Nanotechnology
PAPER
Dynamic imaging of Au-nanoparticles via scanning
electron microscopy in a graphene wet cell
To cite this article: Wayne Yang et al 2015 Nanotechnology 26 315703
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This content was downloaded from IP address 132.216.2.66 on 06/11/2020 at 15:52
Dynamic imaging of Au-nanoparticles via
scanning electron microscopy in a graphene
wet cell
Wayne Yang
1,2,3
, Yuning Zhang
1,2,3
, Michael Hilke
1
and Walter Reisner
1
1
Department of Physics and RQMP, McGill University, Montreal, Canada
E-mail: wayne.yang2@mail.mcgill.ca and yuning.zhang2@mail.mcgill.ca
Received 11 February 2015, revised 21 May 2015
Accepted for publication 1 June 2015
Published 16 July 2015
Abstract
High resolution nanoscale imaging in liquid environments is crucial for studying molecular
interactions in biological and chemical systems. In particular, electron microscopy is the gold-
standard tool for nanoscale imaging, but its high-vacuum requirements make application to in-
liquid samples extremely challenging. Here we present a new graphene based wet cell device
where high resolution scanning electron microscope (SEM) and energy dispersive x-rays (EDX)
analysis can be performed directly inside a liquid environment. Graphene is an ideal membrane
material as its high transparancy, conductivity and mechanical strength can support the high
vacuum and grounding requirements of a SEM while enabling maximal resolution and signal. In
particular, we obtain high resolution (
5<
nm) SEM video images of nanoparticles undergoing
Brownian motion inside the graphene wet cell and EDX analysis of nanoparticle composition in
the liquid enviornment. Our obtained resolution surpasses current conventional silicon nitride
devices imaged in both a SEM and transmission electron microscope under much higher electron
doses.
S Online supplementary data available from stacks.iop.org/nano/26/315703/mmedia
Keywords: graphene, wet cell, electron microscopy
(Some figures may appear in colour only in the online journal)
1. Introduction
Imaging in a liquid environment is important across a wide
range of research fields from physics to biology. The nanos-
cale imaging of such systems drives new insights in molecular
and biological theory [1, 2]. New experiments enabled by
wet-cell technology include live imaging of antibodies and
bacteria to understand immune response, and in situ imaging
of crystals to understand growth kinetics [3]. Most of the
imaging is performed with electron microscopy such as SEM
(scanning electron microscope) or TEM (transmission elec-
tron microscope) [4, 5]. In particular, SEMs are widely
available and accessible to most researchers. While SEMs
offer quick and high resolution nanoscale (2–10 nm) imaging,
the high vacuum operation conditions (
10 Torr
4
<
−
) of these
instruments make the imaging of liquid environments chal-
lenging [6]. Systems that operate at high pressures such as
environmental SEMs (E-SEMs) are specialized tools requir-
ing the use of water vapour to purge and replace air in the
specimen chamber. Moreover, the electron beam in such
systems scatters from the introduced vapour resulting in
limited resolution [7–9].
Conventional wet cells are based on sealing liquid sam-
ples behind a 30–150 nm silicon nitride window [10, 11].
While this approach has proved effective, the resolution is
fundamentally limited by the necessity of using relatively
thick nitride membranes. Experiments have obtained a reso-
lution of only around 20 nm for a membrane thickness of
50 nm in an SEM [12]. The fabrication of thinner nitride
windows with thickness below 50 nm is challenging, requir-
ing special techniques to control the etching rate and achieve
Nanotechnology
Nanotechnology 26 (2015) 315703 (7pp) doi:10.1088/0957-4484/26/31/315703
2
To whom correspondence should be addressed.
3
These authors contributed equally.
0957-4484/15/315703+07$33.00 © 2015 IOP Publishing Ltd Printed in the UK1
etching uniformity [13]. As the nitride membrane becomes
thinner, the windows become too fragile to handle. Silicon
nitride wafers are also electrically insulating, requiring the
sputtering of a thin layer of conductive material such as gold
for electrical leads or to ground the sample [14]. Ultimately,
nitride based windows cannot be extended to thicknesses
below a few nanometres. This is a very crucial technical
limitation, limiting not just resolution but signal. For example,
the need for relatively thick nitride windows obviates appli-
cation of standard SEM techniques such as energy dispersive
x-ray (EDX) due to the absorption of signal by the thick
membrane.
Here we present a graphene wet cell for SEM imaging
under a high vacuum environment. Graphene is an atomically
thick layer of carbon atoms (0.34 nm thickness) [15] with
exceptional properties including high mechanical strength,
high thermal and high electrical conductivity. Graphene’s
atomic thickness makes the material an optimal imaging
window enabling maximum resolution and signal. In parti-
cular, graphene allows for the collection of low energy sec-
ondary electrons as opposed to just backscattered electrons
performed in most SiN wet cell imaging studies [16]. This
greatly improves the signal and resolution of the images.
Graphene’s mechanical strength prevents breakage of ∼5 μm
membranes under vaccum conditions. Graphene’s high ther-
mal conductivity allows excess heat generated from the beam
to dissipate quickly without damaging the sample. Finally,
graphene’s high electrical conductivity obviates the need for
an additional metal coating for grounding. The graphene
membrane also provides convenient electrical leads for vol-
tage and current inputs for adding electrical bias in experi-
ments. Previous groups have used graphene oxide membranes
for imaging [17]. However it is challenging to control the
homogeneity in the graphene oxide membrane across the
window and, at around 20 nm thick, they are comparable in
thickness to nitride. Using chemical vapour deposition (CVD)
with carefully controlled growth conditions we can ensure
that there is a single layer graphene membrane [18, 19].
Our single-layer graphene wet cell device enables
dynamic imaging in a SEM. In particular, we observe
Brownian dynamics of Au-NP’s transiently binding and
unbinding at the surface of the graphene. While Brownian
motion of Au-NP’s has been observed previously in a TEM
using a graphene sandwich assay, developing a molecular in-
liqiuid imaging capability in an SEM has key practical and
fundamental benefits [20]. SEM’s are more available, cheaper
and more versatile tools that permit introduction of much
larger samples. For example, large (1–10 cm size) micro/nano
fluidic devices could be easily introduced into an SEM and
wet cell imaging could then be performed as part of routine
device operation. In particular, as there is no constraint on
sample thickness in an SEM, an SEM-based wet cell can
incorporate much deeper fluidic channels without loss of
signal, significantly simplifying wet-cell design. Moreover,
additional sample material can be potentially pulled in from
deeper in the cell. For example, we show that continuous
scanning attracts Au-NP’s to the graphene interface. Finally,
SEMs are outfitted with a wide range of surface
characterization tools (for example, EDX). We show that,
using our graphene wet cell device, these tools can then be
adapted to study the wet cell environment. As an example, we
able to obtain an EDX spectrum of Au NPs in liquid.
2. Sample preparation
Our fabrication process is divided into three steps, the fabri-
cation of the silicon nitride substrate, the growth and transfer
of the graphene and the wetting and sealing of the device for
SEM imaging. An illustration of the device is shown in
figure 1.
The first step is substrate fabrication. Our substrate is a
4
00
m
μ
thick (110) silicon wafer coated with a 180 nm thick
nitride membrane and divided into 2 × 2 mm dies. The wafer
was patterned with photolithography and etched in KOH from
the back to produce a 70 × 70 μm residual nitride membrane
in the middle of each die. The KOH etched apertaure also
serves as a reservoir for the liquid sample. Lastly, a 2 μm
diameter hole was etched through the middle of the free
standing nitride membrane to form the graphene viewing
window.
Graphene was grown using CVD on a 25 μm thick
copper foil with a growth temperature of 1050
o
C at a pres-
sure of 100 mTorr and a flow of 4 sccm of CH
4
[21]. Our
custom-built CVD system is based on a vertical furnace. Two
gas tubes feeds into the top of a 2.5 cm wide vertical quartz
tube to provide the flow of gases. The quartz tube is lowered
into the oven during the growth and the growth time is
approximately 1 hour. Before the growth, the copper foil was
first annealed in a flow of 12 sccm of hydrogen for an hour to
strip the oxide layer on the foil. The CVD synthesized gra-
phene was then spin coated with a thin supporting layer of
polymethylmethacrylate (PMMA) layer and the Cu substrate
was etched away in a solution of 0.1 M ammonium persulfate
((NH
4
)
2
S
2
O
8
). The sample was transferred by inserting a
glass slide into the ammonium persulfate solution, using the
slide to scoop out the freely floating graphene membrane and
depositing the graphene bearing slide into a beaker of de-
ionized water. To completely remove the ammonium per-
sulfate residues, the sample was transferred into another clean
beaker of de-ionized water before being transferred onto the
top side of the silicon nitride wafer sample to cover the 2 μm
holes. Graphene produced using the same growth conditions
was transferred onto Si0
2
wafers for Raman spectroscopy to
confirm that the graphene was indeed monolayer.
Finally, the sample was ready to be wetted and sealed.
Gold nanoparticles (Au-NP’s) 20 and 50 nm in diameter were
used to characterize the fluid cell. We chose Au particles as
they are commercially available in a wide variety of sizes and
can potentially be used as conductive biological labels [22].
The Au-NP’s were diluted 1:20 from stock solution in DI and
then the nanoparticle containing solution was degassed for an
hour. Degassing was crucial to ensure proper wetting and to
decrease the formation of gas bubbles during imaging. After
degassing, several microliters of solution was pipetted into the
reservoirs and the wafer sample was sealed with Kapton tape
2
Nanotechnology 26 (2015) 315703 W Yang et al
on the back side. The device was then rinsed in acetone and
isopropanol to dissolve the PMMA supporting layer on the
graphene. The imaging of the device was then done using a
FEI-F-50 SEM in the standard high vacuum mode at 10
−6
Torr using a secondary electron detector of the Everhart-
Thornley type. The graphene membrane remained intact at
this operating pressure of 2.2 × 10
−6
Torr. The primary
electron energy used for imaging is 10 KeV. Under these
imaging conditions, the escape depth of secondary electrons
in water should be in the order of 10 s of nm [23].
3. Observation of nanoparticle dynamics
While many Au-NPs are non-specifically bound to the
membrane, we observe Brownian dynamics of Au-NPs
floating in solution below and undergoing transient interac-
tions with the membrane. These dynamics are recorded over
several minutes using a screen capture program. Figure 2
gives an example of bead motion. Beads are observed to be
diffusing in and out of contact with the membrane surface,
confirming that they are indeed contained in a liquid envir-
onment. The particle trajectories are recorded using a custom
tracking program [11].
In the absence of confinement, the gold nanoparticles are
expected to undergo Brownian motion in water, characterized
by a diffusion constant :
D
kT
r6
,(1)
b
πη
=
where k
b
is the Boltzmann constant, T is the temperature
(300 K), η is the viscosity of DI water (1 × 10
−3
Pa S), r is the
radius of the beads (25 nm). For our image frame rate of
t129
s
=
Hz, this leads to a corresponding mean diffusion
length of
L
Dt 550
Ds
=≃
nm at room temperature (300 K).
Hence, within one image frame the particles are expected to
approximately hop 1/4 of the length of the nanopore. Figure 2
suggests that we indeed see fluctuations on that scale.
However we also observe two additional types of behaviours.
Particles can be permanently bound to the membrane over the
course of the imaging time and can also diffuse in and out of
contact with the membrane, interacting transiently with what
appears to be ‘sticky sites’. This sticking behaviour can be
quantified by a plot of occupation probability
px y(, )
. The
occupation probability is taken by integrating the total
number of frames a bead appears at a certain location
normalized over the total number of frames of the video.
Figure 3 shows the occupation probability for the same device
with a spatial resolution of 10 nm and time resolution of
2
5ms
, clearly indicating the existence of strong trapping sites
that permanently bind beads and weaker trapping sites that
give rise to transient interactions.
The non-uniformity of the occupation probability sug-
gests that the graphene membrane varies with regards to its
physical and chemical reactivity towards nanoparticles. One
possible source of non-unformity are the existence of grain-
boundaries in the graphene layer [24]. These grain boundaries
are imperfections in the graphene lattice due to differently
orientated growth directions. The grain boundaries from
previous studies are spaced roughly the same distance apart
Figure 1. (a) A schematic of our graphene wet cell device. (b) SEM image of the liquid environment imaged through the graphene membrane
micropore. The graphene is positioned on top of a circular aperture etched through the SiN membrane. (c) Schematic of device as viewed
from the side. The liquid sample, held in the 400 μm fluid reservoir sandwiched between the graphene membrane and kapton tape, consists of
deionized water with Au nano particles. The figure is not drawn to scale.
3
Nanotechnology 26 (2015) 315703 W Yang et al
(
1mμ
∼
) as the observed sticky sites. Another possible source
of non-uniformity is the presence of graphene ‘wrinkles’
arising from the growth conditions on the inhomogeneous
surface of the copper foils [18]. The wrinkles form valleys in
the graphene sheet allowing beads to be drawn in through
attraction by van der Waals forces (which has also been
observed in other wet cell applications) [25]. To reduce this
effect, we repeated the experiment with polyethylene glycol
Figure 2. Image time-series showing Au-NP dynamics in our graphene wet-cell device. Beads are observed to be diffusing in and out of
contact of the graphene nanopore. The white bar indicates 500 nm.
Figure 3. (a) Plot of the integrated (over 15 s) normalized nanoparticle occupation probability across the graphene membrane. The
nanoparticle occupation is defined as a brightness of 70% or more. Some spots show an occupation of unity, meaning that beads are bound to
the membrane at these positions for the entire duration of the movie. (b) Zoomed image of the upper left corner with arrows indicating the
positions of the time traces in (c). Each pixel shown corresponds to an integrated area of 30 × 30 nm
2
at a frame rate of 29 Hz. The scale bar
denotes 500 nm in length.
4
Nanotechnology 26 (2015) 315703 W Yang et al
