell 200 can be external to sample cell 200, internal
to sample cell 200 or a combination thereof. An external
temperature or external pressure can be subjected to sample
cell 200, e.g., at first surface 8, second surface 32, or a
combination from an outside of viewing reservoir 10. Inter-
nal temperature or internal pressure can be present in view-
ing reservoir 10. Moreover, the pressure or temperature can
be an absolute pressure or absolute temperature based on a
reference scale or can be a differential pressure or a differ-
ential temperature across view membrane (34 or 36). Fur-
ther, the pressure or temperature can be a change in the
external pressure or external temperature; or internal pres-
sure or internal temperature of viewing reservoir 10. Accord-
ingly, pillar 14 separates first view membrane 34 from
second view membrane 36 at substantially constant separa-
tion distance D1 such that separation distance D1 is invari-
able with respect to a temperature and invariable with
respect to a pressure to which sample cell 200 is subjected.

Sample cell 200 can include a number of layers of a same

or different material. Substrate 2 can include silicon, silicon 3

dioxide, SiO,, glass, fused silica, SiC, sapphire, GaAs, InP,
ora combination thereof. An oxide layer can be disposed on
substrate and can include silicon dioxide, aluminum oxide,
cerium oxide, hafnium oxide, lanthanum oxide, or other

transition metal, or lanthanide oxides, or a combination 3

thereof. A structural layer can be included in sample cell 200
and can include, e.g., silicon nitride, Si, SiO,, SiC, BN,
graphene, diamond, or a combination thereof. First view
membrane and the second view membrane independently
comprise silicon nitride, Si, SiO,, SiC, BN, graphene, dia-
mond, or a combination thereof. Electrodes (e.g., 50, 52) are
disposed on substrate 2 and can include an electrically
conductive material such as a transition metal (e.g... tantalum
and the like), conductive oxide (indium tin oxide and the
like), Au, Pt, W, glassy carbon, graphene or a combination
thereof.

It is contemplated that viewing reservoir 10 interposed
between first view membrane 34 and second view mem-
brane 36 is formed by removal (e.g., etching) of sacrificial
member 68. Sacrificial member 68 can include chromium
oxide that is selectively removed by a chromium oxide
etchant. The chromium oxide etchant can include cerium IV,
hydrochloric acid, nitric acid, perchloric acid, or a combi-
nation thereof.

The fluid can be disposed in viewing reservoir 10 of
sample cell 200 and can include a gas, a liquid, or a
combination thereof. In an environment, the fluid is the
liquid. In a certain embodiment, particles (e.g., solid, col-
loidal, gel, nanoparticles, microparticles, and the like) are
disposed in the liquid that flow in viewing reservoir 10. The
fluid can be hydrophobic, hydrophilic, organic, inorganic,
biological, ionically charged, zwitterionic, and the like. A
pressure of the fluid can be from 10° Pascals (Pa) to 10” Pa,
specifically from 10°? Pa to 10° Pa, and more specifically
from 1 Pa to 10° Pa. A temperature of the fluid can be from
-269° C. to 1200° C., specifically from -196° C. to 1000°
C., and more specifically from -100° C. to 1000° C.

S

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40

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Sample cell 200 is disposed in holder 202. Holder 202 can
be made from a variety of materials, and elements (e.g., lid
206, lid 210, body 204, armature receiver 212, and the like)
independently can be a plastic, metal, ceramic, glass, or a
combination thereof. In an embodiment, holder 202 includes
Titanium. To allow the device to be used in SEM and TEM
systems, the components of the Sample cell 200 and Holder
202 are non-magnetic to not distort the electron probe
beams.

Holder 202 is disposed on transfer arm 250. Transfer arm
250 can be made from a variety of materials, and elements
(e.g., armature 252, handle 264, and the like) independently
can be a plastic, metal, ceramic, glass, or a combination
thereof. In an embodiment, holder 202 includes copper and
receives an elastomeric gasket at gasket receiver 258.

The fluid can be communicated in conduits (20, 28)
hydrostatically, pneumatically, electrokinetically, under cap-
illary flow, and the like, or a combination thereof.

In an embodiment, a process for making fluid sampler 100
includes providing sample cell 200 disposed sample cell 200
in holder 202; disposing holder 202 on transfer arm 250.

Sample cell 200 can be made in various ways including
selective removal of a sacrificial member from between
view membranes (34, 36). According to an embodiment, a
process for selectively removing a sacrificial member from
a composite structure includes: providing a first structural
layer; disposing the sacrificial member on the first structural
layer, the sacrificial member including chromium oxide;
disposing a second structural layer on the sacrificial member
such that: the sacrificial member is interposed between the
first structural layer and the second structural layer, and a
composite structure is formed by the first structural layer and
the second structural layer; contacting the sacrificial member
with an etchant, the etchant being selective to etch chro-
mium oxide and substantially inert with respect to etching
the composite structure; and selectively etching the sacrifi-
cial member by the etchant to selectively remove the sac-
rificial member from the composite structure, wherein the
first structural layer and the second structural layer are
spaced apart by a separation distance by removal of the
sacrificial member.

The process for selectively removing the sacrificial mem-
ber from the composite structure further can include: dis-
posing the first structural layer on a substrate; disposing an
oxide layer on the first structural layer; disposing an elec-
trode on the oxide layer such that the sacrificial member is
partially disposed on the electrode. The process for selec-
tively removing the sacrificial member from the composite
structure further can include patterning the sacrificial mem-
ber with a plurality of apertures prior to disposing the second
structural layer. The process for selectively removing the
sacrificial member from the composite structure further can
include forming a plurality of pillars by disposing the second
structural letter in the apertures of the sacrificial member.
The process for selectively removing the sacrificial member
from the composite structure further can include etching a
fluid port in the substrate. The process for selectively
removing the sacrificial member from the composite struc-
ture further can include forming a viewing reservoir by
selectively removing the sacrificial member; and connecting
the fluid port in the substrate to the viewing reservoir by
selectively removing the sacrificial member such that the
fluid port is in fluid communication with the viewing res-
ervoir. In a certain embodiment, in the process for selec-
tively removing the sacrificial member from the composite



US 10,639,634 B2

11

structure, the composite structure is sample cell 200 in
which viewing reservoir 10 and fluid port (¢.g., 16, 24, and
the like) receive a fluid.

In an embodiment, with reference to FIG. 34, FIG. 35,
FIG. 36, FIG. 37, FIG. 38, FIG. 39, FIG. 40, FIG. 41, FIG.
42, FIG. 43, FIG. 44, FIG. 45, FIG. 46, FIG. 47, FIG. 48,
FIG. 49, FIG. 50, FIG. 51, FIG. 52, FIG. 53, FIG. 54, and
FIG. 55, a process for making sample cell 200 includes:
providing substrate 2 (FIG. 34); disposing first structural
layer 62 on second surface 32 of substrate 2 (FIG. 35);
disposing third structural layer 64 on first surface 8 of
substrate 2 (FIG. 35); disposing first oxide layer 66 on first
structural layer 62 (FIG. 36); disposing a plurality of elec-
trodes (e.g., 50, 52) on first oxide layer 66 (FIG. 37, FIG.
38); disposing sacrificial member (68, 70, 72) on first oxide
layer 66 (FIG. 39, FIG. 40 (panel A: cross-section along line
A-Ashown in FIG. 39; panel B: cross-section along line B-B
shown in FIG, 39), FIG. 41, FIG. 42 (cross-section along
line A-A shown in FIG. 41), sacrificial member (68, 70, 72)
including: chromium oxide, a first thickness of sacrificial
member (68, 70) in contact with a portion of each electrode
(50, 52), and a second thickness of sacrificial member (72)
that is less than then the first thickness in an area on substrate
2 that corresponds to viewing reservoir 10 (to be formed
upon removal of sacrificial member 72); forming a plurality
of apertures 76 bounded by wall 78 in sacrificial member
(68, 70, 72) (FIG. 43, FIG. 44 (cross-section along line A-A
shown in FIG. 43), FIG. 45, FIG. 46 (cross-section along
line A-A shown in FIG. 45)) by: disposing photoresist mask
74 on sacrificial member (68, 70, 72), patterning photoresist
mask 74 with a plurality of apertures 80, and removing a
portion of sacrificial member (68, 70, 72) coincident with
apertures 80 to form apertures 76 in sacrificial member (68,
70, 72): disposing second oxide layer 84 on sacrificial

member (68, 70, 72) such that sacrificial member (60, 70, 3

72) is interposed between second oxide layer 84 and first
structural layer 62 (FIG. 47, cross-sectional view); disposing
second structural layer 86 on second oxide layer 84 such that
the sacrificial member (68, 70, 72) is interposed between
second structural layer 86 and first structural layer 62;
etching third structural layer 64 disposed on first surface 8
of substrate 2 to expose a portion of substrate 2 at first
surface 8 (FIG. 48); forming etchant trench 90 on second
surface 32 to expose a portion of oxide layer 84 (FIG. 49);
etching a portion of substrate 2 from first surface 8 to second
surface 32 to expose a portion of first structural layer 62 in
an area that corresponds to viewing reservoir 10 (to be
formed) and a fluid port (to be formed) (FIG. 50); and
selectively etching sacrificial member (68, 70, 72) to remove
sacrificial member (60, 70, 72) from between first structural
layer 62 and second structural layer 86 to form sample cell
200 (FIG. 51).

The process for making sample cell 200 further can
include disposing third oxide layer 98 on second surface 32
to fill etchant trench 90 (FIG. 52: panel A top view, and panel
B cross-section along line A-A shown in panel A). The
process for making sample cell 200 further can include
disposing protective layer 310 on third oxide layer 98 (FIG.
53). The process for making sample cell 200 further can
include (FIG. 54): patterning protective layer 310; etching
third oxide layer 98 to expose viewing reservoir 10; and
etching third oxide layer 98 to expose electrodes (e.g., 50,
52).

In an embodiment of the process for making sample cell
200, substrate 2 can include silicon; first structural layer 62
can include silicon nitride; third structural layer 64 can
include silicon nitride: first oxide layer 66 can include

0

30

40

&

0

12

silicon dioxide; electrodes (e.g., 50, 52) can include tanta-
lum; sacrificial member (68, 70, 72) can include chromium
oxide; photoresist mask 74 can include a polymer; second
oxide layer 84 can include silicon dioxide; second structural
layer 86 can include silicon nitride; third oxide layer 98 can
include silicon dioxide; and protective layer 310 can include
a polymer such as a polyimide.

In an embodiment of the process for making sample cell
200, disposing first structural layer 62 on second surface 32
of substrate 2 (FIG. 35) can include physical vapor deposi-
tion, chemical vapor deposition, low-pressure chemical
vapor deposition, plasma-enhanced chemical vapor deposi-
tion, atomic layer deposition, plasma-enhance atomic layer
deposition.

Disposing third structural layer 64 on first surface 8 of
substrate 2 (FIG. 35) can include physical vapor deposition,
chemical vapor deposition, low-pressure chemical vapor
deposition, plasma-enhanced chemical vapor deposition,
atomic layer deposition, plasma-enhance atomic layer depo-
sition,

Disposing first oxide layer 66 on first structural layer 62
(FIG. 36) can include physical vapor deposition, chemical
vapor deposition, low-pressure chemical vapor deposition,
plasma-enhanced chemical vapor deposition, atomic layer

5 deposition, plasma-enhance atomic layer deposition.

Disposing electrodes (e.g., 50, 52) on first oxide layer 66
(FIG. 37, FIG. 38) can include physical vapor deposition,
chemical vapor deposition, low-pressure chemical vapor
deposition, plasma-enhanced chemical vapor deposition,
atomic layer deposition, plasma-enhance atomic layer depo-
sition, and electrodeposition.

With reference to FIG. 55, disposing sacrificial member
(68, 70, 72) on first oxide layer 66 (FIG. 39) can include
physical vapor deposition, chemical vapor deposition, low-
pressure chemical vapor deposition, plasma-enhanced
chemical vapor deposition, atomic layer deposition, plasma-
enhance atomic layer deposition.

Forming a plurality of apertures 76 bounded by wall 78 in
sacrificial member (68, 70, 72) can include (FIG. 43):
disposing photoresist mask 74 on sacrificial member (68, 70,
72) by spin or spray coating, patterning (by optical, electron-
beam or ion-beam lithography) photoresist mask 74 with a
plurality of apertures 80, and removing (by application of a
selective wet chemical etch, or selective reactive ion etch, or
downstream plasma) a portion of sacrificial member (68, 70,
72) coincident with apertures 80 to form apertures 76 in
sacrificial member (68, 70, 72).

Disposing second oxide layer 84 on sacrificial member
(68, 70, 72) such that sacrificial member (60, 70, 72) is
interposed between second oxide layer 84 and first structural
layer 62 (FIG. 47, cross-sectional view) can include physical
vapor deposition, chemical vapor deposition, low-pressure
chemical vapor deposition, plasma-enhanced chemical
vapor deposition, atomic layer deposition, plasma-enhance

5 atomic layer deposition.

Disposing second structural layer 86 on second oxide
layer 84 such that the sacrificial member (68, 70, 72) is
interposed between second structural layer 86 and first
structural layer 62 can include physical vapor deposition,
chemical vapor deposition, low-pressure chemical vapor
deposition, plasma-enhanced chemical vapor deposition,
atomic layer deposition, plasma-enhance atomic layer depo-
sition.

Etching third structural layer 64 disposed on first surface

5 8 of substrate 2 to expose a portion of substrate 2 at first

surface 8 (FIG. 48) can include the use of a selective, or
timed wet chemical etch, or selective, or timed reactive ion


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US010639634B2

(10) Patent No.: US 10,639,634 B2

Liddle et al. (45) Date of Patent: May 5, 2020
(54) VACUUM COMPATIBLE FLUID SAMPLER (58) Field of Classification Search
CPC esses BOIL 3/50273; BOIL 3/502707; GOIN
(71) Applicant: Government of the United States of 23/02; GOIN 23/2251; GOIN 27/4473:
America, as Represented by the (Continued)
Secretary of Commerce, Gaithersburg,
MD (US) (56) References Cited
(72) Inventors: J. Alexander Liddle, Rockville, MD U.S. PATENT DOCUMENTS
(US); Samuel M. Stavis, North
Potomac, MD (US); Glenn E. Holland, 6,001,229 A 12/1999 Ramsey
Rockville, MD (US) 8,059,271 B2 11/2011 Marsh et al.
(Continued)
(73) Assignee: GOVERNMENT OF THE UNITED
SEP RRSeM LED ett : OTHER PUBLICATIONS
SECRETARY OF COMMERCE, J. M. Grogan, “The Nanoaquarium: A Nanofluidic Platform for In
Gaithersburg, MD (US) Situ Transmission Electron Microscopy in Liquid Media”, Doctral
Dissertation, University of Pennsylvania, 178 pgs. (Year: 2011).*
(*) Notice: — Subject to any disclaimer, the term of this (Continued)
patent is extended or adjusted under 35
USC. 154(b) by 426 days. Primary Examiner — J. Christopher Ball
(74) Attorney, Agent, or Firm — Office of Chief Counsel
(21) Appl. No.: 15/611,557 for National Institute of Standards and Technology
(22) Filed: Jun. 1, 2017 67) ABSTRACT
(65) Prior Publication Data A fluid sampler includes: a sample cell that includes: a
substrate comprising: a first port; a second port in fluid
US 2017/0348686 Al Dee. 7, 2017 communication with the first port; a viewing reservoir in
Related U.S. Application Data fluid communication with the first port and the second port
. eae and that receives the fluid from the first port and commu-
(60) provisional application No. 62/343,909, filed on Jun. cates the fluid to the second port, the viewing reservoir
oie including: a first view membrane; a second view membrane;
(51) Int. CL and a pillar interposed between the first view membrane and
BOIL 9/00 (2006.01) second view membrane, the pillar separating the first view
BOIL 3/00 (2006.01) membrane from the second view membrane at a substan-
tially constant separation distance such that a volume of the
(Continued) viewing reservoir is substantially constant and invariable
(52) U.S. CL with respect to a temperature and invariable with respect to
CPC ...... BOLL 3/50273 (2013.01); BOLL 3/502707 4 pressure to which the sample cell is subjected.

(2013.01); BOIL 9/00 (2013.01);
(Continued)

28

20 Claims, 63 Drawing Sheets



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FIG. 20 shows a top view and bottom view of the fluid
sampler shown in FIG. 18;

FIG. 21 shows a side views of the fluid sampler shown in
FIG. 18 according to an embodiment;

FIG, 22 shows an exploded view of the fluid sampler
shown in FIG. 18;

FIG. 23 shows a perspective view of a fluid sampler that
includes a sample cell and fluid line;

FIG. 24 shows a perspective view of a fluid sampler that
includes a sample cell and fluid line;

FIG, 25 shows a perspective view of a holder;

FIG. 26 shows an exploded view of the holder and
components shown in FIG. 25:

FIG. 27 shows a top view of the holder shown in FIG. 25;

FIG. 28 shows a side view of the holder shown in FIG. 25;

FIG. 29 shows a cross-section along line A-A of the
holder shown in FIG. 27:

FIG. 30 shows a perspective view of a transfer arm;

FIG, 31 shows an exploded view of the transfer arm
shown in FIG. 30;

FIG. 32 shows a top view of the transfer arm shown in
FIG. 30;

FIG. 33 shows a cross-section along line A-A of the
transfer arm shown in FIG. 32:

FIG. 34 shows structures formed in making a sample cell;

FIG. 35 shows structures formed in making a sample cell;

FIG. 36 shows structures formed in making a sample cell;

FIG. 37 shows structures formed in making a sample cell;

FIG. 38 shows structures formed in making a sample cell;

FIG. 39 shows structures formed in making a sample cell;

FIG. 40 shows structures formed in making a sample cell;

FIG. 41 shows structures formed in making a sample cell;

FIG. 42 shows structures formed in making a sample cell;

S

FIG. 43 shows structures formed in making a sample cell; ~

FIG. 44 shows structures formed in making a sample cell;
FIG. 45 shows structures formed in making a sample cell;
FIG. 46 shows structures formed in making a sample cell;
FIG. 47 shows structures formed in making a sample cell;
FIG. 48 shows structures formed in making a sample cell;
FIG. 49 shows structures formed in making a sample cell;
FIG. 50 shows structures formed in making a sample cell;
FIG. 51 shows structures formed in making a sample cell;
FIG. 52 shows structures formed in making a sample cell;
FIG. 53 shows structures formed in making a sample cell;
FIG. 54 shows structures formed in making a sample cell;
FIG. 55 shows structures formed in making a sample cell;
FIG. 56 shows a graph of signal-to-noise ratio versus

liquid thickness;

FIG. 57 shows a graph of deflection versus spacing in

panel A and a graph of deflection versus pressure in panel B;
FIG. 58 shows structures formed in making a sample cell;
FIG. 59 shows a micrograph of a viewing reservoir in

panel A and a graph of thickness there is a distance and panel

B

FIG. 60 shows a transmission electron microscope image
ofa nanorod in panel A and a Fourier Transform of the image
in panel B;

FIG. 61 shows a transmission electron microscopic image
of a nanoparticle cluster in panel A and a high-resolution
scanning transmission electron microscope image of the
nanoparticle cluster;

FIG. 62 shows a graph of counts versus energy loss in
panel A and a graph of counts versus energy loss in panel B;
and

FIG. 63 shows a graph of counts versus energy loss

40

50

4
DETAILED DESCRIPTION

A detailed description of one or more embodiments is
presented herein by way of exemplification and not limita-
tion.

It has been discovered that a fluid sampler herein includes
a monolithic sample cell for imaging and spectroscopy, of a
fluid, e.g., a thin liquid layer. The fluid sampler provides for
encapsulating and obtaining high-resolution imaging and
spectroscopic measurements of the fluid, e.g., in a transmis-
sion electron microscope (TEM). The sample cell can be
nanofabricated so that the fluid is separated from vacuum of
the TEM. Moreover, the fluid sampler provides fluid flow,

s combining, heating, and application of voltage for, e.g.

electrochemical studies. Additionally, the fluid sampler is
vacuum compatible and includes a plurality of inputs (e.g.,
an electrical feedthrough) for communication with an exte-
rior of the TEM’s.

In an embodiment, with reference to FIG. 1, FIG. 2, FIG.
3, FIG. 4, and FIG. 5, fluid sampler 100 includes sample cell
200 that includes: substrate 2 including: first end 4; second
end 6 opposing first end 4; first surface 8 traversing a length
of substrate 2 from first end 4 to second end 6; and second

surface 32 opposing first surface 8 and traversing the length

of substrate 2 from first end 4 to second end 6; first port 16
bounded by wall 18 disposed in substrate 2 and that receives
a fluid; second port 24 bounded by wall 26 disposed in
substrate 2 and in fluid communication with first port 16;
viewing reservoir 10 bounded by wall 12 disposed on
substrate 2 in fluid communication with first port 16 and
second port 24 and that receives the fluid from first port 16
and communicates the fluid to second port 24, viewing
reservoir 10 including: first view membrane 34 disposed on
first surface 8 of substrate 2; and second view membrane 36
disposed on second surface 32 of substrate 2, wherein the
fluid when present is interposed between first view mem-
brane 34 and second view membrane 36; and pillar 14
interposed between first view membrane 34 and second view
membrane 36, pillar 14 separating first view membrane 34
from second view membrane 36 at a substantially constant
separation distance D1 such that a volume of viewing
reservoir 10 is substantially constant and invariable with

5 respect to a temperature and invariable with respect to a

pressure to which sample cell 200 is subjected. Here, pillar
14, first view membrane 34, and second view membrane 36
are monolithic.

Sample cell 200 also can include first conduit 20 bounded
by wall 22 such that first conduit 20 is in fluid communi-
cation with first port 16 and viewing reservoir 10 and
interposed between first port 16 and viewing reservoir 10,
wherein first conduit 20 communicates the fluid from first
port 16 to viewing reservoir 10. Sample cell 200 also can

5 include second conduit 28 bounded by wall 30. Second

conduit 28 is in fluid communication with second port 24
and viewing reservoir 10 and interposed between second
port 24 and viewing reservoir 10, wherein second conduit 28
communicates fluid from viewing reservoir 10 to second
port 24.

In an embodiment, as shown in FIG. 6 (an exemplary
cross-section through line A-A of FIG. 2), cell 200 includes
recess 42 bounded by wall 44 at viewing reservoir 10 to
provide a selected thickness for viewing reservoir 10. Here,

Ss first end 4 can have thickness D2 that is different than

thickness D1 of viewing reservoir 10. Moreover, first view
membrane 34 and second view membrane 36 of viewing


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communicates the fluid with the first port; and
a second fluid reservoir in fluid communication with
the second port and that:
stores the fluid for communication with the second
port; and
communicates the fluid with the second port;
wherein the fluid comprises a gas, and the fluid sampler
further comprises:

a first gas line in fluid communication with the first fluid
reservoir and that supplies the gas to the first reservoir;
and

a second gas line in fluid communication with the second
fluid reservoir and that receives the gas from the second
reservoir, such that the gas flows from the first gas line
to the first fluid reservoir, from the first fluid reservoir
to the viewing reservoir, from the viewing reservoir to
the second port, and from the second port to the second
gas line.

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US 10,639,634 B2

1
VACUUM COMPATIBLE FLUID SAMPLER

CROSS REFERENCE TO RELATED
APPLICATIONS

This application claims the benefit of U.S. Provisional
Patent Application Ser. Nos. 62/343,909, filed Jun. 1, 2016,
the disclosure of which is incorporated herein by reference
in its entirety.

STATEMENT REGARDING FEDERALLY
SPONSORED RESEARCH

This invention was made with United States Government
support from the National Institute of Standards and Tech-
nology, an agency of the United States Department of
Commerce. The Government has certain rights in the inven-
tion.

BRIEF DESCRIPTION

Disclosed is a fluid sampler comprising: a sample cell that
comprises: a substrate comprising: a first end; a second end
opposing the first end; a first surface traversing a length of
the substrate from the first end to the second end; and a
second surface opposing the first surface and traversing the
length of the substrate from the first end to the second end;
a first port disposed in the substrate and that receives a flui
a second port disposed in the substrate and in fluid commu-

nication with the first port; a viewing reservoir disposed on 3

the substrate in fluid communication with the first port and
the second port and that receives the fluid from the first port
and communicates the fluid to the second port, the viewing
reservoir comprising: a first view membrane disposed on the

first surface of the substrate; and a second view membrane 3

disposed on the second surface of the substrate, wherein the
fluid is interposed between the first view membrane and the
second view membrane; and a pillar interposed between the
first view membrane and the second view membrane, the
pillar separating the first view membrane from the second
view membrane at a substantially constant separation dis-
tance such that a volume of the viewing reservoir is sub-
stantially constant and invariable with respect to a tempera-
ture and invariable with respect to a pressure to which the
sample cell is subjected, wherein the pillar, the first view
membrane, and the second view membrane are monolithic.

Disclosed also is a process for selectively removing a
sacrificial member from a composite structure, the process
comprising: providing a first structural layer; disposing the
sacrificial member on the first structural layer, the sacrificial
member comprising chromium oxide; disposing a second
structural layer on the sacrificial member such that: the
sacrificial member is interposed between the first structural
layer and the second structural layer, and a composite
structure is formed by the first structural layer and the
second structural layer; contacting the sacrificial member
with an etchant, the etchant being selective to etch chro-
mium oxide and substantially inert with respect to etching
the composite structure; and selectively etching the sacrifi-
cial member by the etchant to selectively remove the sac-
rificial member from the composite structure, wherein the
first structural layer and the second structural layer are
spaced apart by a separation distance by removal of the
sacrificial member.

Further discloses is a process for making a sample cell,
the process comprising: providing a substrate; disposing a
first structural layer on a second surface of the substrate;

20

40

&

0

2

disposing a third structural layer on the first surface of the
substrate; disposing a first oxide layer on the first structural
layer: disposing a plurality of electrodes on the first oxide
layer; disposing a sacrificial member on the first oxide layer,
the sacrificial member comprising: chromium oxide; a first
thickness in contact with a portion of each electrode; and a
second thickness that is less than then the first thickness in
an area on the substrate that corresponds to a viewing
reservoir; forming a plurality of apertures in the sacrificial
member; disposing a second oxide layer on the sacrificial
member such that the sacrificial member is interposed
between the second oxide layer and the first structural layer;
disposing a second structural layer on the second oxide layer
such that the sacrificial member is interposed between the
second structural layer and the first structural layer; etching
the third structural layer to expose the substrate at the first
surface; forming an etchant trench on the second surface;
etching a portion of the substrate from the first surface to the
second surface to expose a portion of the first structural layer
in an area that corresponds to a viewing reservoir and a fluid
port; and selectively etching the sacrificial member remov-
ing the sacrificial member from between the first structural
layer and the second structural layer to form the sample cell.

BRIEF DESCRIPTION OF THE DRAWINGS.

The following descriptions should not be considered
limiting in any way. With reference to the accompanying
drawings, like elements are numbered alike.

FIG. 1 shows a perspective view of a fluid sampler that
includes a sample cell;

FIG. 2 shows a top view of the fluid sampler shown in
FIG. 1;

FIG. 3 shows a bottom view of the fluid sampler shown
in FIG. 1;

FIG. 4 shows a cross-section along line A-A of the fluid
sampler shown in FIG. 2 according to an embodiment;

FIG. 5 shows a cross-section along line B-B of the fluid
sampler shown in FIG. 2;

FIG. 6 shows a cross-section along line A-A view of the
fluid sampler shown in FIG. 2 according to an embodiment;

FIG. 7 shows a cross-section along line B-B of the fluid
sampler shown in FIG. 2;

FIG. 8 shows a perspective view of a fluid sampler that
includes a sample cell;

FIG. 9 shows a top view of the fluid sampler shown in
FIG. 8;

FIG. 10 shows a bottom view of the fluid sampler shown
in FIG. 8;

FIG. 11 shows a cross-section along line A-A of the fluid
sampler shown in FIG. 9 according to an embodiment;

FIG. 12 shows a cross-section along line B-B of the fluid
sampler shown in FIG. 9;

FIG. 13 shows a perspective view of a fluid sampler that

5 includes a sample cell having a fluid reservoir;

FIG. 14 shows a top view of the fluid sampler shown in
FIG. 13;

FIG. 15 shows a bottom view of the fluid sampler shown
in FIG. 13;

FIG. 16 shows a cross-section along line A-A of the fluid
sampler shown in FIG. 14 according to an embodiment;

FIG. 17 shows a cross-section along line B-B of the fluid
sampler shown in FIG. 14;

FIG. 18 shows a top perspective view of a fluid sampler

5 that includes a sample cell having a fluid reservoir;

FIG. 19 shows a bottom perspective view of the fluid
sampler shown in FIG. 18:


U.S. Patent May 5, 2020 Sheet 1 of 63 US 10,639,634 B2

Figure 1



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cell 200 is disposed. Lid 206 of lidded compartment 214 can
include transmission aperture 208 to transmit the probe
beam there is through to viewing reservoir 10 of sample cell
200; fastener hole 216 to receive a fastener (e.g,, a screw);
prong the 236 disposed at and projecting from a first end of
lid 206 to mechanically engage body 204; and hook 218
disposed at a second end of lid 206, wherein hook 218
mechanically engages lid 210 by inserting tab 240 of lid 210
between hook 218 and hook 218 of lid 206. Additionally, lid
206 can include a spring member (e.g., a leaf spring) to
mechanically engage first surface 8 of sample cell 200 and
to impart a force on sample cell 200 so that electrodes (50,
52) of sample cell 200 are pressed against electrical con-
tactors 226 of electrode member 224.

Lid 210 can include transmission aperture 208 to be
transmissively coincident with transmission aperture 208 of
lid 206; and tab 240 to mechanically engage with hook 218
of lid 206. Body 204 receives electrode member 224.
Electrode member 224 includes electrical contactors 226
disposed in electrode receivers 228 that are disposed on
platform 230. Platform 230 can be offset from platform 232
at step edge 234. Step edge 234 can engage first end 4 of
sample cell 200. Electrical contactors 226 received in elec-
trode receivers 228 of platform 230 can be held there in by
electrode stay 226. Electrode stay 226 can be connected to
(e.g., adhered, fastened, and the like) to electro member 224
and can include aperture is 227 to receive a portion of
electrical contactors 226. Further, Armature receiver 212 can
include receptacle 214 that receives transfer arm 250. It is
contemplated that receptacle 214 can include a feature (e.g.,
threading, slot, groove, alignment pin, and the like) that
engages with transfer arm 250.

It is contemplated that holder 202 receives elements (¢.g.,
electrical wiring, fluid line 300, and the like) to interface

with sample cell 200. In this manner, sample cell 200 3

communicates with external sources (e.g., an electrical
source, fluid source, and the like). Moreover, holder 202
provides an off-set or non-symmetric placement of sample
cell 200 disposed in holder 202 with respect to transfer arm
250 and provide a space between pole pieces in a TEM so
that space is available below sample holder 202 to allow
other diagnostic tools to be installed in a TEM vacuum
chamber. Lidded compartment 214 provides engagement of
tab 240 with hook 218 so that sample cell 200 maintains
alignment while lids (210, 206) are installed. Lidded com-
partment 214 can include a leaf spring for a force to maintain
a position of sample cell 200 in internal compartment 222
and during probing of fluid in viewing reservoir 10 of
sample cell 200. The interlocking design allows for the
installation of a locking screw to secure the lid without the
risk of sample misalignment. Since clamping and interlock-
ing of lid (206, 210) is offset from a centerline of transfer
arm 250, a central region of transfer arm 250 provides
communication of electrical wire and fluid lines and being
vacuum tight and compatible.

In an embodiment, with reference to FIG. 30, FIG. 31,
FIG. 32, and FIG. 33, fluid sampler 100 includes transfer
arm 250 on which holder 202 is disposed. Transfer arm 250
includes armature 252 including: first armature end 254 that
is received by armature receiver 212 of holder 202; second
armature end 256 that is arranged opposite first armature end
254 and distal to armature receiver 212; electrical
feedthrough 258 disposed at second armature end 256; and
gasket receiver 258 that receives a gasket (e.g., an O-ring)
for producing a vacuum seal in combination with a vacuum
chamber. Transfer arm 250 also can include alignment pin
260 disposed on armature 252, handle 264 disposed at

30

40

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0

8

second armature end 256, and the like. According to an
embodiment, transfer arm 250 further includes: a plurality of
wires 262 in electrical communication with electrical
feedthrough 258. Wire 262 can include a first wire to
communicate the first electric potential from electrical
feedthrough 258 to first electrical contactor 226A of holder
202; and a second wire to communicate the second electric
potential from electrical feedthrough 258 to second electri-
cal contactor 226B. A number of wires 262 can be selected
based on a number of electrical contactors 226 or electrodes
(e.g, 50, 52). Electrical wires 262 can be independent
components or can be coupled together as a group, e.g., in
a printed flexible cable, a printed circuit board, a coaxial
cable, and the like. It is contemplated that transfer arm 250
can include a radiation shield disposed therein. The radiation
shield can include a material (e.g., a metal or alloy that
includes an element with a high atomic number such as 82)
that blocks certain radiation (e.g., X-rays) from propagating
from sample cell 200 to second armature end 256. The
radiation shield can be a foil, block, and the like.

‘Transfer arm 250 can be monolithic or a plurality of parts
and have a length selected for inserting sample cell 200 into
a vacuum chamber while handle 264 extends external to the
vacuum chamber. Moreover, first Armature end 254 of
transfer arm 250 can include a threaded portion to mechani-
cally engage with receptacle 214 of holder 202.

In fluid sampler 100, sample cell 200 includes viewing
reservoir 10 that can have a shape (e.g., circular, square,
polygonal, and the like) effective for viewing fluid disposed
therein from first view membrane 34 to second view mem-
brane 36. In an embodiment, viewing reservoir 10 has a
rectangular shape (e.g., as shown in FIG. 2) with a length L1
and width D2, wherein sample cell 200 has width W1
Moreover, length L1 can be from 10 micrometers (jum) to
2000 jum, specifically 20 1m to 500 um, and more specifi-
cally from 100 ym to 200 jm. Width W1 and width W2
independently can be from 2 millimeters (mm) to 10 mm,
specifically 4 mm to 8 mm, and more specifically from 4 mm
to 6 mm.

Anumber of pillars 14 is selected to provide substantially
constant separation distance D1. Separation distance D1 can
be from 10 nm to 1000 nm, specifically 20 nm to 500 nm,
and more specifically 50 nm to 200 nm. The number of
pillars 14 can be, e.g.. from 1 to 1,000,000 and selected
based on a surface area of first view membrane 34 or second
view membrane 36. A cross-sectional thickness of pillars
can be from 10 nm to 1000 nm, specifically 20 nm to 500
nm, and more specifically 25 nm to 50 nm. Neighboring
pillars 14 can have pitch P from 400 nm to 50000 nm,
specifically 1000 nm to 4000 nm, and more specifically 1000
nm to 2000 nm. Pillars 14 can have a same shape or different
shapes from one another. The shape of pillars 14 indepen-
dently can be columnar, cylindrical, frustoconical, polygo-
nal, irregular, contoured, and the like. Pillar 14 can be solid
or have an internal cavity that provides a structure to pillar
14 such as an annular shape (e.g.. wherein pillar 14 is a
frustocone or an annular frustocone (i.e., a frustocone with
an annular cross-section). An aspect ratio (i.e., D1:T (see,
e.g., FIG. 4) of pillar 14 can be any ratio effective to provide
a substantially constant separation D1 between view mem-
branes (34, 36). Exemplary aspect ratios (D1:T) of pillar 14
is from 1:10° to 10°:1, specifically 10:1 to 0.01:1, and more
specifically, from 1:1 to 0.1:1.

Pillars 14 provide substantially constant separation dis-

5 tance D1 between first view membrane 34 and second view

membrane 36, wherein under a compression force (due to a
compressive stress) subjected to pillar 14 across first view


U.S. Patent May 5, 2020

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Page 3

(56) References Cited
OTHER PUBLICATIONS

Zheng, H., et al., Observation of Single Colloidal Platinum Nanocrystal
Growth Trajectories, Science, 2009, 1309-1312, 324(5932).

Wu, F., et al., Advances in sealed liquid cells for in-situ TEM
electrochemical investigation of lithium-ion battery, Science Direct,
2014, 1-15

Dukes, M., et al., Improved microchip design and application for In
situ transmission electon microscopy of macromolecules, Micros-
copy and Microanalysis, 2014, 338-345.

Egawa, M., et al., In-situ realtime monitoring of nanoscale gold
electroplating using micro-electro-mechanical systems liquid cell
operating in transmission electron microscopy, Applied Physics
Letters, 2016, 023104-1-023104-4, 108.

Grogan, J.M., et al., The nanoaquarium: A new paradigm in electron
microscopy, Journal of the Indian Institute of Science, 2012, 295-
308, 92(2)

Klein, K.L..et al., Transmission electron microscopy with a liquid
flow cell, Journal of Microscopy, 2011, 117-123, 242.

Leenheer, A.J., et al., A sealed liquid cell for in situ transmission
electron microscopy of controlled electrochemical process, Journal
of Microelectromechanical Systems, 2015, 1061-1068, 24(4).
Tanase, M., et al., High-resolution imaging and spectroscopy at high
pressure: a novel liquid cell for the transmission electron micro-
scope, Microscopy and Microanalysis, 2015, 1629-1638, 21

Ring. E.A., et al., Mircrofluidic system for transmission electron
microscopy, Microscopy and Microanalysis, 2010, 622-629, 16.
Bartholomew, C.H., et al., Fundamentals of industrial catalytic
processes, 2006, Second Edition

* cited by examiner


U.S. Patent May 5, 2020

{3

Sheet 35 of 63 US 10,639,634 B2

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Figure 35


U.S. Patent

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May 5, 2020 Sheet 23 of 63 US 10,639,634 B2
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Figure 28


US 10,639,634 B2
Page 2

(51) Int. CL

GOIN 1/28 (2006.01)
GOIN 35/00 (2006.01)
GOIN 27/447 (2006.01)
GOIN 23/04 (2018.01)
HOIL 21311 (2006.01)
HOIL 21/3065 (2006.01)
HOI 21/4757 (2006.01)
GOIN 23/2251 (2018.01)
HOLS 37/16 (2006.01)
HOIST 37/20 (2006.01)
GOIN 23/02 (2006.01)
(52) U.S. Cl.
CPC veeeecteeee GOIN 1/28 (2013.01), GOIN 23/02

(2013.01); GOIN 23/04 (2013.01); GOIN
23/2251 (2013.01); GOIN 27/4473 (2013.01);
GOIN 27/44791 (2013.01); GOIN 35/0099
(2013.01); HOLS 37/16 (2013.01), HOLS 37/20
(2013.01); HOIL 21/3065 (2013.01); HOIL
21/31122 (2013.01); HOIL 21/47573
(2013.01); BOIL 2200/12 (2013.01); BOIL
2300/046 (2013.01); BOIL 2300/06 (2013.01);
BOIL 2300/0609 (2013.01); BOIL 2300/0681
(2013.01); BOIL 2300/0867 (2013.01), BOIL
2300/0887 (2013.01); BOIL 2300/0896
(2013.01); BOIL 2400/0415 (2013.01); GOIN
2223/307 (2013.01); GOIN 2223/418
(2013.01); GOIN 2223/638 (2013.01); HOLT
2237/2002 (2013.01); HOLL 2924/1067
(2013.01)
(58) Field of Classification Search

CPC veeeecstteeee GOIN 27/44791; GOIN 1/28; GOIN
2223/418; GOIN 2223/307; HO1J 37/16

See application file for complete search history.

(56) References Cited

U.S. PATENT DOCUMENTS
1/2012 Marsh et al.
12/2012 Cao et al.

7/2012 Harb et al
2012/0298883 Al* 11/2012 Grogan

HO1J 37/20
250/440.11

OTHER PUBLICATIONS

Abrams, I.M., et al., A Closed Cell for Electron Microscopy. Journal
of Applied Physics, 1944, 607-609,15(8)

Baker, R.TK., et al., Controlled atmosphere electron microscopy.
Journal of Physics E: Scientific Instruments,1972, 793, 5(8).

Bell, A-T., The Impact of Nanoscience on Heterogeneous Catalysis.
Science,2003, 1688-1691, 299(5613).

Chen, Q., et al., 3D Motion of DNA-Au Nanoconjugates in Graphene
Liquid Cell Electron Microscopy. Nano Letters, 2013, 4556-4561,
13(9).

Creemer, LF, et al., Atomic-scale electron microscopy at ambient
pressure, Ultramicroscopy, 2008, 993-998, 108(9).

De Jonge, N., et al., Electron microscopy of whole cells in liquid
with nanometer resolution, Proceedings of the National Academy of
Sciences, 2009.

De Jonge, N., et al., Nanometer-resolution electron microscopy
through micrometers-thick water layers, Ultramicroscopy, 2010,
1114-1119, 110(9).

De Jonge. N., et al., Electron microscopy of specimens in liquid. Nat
Nano, 2011, 695-704, 6(11).

Dukes, M.J., et al., Visualizing nanoparticle mobility in liquid at
atomic resolution, Chemical Communications, 2013, 3007-3009,
49(29).

Gai, P., Developments in in situ Environmental Cell High-
Resolution Electron Microscopy and Applications to Catalysis,
‘Topics in Catalysis, 2002, 161-173, 21(4).

Grogan, J.M., et al., The Nanoaquarium: A Platform for In Situ
Transmission Electron Microscopy in Liquid Media, Journal of
Microelectromechanical Systems, 2010, 885-894, 19(4).

Gu, M,, et al., Demonstration of an Electrochemical Liquid Cell for
Operando Transmission Electron Microscopy Observation of the
Lithiation/Delithiation Behavior of Si Nanowire Battery Anodes,
Nano Letters, 2013, 6106-6112, 13 (12).

Holtz, ME., et al., In Situ Electron Energy-Loss Spectroscopy in
Liquids, Microscopy and Microanalysis, 2013, 1027-1035, 19(04),
Jeangros, Q., et al., In situ redox cycle of a nickel-YSZ fuel cell
anode in an environmental transmission electron microscope, Acta
Materialia, 2010, 4578-4589, 58(14).

Jensen, E., et al., Monolithic Chip System with a Microlluidic
Channel for In Situ Electron Microscopy of Liquids, Microscopy
and Microanalysis, 2014, 445-451, 20(02)

Jungjohann, K.L., et al., Atomic-Scale Imaging and Spectroscopy
for In Situ Liquid Scanning Transmission Electron Microscopy,
Microscopy and Microanalysis, 2012, 621-627, 18(03)

Li, D.,, et al., Direction-Specific Interactions Control Crystal Growth
by Oriented Attachment, Science, 2012, 1014-1018, 336(6084).
Liao, H.-G., et al., Facet development during platinum nanocube
growth, Science, 2014, 916-919, 345(6199).

Liu, K-L., et al., Novel microchip for in situ TEM imaging of living
organisms and bio-reactions in aqueous conditions, Lab on a Chip,
2008, 1915-1921, 8(LL)

Marton, L., La microscopie electronique des objets biologiques,
Bull. de L’Acad. Royale de Belgique, 1935, 553-560, 21

Radisic, A., et al., In Situ Study of the Growth Kinetics of Individual
Island Electrodeposition of Copper, The Journal of Physical Chem-
istry B, 2006, 7862-7868, 110(15).

Radisic, A., et al., Quantifying Electrochemical Nucleation and
Growth of Nanoscale Clusters Using Real-Time Kinetic Data, Nano
Letters, 2006, 238-242, 6(2)

Ross, F.M., Controlling nanowire structures through real time
growth studies, Reports on Progress in Physics, 2010, 114501,
73(11),

Sharma, R., Design and Applications of Environmental Cell Trans-
mission Electron Microscope for In Situ Observations of Gas-Solid
Reactions, Microscopy and Microanalysis, 2001, 494-506, 7(06)
Sharma, R., et al,. Observation of dynamic nanostructural and
nanochemical changes in ceria-based catalysts during in-situ redue-
tion, Philosophical Magazine, 2004, 2731-2747, 84(25-26)
Sharma, R., et al., Dynamic observations of the effect of pressure
and temperature conditions on the selective synthesis of carbon
nanotubes, Nanotechnology, 2007, 125602, 18(12).

Smeets, P.J.M., et al., Calcium carbonate nucleation driven by ion
binding in a biomimetic matrix revealed by in situ electron micros-
copy, Nat Mater, 2015, 394-399, 14(4).

Swifi, J.A., et al., An environmental cell for the examination of wet
biological specimens at atmospheric pressure by transmission scan-
ning electron microscopy, Journal of Physies E: Scientific Instru-
ments, 1970, 924, 3(11).

Vendelbo, $.B., et al., Visualization of oscillatory behaviour of Pt
nanoparticles catalysing CO oxidation, Nat Mater advance online
publication, 2014.

Wang, C.-M., et al., Observation of materials processes in liquids by
electron microscopy, MRS Bulletin, 2015, 46-52, 40(01)

Wang, C., et al., High-Resolution Electron Microscopy and Spec-
troscopy of Ferritin in Biocompatible Graphene Liquid Cells and
Graphene Sandwiches, Advanced Materials, 2014, 3410-3414, 26(2 1).
Wang, R., et al., Structural Transformation in Ceria Nanoparticles
during Redox Processes, The Journal of Physical Chemistry C,
2009, 5700-5704, 113(14).

Williamson, M.J,, et al., Dynamic microscopy of nanoscale cluster
growth at the solid-liquid interface, Nat Mater, 2003, 532-536, 2(8).
Yuk, J.M., et al., High-Resolution EM of Colloidal Nanocrystal
Growth Using Graphene Liquid Cells, Science, 2012, 61-64, 336(6077).


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