nanomaterials

Review
Scanning Tunneling Microscopy of Biological Structures:
An Elusive Goal for Many Years
Andrés Rodríguez-Galván 1, * and Flavio F. Contreras-Torres 2

1 Carrera de Biología, Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional
Autónoma de México, Tlalnepantla 54090, Edo. Mex., Mexico
2 Tecnologico de Monterrey, The Institute for Obesity Research, Monterrey 64849, NL, Mexico
* Correspondence: andres.rodriguez@comunidad.unam.mx

Abstract: Scanning tunneling microscopy (STM) is a technique that can be used to directly observe
individual biomolecules at near-molecular scale. Within this framework, STM is of crucial significance
because of its role in the structural analysis, the understanding the imaging formation, and the
development of relative techniques. Four decades after its invention, it is pertinent to ask how much
of the early dream has come true. In this study, we aim to overview different analyses for DNA, lipids,
proteins, and carbohydrates. The relevance of STM imaging is exhibited as an opportunity to assist
measurements and biomolecular identification in nanobiotechnology, nanomedicine, biosensing, and
other cutting-edge applications. We believe STM research is still an entire science research ecosystem
for joining several areas of expertise towards a goal settlement that has been elusive for many years.

Keywords: scanning tunneling microscopy; nucleic acids; carbohydrates; proteins; lipids





 1. Introduction
Citation: Rodríguez-Galván, A.; Biomolecules are chemical structures produced by living organisms that primarily
Contreras-Torres, F.F. Scanning
carry out essential functionalities such as catalysis, generation and transmission of signals,
Tunneling Microscopy of Biological
and immune defense, thus taking an essential role in the metabolism and reproduction of
Structures: An Elusive Goal for Many
living organisms. The most critical aspect of biomolecules is their fundamental relationship
Years. Nanomaterials 2022, 12, 3013.
between structure and function. DNA stores genetic information about the composition
https://doi.org/10.3390/
of proteins. RNA is typically a single-stranded biopolymer responsible for biochemical
nano12173013
reactions, like enzymes, mainly in cellular protein synthesis. Proteins are macrostructures
Academic Editor: Jose M. Palomo consisting of a typical sequence of amino acids that ultimately determine different func-
Received: 4 March 2022
tions. Lipids and carbohydrates have structural roles and participate in cell signaling and
Accepted: 13 April 2022
as a source and reservoir for energy. Unlike organic engineered structures prepared in
Published: 31 August 2022
laboratories, biological structures are dynamic and self-organize; they sculpt themselves
and change their architecture. Biological structures have structural building blocks that
Publisher’s Note: MDPI stays neutral
generate force and constantly come on and off [1].
with regard to jurisdictional claims in
The structure and function of biomolecules can be influenced by environment condi-
published maps and institutional affil-
tions where biomolecules occur. Therefore, the structural characterization, the identification
iations.
of physical interactions, and the direct imaging of biomolecules are of great value to a better
understanding of the functionalities of biomolecules in different environments. In particu-
lar, the visualization of biomolecules also aims at supporting the rational design of new
Copyright: © 2022 by the authors.
complexes or customized substances with specific properties. X-ray crystallography [2],
Licensee MDPI, Basel, Switzerland. nuclear resonance spectroscopy (NMR) [3], and transmission electron microscopy (TEM) [4]
This article is an open access article are alternative or complementary techniques in identifying the structure of biomolecules.
distributed under the terms and However, in the case of X-ray techniques, several experimental conditions such as effi-
conditions of the Creative Commons cient protein extraction, solubilization, stabilization, and generating diffracting crystals for
Attribution (CC BY) license (https:// some proteins (i.e., membrane proteins) can limit their performance. More importantly,
creativecommons.org/licenses/by/ the information obtained only refers to the average configuration of the molecules inside
4.0/). the crystal and not the information of individual biomolecules [5]. The analyses using

Nanomaterials 2022, 12, 3013. https://doi.org/10.3390/nano12173013 https://www.mdpi.com/journal/nanomaterials

Nanomaterials 2022, 12, 3013 2 of 22

NMR require higher concentrations of biomolecules. The samples need to be stabilized for
long periods, avoiding aggregation because NMR usually takes several days for the data
acquisition (i.e., 3D HNCO) [6]. Finally, biomolecules can be challenging to visualize using
TEM. Sometimes, the electron current required to achieve an acceptable signal-to-noise ratio
can damage the biomolecules. Additionally, heavy metals for staining can give inaccurate
structural information; they are highly mobile under high-energy electron beams, which
leads to fuzzy images [7].
Scanning tunneling microscopy (STM) is a technique that allows the visualization of
atoms at solid surfaces [8]. STM was created as a method for the analysis of conductive
surfaces; however, a time after their development, it was reported that biological samples
could be analyzed by STM when deposited on conductive surfaces [9]. The interest in
using STM for biological characterization traces back to the late 1980s. Former reports
suggested that STM could be operated underwater and with other fluids [10]. In this way,
it was thought that STM could be used to visualize biological molecules in conditions that
can mimic those found in vivo. STM could analyze biomolecules in a liquid environment,
allowing the measurements in the native occurrence. STM can also provide structural
information without significant sample preparation procedures contrary to TEM (i.e.,
avoiding fixation and staining). A few micrograms are required for the analysis, and
usually, samples can be preserved for further analysis. Furthermore, STM showed the
capability to resolve assemblies on flat surfaces or when biomolecules formed larger
complexes. This technique is not limited to analyzing only small individual biomolecules
but also fragments of assemblies [11,12]. The potential for determining the structures of
biomolecules in aqueous environments emphasizes the importance of STM in biology,
technology, and instrumentation. STM and atomic force microscopy (AFM) are part of
the family of scanning probe microscopes (SPM) that are characterized by using a very
sharp tip to sense the surface of a sample [8]. However, AFM is not restricted to conductive
samples, and it is currently most often used for imaging biomolecules [13]. AFM can image
biological samples at the nanoscale. It can help to characterize mechanical properties, such
as elasticity, viscosity, and adhesion [14]. The objective of STM is not to compete with X-ray
crystallography, NMR, TEM, or AFM, which are much more suitable for high-resolution
structures, but rather to use STM for systems in which other functional analyses are needed.
Herein, we focus on the reported applications of STM to study biological systems.
Different studies on biomolecule measurements were scrutinized and organized (e.g., DNA,
lipids, proteins, and carbohydrates). We first reviewed theories about STM imaging forma-
tion. Further, the relevance of STM imaging of biomolecules is presented by comparing
the current state-of-the-technique in the field. Perspectives are manifested considering
that the potential utility of STM has yet to be fully realized in several biological systems.
Within this framework, this review resumes the investigations that continue appealing to
STM to study the structure of biological samples. It is worth mentioning that STM imaging
is operationally supported by developing instrumental accessories to operate in several
conditions, from a vacuum to liquid, allowing it to mimic physiological environments.

2. STM Imaging Formation and Contrast Mechanisms

The principle of STM is illustrated in Figure 1. A typical STM consists of a system
that conducts a current between a conductive tip and a conductive sample. Only at a few
nanometers of the contacting regimen, the sample (conductive specimen) receives primary
electrons through the “tunnel effect” ejected from a metallic tip (Figure 1a). The small
tunneling current (10 pA to 1 nA) depends on the tip-surface distance and the bias voltage;
therefore, these parameters can be optimized to scan the surface at high resolution [15]. A
piezoelectric scanning unit controls the vertical and lateral movement of the tip. Hence,
STM images are acquired by two different modes. The images can be generated for the
constant current mode by adjusting the tip-sample distance (Figure 1b); otherwise, the
image is generated through variations in the tunneling current in the constant height mode
(Figure 1c). The constant height mode is preferably used in flat surfaces to avoid loss of
 voltage; therefore, these parameters can be optimized to scan the surface at high resolu-
tion [15]. A piezoelectric scanning unit controls the vertical and lateral movement of the
tip. Hence, STM images are acquired by two different modes. The images can be generated
for the constant current mode by adjusting the tip-sample distance (Figure 1b); otherwise,
Nanomaterials 2022, 12, 3013
the image is generated through variations in the tunneling current in the constant height 3 of 22

mode (Figure 1c). The constant height mode is preferably used in flat surfaces to avoid
loss of current sensitivity. Alternatively, constant current mode is employed for samples
with high
current roughness
sensitivity. or protrusions
Alternatively, that can
constant damage
current the is
mode tipemployed
while scanning the surface.
for samples with
Finally,
high digital or
roughness feedback collects
protrusions thatthe
canoutput
damage signal from
the tip thescanning
while current pre-amp,
the surface.compares
Finally,
the signal
digital level with
feedback the preset
collects value,
the output calculates
signal from the
the response according
current pre-amp, to the user-defined
compares the signal
feedback parameters, and sends the feedback voltage. The feedback signal and tunneling
level with the preset value, calculates the response according to the user-defined feedback
current signal return to an electronic card in the computer to generate topographic current
parameters, and sends the feedback voltage. The feedback signal and tunneling images.
signal return to an electronic card in the computer to generate topographic images.

Figure 1.1. Schematic

Figure Schematic illustration
illustration of of basic
basic components
components of of STM.
STM. STM
STM collects
collects the
the tunneling
tunneling current
current
between the
between the tip
tip apex
apex and
and thethesample
samplewhen
whenaabias
biasvoltage
voltageisisapplied
applied(a).
(a). Relative,
Relative, precise,
precise, lateral
lateral
sample-lever movement
sample-lever movement isisachieved
achievedbybymounting
mounting either thethe
either sample or the
sample or tip
theon
tipthe
onend
theofend
a piezo-
of a
electric scanner.
piezoelectric The tip
scanner. Theis tip
theisphysical sensing
the physical probe,probe,
sensing a fineawire
fine cut
wireorcut
etched to form
or etched to aform
veryasharp
very
tip. The ideal tips have a single atom at the apex because they can provide images at atomic resolu-
sharp tip. The ideal tips have a single atom at the apex because they can provide images at atomic
tion. Generic STM operating modes in constant-current (b) and constant-height (c).
resolution. Generic STM operating modes in constant-current (b) and constant-height (c).

BecauseSTM
Because STMisisbased
basedon onthe
theflow
flow
ofofanan electrical
electrical current,
current, thethe obtained
obtained images
images repre-
represent
sent a convolution of the topography and electronic properties of
a convolution of the topography and electronic properties of the sample. Hence, STM the sample. Hence, STM
cannot directly
cannot directlyimage
image insulating
insulating material.
material. Only
Only organic
organic samples
samples thinner
thinner than
than about
about 11 nm,
nm,
which allow tunneling through the material, can be imaged with good
which allow tunneling through the material, can be imaged with good results. Therefore, results. Therefore,
STM images
STM imagesof ofaanonconducting
nonconductingsample samplemay maynotnotbebeinterpreted
interpreted asasthethe surface
surface topology.
topology. It
It is noteworthy that the interpretation of STM images is not straightforward;
is noteworthy that the interpretation of STM images is not straightforward; the resolved the resolved
images usually
images usually do
do not
not reflect
reflect aanatural
natural topographic
topographic position
position ofof atoms
atoms into
into the
the molecular
molecular
system but
system but rather
rather constitute
constitute thethe response
response from
from the
the electronic
electronic states
states contributing
contributing to to the
the
tunneling between
tunneling betweentiptipand
andsample
sample[16].[16].
Many theories were proposed to explain the contrast formation in STM. In 1989, Spong
et al. [17] suggested a contrast mechanism for resolving organic molecules with tunneling
microscopy. The authors, revealed that STM could resolve the individual molecules and
distinguish between the different functional groups within the molecules. The contrast
mechanism responsible for the well-resolved images was proposed to be a modulation of
the local work function of the substrate by the polarizable molecular adsorbates. Hence,
contrast mechanisms for detecting individual segments in large molecules were related to
Nanomaterials 2022, 12, 3013 4 of 22

the dependence of the local surface conductivity on the individual hydrophilicity of the
molecular groups located directly under the tip. In 1990, Miles et al. [18] demonstrated
that STM could be used to image a range of biological molecules. They reported studies on
proteins and polypeptides in extended helical conformation; they presented images from
a sample of ethidium bromide bound to linearized plasmid DNA; they also investigated
α-cyclodextrin when adsorbed onto graphite substrate that appears to form lattices. It
was suggested that the contrast mechanism is due to the dipole moment of the adsorbed
molecule reducing the work function of the substrate (e.g., graphite). Subsequent studies
suggested that STM can obtain images for non-conductive samples in humid air atmosphere,
implying that the mechanism may be indirectly related to the physisorbed water molecules.
Several years ago, STM imaging analysis noted dependence on humidity. Gucken-
berger et al. [19] carried out experimental STM measurements to show that one monolayer
of water at the investigated surface is sufficient for successful STM imaging of insulating
material at low current. Two types of experiments were performed: measuring the surface
conductivity of bulk insulators as a function of the ambient air’s humidity and imaging such
surfaces and several biological samples prepared on mica. These showed the dependence
of the steady-state current on ambient humidity. The average thickness of the water layer
on a mica surface depends on the ambient air’s relative humidity (RH), which ranges from
0.15 nm at 45% RH to 0.4 nm at 70% RH. Hence, it was proposed that such a monolayer acts
as a conductive coating, similar to the metal coating commonly used for insulating materials
for scanning electron microscopy (SEM) [19]. Therefore, a monolayer film of water covering
the sample can explain STM imaging formation of electrically insulating biomolecules
such as proteins (with resistivity typically in about 1015 –1018 Ωm). This explication can be
supported by experimental evidence of the electrical conductance observed in hydrated
proteins [20]. Typically, the wool can show a semiconductive behavior in a hydrated
state. Similarly, the conductivity of crystalline bovine hemoglobin increases when it is
hydrated [21]. These observations suggest that thicknesses of nonconducting materials are
limited to very thin preparations in the range of just a few nanometers.
Another question concerns the gap between the tip and the biological sample. Water
molecules might bridge this distance as a mechanism for imaging biological material [22].
Assuming that the thin film of water is connected with an infinitely large reservoir to
ensure a moist surface on the sample, the formation of the water bridge can validate the
idea that the water molecules provide a closed circuit for ion conduction. According to
the authors [22], this expectation is incorrect because this idea has implicitly assumed that
the tip surface is dry around the contact area. The tip materials are not easy to wet due to
cleaning before measurements; a tip with a small radius is difficult to wet. They concluded
that the apical region of the tip should be dry at every moment, even if the whole tip is wet.
The tip oscillated at higher humidity, while no images could be obtained at lower humidity.
There is a possibility that the critical radius of the tip becomes small enough when humidity
is sufficiently high; in this way, the water film merges with the bridge on the tip, and the
contact originates the current. Nevertheless, the STM tip is a macroscopic object that is
rarely in equilibrium with the surface. The exact geometry of the tip is commonly unknown
except for some outstanding STM measurements, where the tip structure was determined
before and after a scan by field-ion microscopy [23].
Ultra-high vacuum (UHV) can assist in obtaining images of individual atoms and
determining energy states on an atom-by-atom basis [10]. At such conditions, the tunneling
current is an exponentially decaying function of the separation between the tip and the
surface. This fact enhances the tunneling, creating contiguous segments of the conductively
coated surface, mainly using platinum/carbon (Pt/C). Thus, the coated segments of biolog-
ical samples are conductive and mechanically stable to allow easy identification by STM.
This method has been primarily reported for DNA imaging, commonly deposited on HOPG
and mica substrates. It was reported that the micrometer-sized conductive regions created
by masking might be influenced by the state of hydration of the molecules, which may
alter their conductivity and change their contrast [24]. The analysis of biological samples in
Nanomaterials 2022, 12, 3013 5 of 22

UHV conditions shows reproducibility in the terminus of macromolecular structure. So far,

UHV maintains the samples free of temperature changes and free contaminants.
The mechanism for contrast formation for non-conductive samples remains unsuccess-
fully understood. The difficulty relies on the nature of the phenomenon. The tunneling
junction is transported from a metal tip towards the substrate and through the sample
(non-conductive). Generally, for small organic molecules, it is needed to immobilize the
samples to achieve atomic resolution. Since the current in the tunneling junction is always
due to the same physical process, this process is determined by (i) the distance between
the leads, (ii) the chemical composition of surface and tip, (iii) the electronic structure of
both systems, (iv) the chemical interactions between surface and tip atoms, and (v) the
electrostatic interactions of sample and tip [16]. Hypothetically, a single atom at the apex of
clean and sharp tips is ideal for providing atomic resolution. However, it is not feasible
in experiments due to salts, surfactants, or biological impurities in the solutions where
biological samples are analyzed, which usually contaminate the tip or promote flattening
that affects the imaging resolution [25].
The poor electron conductivity of most biological samples is another factor that restricts
the application of STM for imaging biological materials. Hence, the development of
substrates to allocate the biological samples has facilitated the preparation and identification
of DNA, lipids, and proteins (see Section 3). In this regard, metal substrates and HOPG
are commonly chosen to attach biological samples. It should be noted that the presence of
defects which can mimic biological samples in HOPG, promote the denaturation of proteins
because of their hydrophobic nature [26]. Gold substrates can be a more feasible option to
attach proteins because of the strong interactions; thiol-functionalized Au substrates favor
image acquisition and decrease the mobility of the samples. The methodology employed
for the deposition of samples commonly uses drop casting [27]. This deposition way is
easy and rapid for sample preparation despite its limitations related to the lack of ability to
control uniformity deposits [28]. For instance, areas of high concentration can be difficult
to scan because the tip is usually prone to contamination or to disrupt the fragile structure
of biomolecules. It has been suggested that well-dispersed thin samples consisting of a
single layer of biomolecules are critical in achieving good reproducibility [8]. Finally, the
electrospray ion beam deposition (ES-IBD) is a technology that can be key and necessary
to immobilize molecules aiming for reproducible images. ES-IBD is a proven technique
for depositing carbohydrates, peptides, and proteins without damaging the integrity of
biomolecules [29].
The most significant challenge and a primary goal of STM, AFM, and other surface
microscopies is the single atoms imaging within a molecule. Several relationships and
differences between STM and AFM have been proposed for several decades. In particular,
the existence of a direct correlation between atomic forces and tunneling has been the
most significant paradigm among these two techniques. On the one hand, atomic forces
are subject to pure mechanical measurements. The short-range forces can be repulsive
(Pauli) or attractive (van der Waals and electrostatic forces). On the other hand, tunneling
is subject to pure electrical measurements. The tunneling current in STM is sensitive to
the local electron density of states close to the Fermi level. The coexistence of tunneling
conductance and attractive atomic forces was first discovered by Dürig, Züger, and Pohl in
1988 [30,31]. It has been demonstrated that as a consequence of the Schrödinger equation,
tunneling and covalent bonding are equivalent. It was found that the attractive force varies
exponentially with tip-sample separation, with a decay length roughly twice the decay
length of tunneling conductance. Alternatively, tunneling conductance is proportional to
the square of covalent-bond energy. The tunneling conductance varies with tip-sample
separation by a decay length of about 0.043 nm. The force varies with such separation by a
decay length of about 0.086 nm. This fact represents the equivalence between tunneling
and interaction energy for two interacting systems. Today, the principle of reciprocity is
well established and verified in terms of STM [32]. Several experiments have confirmed
such equivalence [33–36].
Nanomaterials 2022, 12, 3013 6 of 22

The atomic resolution has also been achieved in several AFM investigations [36].
Gross et al. [37] demonstrated that modifications of the AFM tip lead to dramatically
enhanced atomic-scale contrast. They showed that the exact atomic composition and the
geometry of the tip are the main factors influencing the atomic resolution. It is necessary to
operate in the short-range forces regime where chemical interactions result in substantial
contributions for imaging. Operationally, STM imaging at the atomic scale is more chal-
lenging because the tunneling current is sensitive to the local electron density of states.
STM can image features on the molecular surface, but resolving single atoms within an
adsorbed molecule can lead to misleading interpretations. For instance, STM images the
molecular orbitals near the Fermi level when molecules are adsorbed onto insulating films.
However, STM produces broadened and distorted molecular orbital when molecules are
adsorbed on metals. The latter is due to coupling to the electronic states of the substrate
and molecules where an energetic coupling of two parties, or two atomic orbitals, can form
a weak adhesion (about 10–16 J). In this way, STM is sensitive to substrate choices, whereas
AFM images are more sensitive to the tip’s functionalization and geometry.

3. Imaging of Biomolecules by STM

3.1. DNA
One of the first biomolecules studied by STM was double-stranded DNA. Images
underwater and air have been reported. DNA is relevant since all living organisms use it
to store information to develop, survive, and reproduce [38]. DNA is constituted by two
paired strands complementary in their nucleotide sequence and assembled in a double
helix. The model for DNA double helix was proposed in 1953 by James Watson and Francis
Crick based on Rosalind Franklin’s and Maurice Wilkin’s X-ray diffraction patterns. Since
discovering the double helix, several attempts have been focused on the direct imaging of
DNA; however, the challenge remains elusive; some works by HRTEM indicate it is difficult
to resolve DNA structure because of the low signal-to-noise ratio (SNR) imaging [4].
As mentioned, DNA is among the first biomolecules visualized by STM. In 1987, DNA
samples were incubated with the recA-protein to form a complex, fixed with glutaraldehyde,
and purified to eliminate the unbound protein [39]. The samples were deposited on highly
oriented pyrolytic graphite (HOPG). STM experiments were performed at air and room
temperature (RT). The images obtained were difficult to interpret; however, the authors
showed that meaningful structural imaging by STM is possible on bare biological samples.
Years later, Driscoll et al. [40] reported the atomic-resolution imaging of duplex DNA
by STM in an ultra-high vacuum and using freshly cleaved HOPG as substrate. They
reported a correlation between the observed STM profiles with data derived from X-ray
crystallography of DNA in the A-form. It was resolved in both the major and minor grooves
of the double helix.
Those first reports showed the capability of STM for imaging DNA. However, skepti-
cism emerged when it was reported that bare HOPG often exhibits defects similar to DNA
fragments. Hence, one must be cautious when analyzing biological samples deposited on
HOPG. However, such local defects on HOPPG are very stable to significant variations of
imaging parameters; thus, this characteristic can be used to identify biological samples [41].
Because of the concerns found with HOPG, the use of alternative substrates was
explored for the analysis of DNA. For example, Tanaka et al. [42] used Cu(111) as substrate
(Figure 2). They analyzed short single-stranded and double-stranded plasmid DNA (of
3 k base-pairs) under ultrahigh vacuum (UHV) conditions. The images exhibited the
detailed structure of DNA. Some short single-stranded DNA molecules formed a double
helix with a pitch of helical periodicity ~3.5 nm and a height of 0.16–0.36 nm. Values that
correspond with the values of Watson–Crick model for DNA in its B-form (~3.4 nm and
~2 nm, respectively). The double-strand exhibited a topographic height of 0.2–0.5 nm
and a strand diameter of about 2–4 nm, and an internal periodicity of 2.6–3.7 nm along.
These values were near the Watson–Crick DNA model; the authors explain the slight
 (Figure 2). They analyzed short single-stranded and double-stranded plasmid DNA (of 3k
base-pairs) under ultrahigh vacuum (UHV) conditions. The images exhibited the detailed
structure of DNA. Some short single-stranded DNA molecules formed a double helix with
a pitch of helical periodicity ~3.5 nm and a height of 0.16–0.36 nm. Values that correspond
with the values of Watson–Crick model for DNA in its B-form (~3.4 nm and ~2 nm, re-
Nanomaterials 2022, 12, 3013 spectively). The double-strand exhibited a topographic height of 0.2–0.5 nm and a strand 7 of 22
diameter of about 2–4 nm, and an internal periodicity of 2.6–3.7 nm along. These values
were near the Watson–Crick DNA model; the authors explain the slight discrepancy in
periodicity in
discrepancy may be due tomay
periodicity the interaction of interaction
be due to the DNA with oftheDNA
substrate andsubstrate
with the the highand
flexibil-
the
ity offlexibility
high DNA chain [42]. chain [42].
of DNA

Figure2.2.STM
Figure STMimages
imagesofof2739
2739base-paired
base-paireddouble-stranded
double-stranded plasmid
plasmid DNA
DNA molecules
molecules deposited
deposited onon
a
a Cu (111) surface. The samples were deposited using the pulse injection method. Typical
Cu (111) surface. The samples were deposited using the pulse injection method. Typical STM images STM im-
ofages of plasmids
plasmids (a–d).(a–d). A magnified
A magnified image
image of aof a fragment
fragment of (d)
of (d) is shown
is shown in in
(e)(e) anda ahigh-resolution
and high-resolution
image of a selected area of (e) shows a periodic structure with a periodicity of 2.6–3.6 nm along the
image of a selected area of (e) shows a periodic structure with a periodicity of 2.6–3.6 nm along the
strand is visible (f). For comparison, one scheme of DNA at the same scale is included. Reprinted
strand is visible (f). For comparison, one scheme of DNA at the same scale is included. Reprinted
with permission from Ref. [42]. Copyright (1999), Elsevier.
with permission from Ref. [42]. Copyright (1999), Elsevier.

Oneofofthe
One themain
mainadvantages
advantagesof of
thisthis
workwork
waswasthethe method
method of deposition.
of deposition. TheyThey
usedused
the
the pulse
pulse injection
injection methodmethod
underunder
UHV UHV conditions,
conditions, avoiding
avoiding samplesample degradation
degradation [43].
[43]. Gold
Gold surfaces
surfaces have
have also also
been been explored
explored as a substrate
as a substrate for DNA for DNA Jing
analysis. analysis.
et al. Jing et al. [44]
[44] analyzed
several synthetic oligonucleotides adsorbed on Au (111) electrode, and Wang etWang
analyzed several synthetic oligonucleotides adsorbed on Au (111) electrode, and al. [45]et
al. [45] examined single-stranded DNA (ssDNA) from denatured
examined single-stranded DNA (ssDNA) from denatured DNA plasmids using gold DNA plasmids using
as
gold as substrate. More recently, Shapir et al. [46] analyzed poly(G)–poly(C)
substrate. More recently, Shapir et al. [46] analyzed poly(G)–poly(C) DNA molecules of DNA mole-
culesbase
4000 of 4000
pairsbase
long pairs long deposited
deposited on gold substrates.
on gold substrates. Thisexplored
This group group explored the elec-
the electrostatic
trostatic deposition
deposition method. Themethod. The gold
gold surface was surface was biased
positively positively
(0.18biased (0.18the
V) during V) incubation
during the
incubation
of DNA solutionof DNA solution
on the on the
substrate. The substrate.
analysisThewasanalysis
performed wasatperformed at room and
room temperature tem-
perature and
ultra-high ultra-high
vacuum vacuum
conditions. STM conditions.
images showed STM images showed DNA
DNA structures structures
like ropes like
that were
clearly distinguished from the substrate. The height of the ropes was around 0.8 nm, and it
showed a globular pattern along the molecule. The longitudinal profile showed an average
length of ∼4 nm, slightly longer than expected for the B-form of DNA (3.4 nm). The
authors suggested this discrepancy could be because poly(G)−poly(C) DNA adopts the
A-form instead of the B-form; the A-form can be adopted by native DNA in low humidity
conditions [47]. The use of metals as substrates for analyses of DNA has permitted over-
coming the artifacts observed in HOPG. The reported images have displayed consistency
in the macromolecular structure of DNA. They have helped differentiate between single
and double-stranded DNA in an intact plasmid [48]. The single-stranded chains seemed
more meandering and lower in apparent topographic height than double-helical regions
(Figure 3). Additionally, the development of efficient deposition techniques has shown
that it is possible to identify guanine base molecules in extended and fixed single DNA
chains (ssDNA). The individual nucleotides were observed as bright points along the single
chain. Some points were brighter than others along the chain. These brightest points were
assigned as guanines by correlating imaging and spectroscopic data of oligomers contain-
ing a remarkable number of guanine nucleotides. More interesting, when STM images
 [48]. The single-stranded chains seemed more meandering and lower in apparent topo-
graphic height than double-helical regions (Figure 3). Additionally, the development of
efficient deposition techniques has shown that it is possible to identify guanine base mol-
ecules in extended and fixed single DNA chains (ssDNA). The individual nucleotides
Nanomaterials 2022, 12, 3013 were observed as bright points along the single chain. Some points were brighter8 of than
22
others along the chain. These brightest points were assigned as guanines by correlating
imaging and spectroscopic data of oligomers containing a remarkable number of guanine
nucleotides.
were Morepart
aligned with interesting, whenbase
of the known STMsequence
images were
of thealigned with
analyzed part of
ssDNA, thethe known
brightest
base sequence of the analyzed ssDNA, the brightest point matched with the position
point matched with the position of the guanines in the sequence [49]. These results opened of
the guanines in the sequence [49]. These results opened the possibility of simultaneous
the possibility of simultaneous sequencing and structural mapping of individual DNA
sequencing
molecules and structural mapping of individual DNA molecules [50].
[50].

Figure3.3.STM
Figure STMimages
imagesof of2871
2871base-paired
base-pairedplasmid
plasmidDNADNAwereweredeposited
depositedon onaaCu
Cu(111)
(111)surface
surfaceusing
using
aapulse
pulseinjection
injectionmethod.
method.Typical
Typical
STMSTM images
images of plasmids
of plasmids (a,c).
(a,c). A higher
A higher magnification
magnification imageimage of
of the
the area indicated with a white arrow in (c) is shown in (b). The white dotted square region
area indicated with a white arrow in (c) is shown in (b). The white dotted square region of (b) shows of (b)
ashows a bubble-like
bubble-like structure,
structure, where strands
where single single strands are indicated
are indicated as SS,
as SS, and and double-stranded
double-stranded seg-
segments
ments are indicated as DS (d). Reprinted with permission from Ref. [48] . Copyright
are indicated as DS (d). Reprinted with permission from Ref. [48]. Copyright (2010), American (2010), American
Chemical Society.
Chemical Society.

As was mentioned, STM can be used for the study of hybrid materials. Recently,
Fardian-Melamed et al. [51,52] analyzed nanowires consisting of silver-containing poly(dG)–
poly(dC) DNA molecules. STM allowed the detailed structural analysis of their morpho-
logical characteristics and electronic properties. Those nanowires are envisioned for appli-
cations in diverse areas such as nanolithography, energy conversion and storage, catalysis,
sensing, and biomedical engineering [53].

3.2. Lipids
Lipids are very diverse biomolecules in terms of structure; however, they share the
property that they are insoluble in water. Lipids play several fundamental biological
functions: energy storage, biological membranes, signal messengers, and molecular recog-
nition [54]. Lipids are among the first biomolecules analyzed by STM. Smith et al. [55]
reported the molecular structure of arachidic acid bilayers on graphite by STM in air. The
samples were deposited with the Langmuir–Blodgett technique. They reported the ob-
servation in the order of the organization of the lipids. Hörber et al. [56] analyzed the
assembly of di-myristoyl-phosphatic acid (DMPA) on graphite, oxidized graphite, and
Nanomaterials 2022, 12, 3013 9 of 22

gold-coated calcite. The Langmuir–Blodgett technique also prepared the samples, and the
analysis was performed in the air. They reported that the lipids formed tightly packed films,
where embedded proteins could be retained and stable for imaging. Matsuda et al. [57]
analyzed fatty acid molecules deposited by the Langmuir–Blodgett technique on highly
oriented pyrolytic graphite and molybdenum disulfide. Interestingly, they observed that
the lipids formed parallel arrangements when the analysis was performed in air conditions.
Woodward et al. [58] explored the use of a modified freeze-fracture replication technique
to overtake the poor conductivity of lipids and analyzed bilayers of phosphatidylcholine
in the ripper phase form. They reported that the images obtained by STM presented a
superior resolution than direct imaging by TEM. They mentioned the images were easier
to interpret and reproduce [58]. In addition to fatty acids, complex structures such as
liposomes were analyzed by STM. Zareýe et al. [59] analyzed cationic liposomes deposited
on a mica covered with gold (111) in air conditions at room temperature. They reported that
STM could be useful for analyzing fragile liposomal samples. The images were permitted
to calculate the diameter of the liposomes.
More recently, STM was employed to study the formation of lipid bilayers on gold
surfaces. The study of lipid bilayers by STM proposes surfaces for biosensing devices.
It investigates biomembrane-related processes such as protein binding, interactions with
peptides or enzymes, and transport phenomena. Xu et al. [60] analyzed the formation
of bilayers by spreading small unilamellar vesicles (SUVs) of DMPC (1,2-dimyristoyl-sn-
glycero-3-phosphatidylcholine) on Au(111) electrode surfaces. According to the as-obtained
STM images, the DMPC molecules were adsorbed with the acyl chains oriented parallel
to the surface and assembled into an ordered monolayer. With time, the molecules were
reoriented, and the monolayers were transformed into hemimicellar films. Sek et al. [61]
analyzed the assembly of suspensions of vesicles of DMPC/Cholesterol on Au(11) elec-
trode surfaces. Remarkably, they used electrochemical STM (EC-STM) for their analysis.
Under these conditions, the self-assembly process was controlled by the hydrocarbon
skeleton−metal surface interaction and observed the formation of ordered domains of
either pure DMPC or pure cholesterol. The formation of these domains was transient
because when more molecules accumulate at the surface, the molecule−molecule interac-
tions start to dominate. The monolayer is then transformed into a bilayer [61]. Pawłowski
et al. [62] described a mechanism for the lipid bilayer formation on an Au(111) based on
SUVs of DMPC/cholesterol. They explained that the molecules are adsorbed with flat-lying
orientation and formed stripe-like domains. Then, the monolayers serve as a template for
developing hemimicellar films; a single planar bilayer is formed due to the fusion between
coupled layers. EC-STM facilitates the molecular resolution imaging of lipid films with
embedded pores formed by gramicidin [63], alamethicin [64], and trichogen [65]. These
studies revealed the differences in aggregation behavior of these peptides within lipid films.
Gramicidin preferentially creates individual channels, whereas alamethicin and trichogyne
tend to form clustered assemblies with the aggregation pattern of the barrel-stave model.
Finally, Matsunaga et al. [66] observed that monolayers of 1,2-dihexanoyl-sn-glycero-3-
phosphocholine (DHPC) formed on alkanethiol-modified gold surfaces in a buffer solution
can suffer phase transitions driven by potential electrochemical control. The monolayers
can shift among fluidic-I, striped fluidic-II, and grainy phases.

3.3. Proteins
Proteins are of paramount importance for living systems because their function pro-
vides the structural stiffness and rigidity to define the distinct shape of each living be-
ing. Proteins also have key roles: catalyzing cellular chemical reactions in the immune
system, signaling, and signal transduction. Proteins are also valuable for biotechnolog-
ical applications. Protein structure is determined by X-ray crystallography or nuclear
magnetic resonance (NMR), which are considered the gold standard because they bring
three-dimensional information at high resolution. STM will never compete with these
techniques, but it can be a complementary tool. For example, some proteins cannot benefit
Nanomaterials 2022, 12, 3013 10 of 22

from X-ray because they cannot form crystals of good quality for X-ray diffraction. Some
proteins are sensitive proteins that degrade upon radiation exposure. Proteins such as
membrane proteins frequently produce only tiny crystals [67]. Additionally, the informa-
tion obtained only refers to the average configuration of all the molecules forming the
crystal. Thus, information about structural changes at the level of individual molecules
is prohibitive. In the case of NMR, it requires higher concentrations of biomolecules than
X-ray crystallography. Further, the samples need to be stable for long periods since the
data acquisition can take weeks [58]. In the case of systems that integrate proteins with
electronic elements for different technological applications, STM can provide an accurate
morphological characterization of single adsorbed proteins, which can be problematic for
X-ray and MNR [68].
Among the first justifications to analyze proteins with STM was to explore alternatives
for the structural analysis. Edstrom et al. [69] reported the direct observation of phos-
phorylase kinase and phosphorylase b by STM using HOPG as substrate. The shape and
subunit organization of proteins were discernible, the dimensions matched with previous
data of X-ray crystallography, and the resolution was better than the previously obtained
by electron microscopy. Years later, Miles et al. [70] analyzed the structure of the high
molecular weight (HMW) subunit of wheat gluten protein. Arakawa et al. [71] analyzed
the turtle α-macroglobulin deposited in HOPG and the analysis was performed under
air conditions, showing that STM can resolve the structure of proteins at submolecular
resolution with a comparable or better resolution to the obtained by TEM.
Nanomaterials 2022, 11, x FOR PEER REVIEW 20 of 23
After these first studies, several proteins were analyzed underwater or in air condi-
tions, including cytochrome c551 [72], glucose oxidase [73], hemoglobin [74], catalase [75],
cytochrome c [75], azurin [76,77], rubredoxin [78], and albumin [79]. The advantage of STM
air the
for conditions.
analysisThe holo
of one form
kind of contains copper,metalloenzymes
enzyme named while the apo form wasdoes not.
tested; In this
these analy-
enzymes
sis, the authors did not observe any influence of the Cu ions in the holo form,
have metal ions that play key roles in their biological activity [80]. The blue copper protein and no
significant difference was observed between the two forms. However, several
azurin is a metalloenzyme and contains one copper atom in its structure. This protein was reports
have statedonthat
deposited water and
Au(111) can analyzed
play an essential
by STMrole in forming
in water STM images.
conditions by FriisStudies of dehy-
et al. [77]. STM
drated proteins have shown that the images of these samples display a shallow
images exhibited a central area of high contrast, with a 2- to 4-fold current enhancement image
contrast, which
compared improves when
with peripheral parts, the samples
which are hydrated
was assigned as the[84].
copper atom (Figure 4).

Figure 4.
Figure 4. STM
STM images
images of
of protein
protein azurin
azurin deposited
deposited on
on Au(111).
Au(111). The
The spots
spots represent individual azurin
represent individual azurin
molecules(a).
molecules (a).The
The3D
3Dimage
image shows
shows thethe proteins
proteins exhibited
exhibited a high
a high central
central contrast
contrast thatinterpreted
that was was inter-
preted
as as of
an area anhigh
areatunneling
of high tunneling conductivity
conductivity (b). STM
(b). STM analysis analysis
was was in
performed performed in liquid.with
liquid. Reprinted Re-
printed with
permission permission
from Ref. [77].from Ref. [77]
Copyright . Copyright
(1999), National(1999), National
Academy Academy of Sciences.
of Sciences.

Recently,
Davis several
et al. globularthe
[76] analyzed proteins
proteinsuch asmetallothionein
Zn7 streptavidin, antibodies, and liquid
on gold and EGFR kinase
condi-
domain
tions. as monomers
This and dimers
protein contains seven were studiedarranged
zinc atoms, by STM in
in two
solution [85,86].
clusters, oneSTM images
containing
showed
four theatoms
metal samples andhave
the high
othermobility,
with threeand the proteins
metal atoms. Itadopted different
is thought orientations.
that dry samples
In some cases, the orientations resembled the reported crystal structures. The high on
are less conductive. Interestingly, STM images showed a current enhancement mobil-
the
ity was explained because of the experimental conditions; it was performed in solution.
expected positions of the metal centers. These studies demonstrated that STM can be used
The samples were not fixed to the substrate; thus, the proteins could adopt different ori-
entations and move laterally and vertically.
STM was used to analyze proteins of medical importance. Aggregated amyloid β
(Aβ) peptides in plaques and tangles are the histopathological hallmarks of Alzheimer’s
disease. The aggregation pathway of precursor amyloid peptides to mature fibrils is of
Nanomaterials 2022, 12, 3013 11 of 22

to analyze the structure of proteins and their electronic properties. In contrast, Contera
et al. [81–83] compared the metalloenzyme pseudoazurin (pAz) in its holo and apo forms
in air conditions. The holo form contains copper, while the apo form does not. In this
analysis, the authors did not observe any influence of the Cu ions in the holo form, and no
significant difference was observed between the two forms. However, several reports have
stated that water can play an essential role in forming STM images. Studies of dehydrated
proteins have shown that the images of these samples display a shallow image contrast,
which improves when the samples are hydrated [84].
Recently, several globular proteins such as streptavidin, antibodies, and EGFR kinase
domain as monomers and dimers were studied by STM in solution [85,86]. STM images
showed the samples have high mobility, and the proteins adopted different orientations. In
some cases, the orientations resembled the reported crystal structures. The high mobility
was explained because of the experimental conditions; it was performed in solution. The
samples were not fixed to the substrate; thus, the proteins could adopt different orientations
and move laterally and vertically.
STM was used to analyze proteins of medical importance. Aggregated amyloid β
(Aβ) peptides in plaques and tangles are the histopathological hallmarks of Alzheimer’s
disease. The aggregation pathway of precursor amyloid peptides to mature fibrils is of
great value. There is an emerging consensus that soluble oligomeric species such as soluble
dimers, trimers, and small oligomeric aggregates but not monomers are more pathogenic
species in Alzheimer’s than insoluble mature fibrils and dense fibril meshes. Thus, un-
derstanding the structural details of oligomers is an essential first step toward elucidating
the aggregation pathway involved in plaques. Detailed structural information obtained
by STM can help rational design therapeutic tools and methods for diagnostics. Studies
by STM have been conducted to characterize the morphology of β-amyloid fibrils [87,88]
and oligomers [89,90] at the submolecular level of resolution. Notably, Losic et al. [89]
provided high-resolution STM images of Aβ monomers, dimers, and oligomers for the first
time (Figure 5). Remarkably, with the high level of resolution, they suggested a possible
mechanism for oligomerization, which consisted of the sequential addition of monomers to
form dimers and oligomers. Forman et al., in 2013, analyzed amyloid fibers decorated with
monomers of cytochrome b562 and the monomeric multi-haem protein, GSU1996. These
structures were engineered as conducting fibers for new devices. STM images allowed the
visualization of the amyloid fibers as dark stripes and the attached proteins as conducting
peaks [91]. STM is not restricted to small proteins [11]; complexes of proteins such as
collagen I [92] and microtubules (Figure 6) [12] were studied.
Recently Deng et al. [93] utilized electrospray ion beam deposition (ES-IBD) to deposit
cytochrome C proteins in different substrates such as Cu(001), Au(111), and BN/Rh(111)
substrates. With this approach, they folded or unfolded proteins on the substrates control-
ling the pH of the electrospray solution and filtering the charge states (unfolded proteins
were found mainly as high-charge state ions (z > +10). In contrast, folded proteins were
found as low-charge states (z < +8)). Notably, STM images of folded proteins displayed a
globular structure (Figure 7). Meanwhile, unfolded proteins were observed as extended
polymer strands. STM images of high resolution revealed internal lobes structures inferred
as groups of amino acids. The reports indicated can give direct information for the structure
and organization of these types of assemblies and complement the classical methods of
electron microscopy and X-ray diffraction.

3.4. Carbohydrates
Carbohydrates play various biological functions, including a source of chemical energy,
energy storage, communication messenger, a structural component of cell membranes,
and functions as the key structural component in plants [94]. Carbohydrates can be
found in nature as simple molecules (monosaccharides), joined together (disaccharides,
oligosaccharides, and polysaccharides), or as components of biopolymers (glycolipids,
glycoproteins) [95]. The analysis of carbohydrates by STM can be of great value since
 Nanomaterials 2022, 12, 3013 12 of 22

Nanomaterials 2022, 11, x FOR PEER REVIEW 20 of

compounds with molecular weights ranging from 1000 to 5000 are difficult to crystallize [94].

Figure5.5. STM
Figure STMimages
imagesofofmonomers,
monomers, dimers,
dimers, andand oligomers
oligomers of Aβ1–40
of Aβ1–40 on atomically
on atomically flat gold s
flat gold
faces. Monomers contain three or four parallel strands (arrows) (a). Dimers are constituted
surfaces. Monomers contain three or four parallel strands (arrows) (a). Dimers are constituted by by tw
monomers
two monomers (b).(b).
Oligomers
Oligomersresulted
resulted from thefusion
from the fusionofofmonomers
monomers or dimers
or dimers (c),arrows
(c), the the arrows
are are in

cating the parallel strands (d). Reprinted with permission from Ref. [89]. Copyright (2006), Elsevi
indicating the parallel strands (d). Reprinted with permission from Ref. [89]. Copyright (2006), Elsevier.

Additionally, many carbohydrates are epimers, anomers, and regioisomers, and are
challenging to identify without additional chemical steps. As an alternative, the use of
STM was explored to analyze carbohydrates. Among the first studies were the analysis
of β-cyclodextrin [18,96], cellulose [97], glycogen [98], and polysaccharides [99]. Miyake
et al. [100] analyzed the structure of the cyclodextrin (CyD) necklace, which consist of cy-
clodextrin units threaded onto a poly(ethylene glycol) (PEG) chain. Notably, they analyzed
one necklace of 22 α-CyDs and one of 15 α-CyDs on HOPG and MoS2 . They reported
that the molecular necklaces were stable and observed during the analysis (Figure 8a). In
the case of 22 α-CyDs, cyclodextrins were observed separately on the PEG chain, and in
15 α-CyD, the molecules were observed closely packed (Figure 8b). Their results found
that nearly 20% of the cyclodextrins were in a head-to-tail conformation. A few years after,
Jia et al. [101] analyzed a cyclodextrin necklace based on cyclodextrin units threaded onto
biodegradable and thermoresponsive polyurethanes derived from bile acids. In their STM
images, the cyclodextrins were observed as aligned bright spots on the Au(111) substrates.
Abb et al., in 2019 [102,103] analyzed the disaccharides sucrose and trehalose. They em-
ployed soft-landing electrospray ion beam deposition (ES-IBD) to deposit the molecules on
Cu(100) substrates. In their analysis, sucrose was observed as elongated double lobes with
dimensions of 1.0 ± 0.2 nm in length and 0.5 nm width; these dimensions fit the expected
Nanomaterials 2022, 12, 3013 13 of 22

size of a sucrose molecule. Moreover, the trehalose molecules showed a globular structure
with 0.9 ± 0.1 nm in length and 0.6 ± 0.1 nm in width. These dimensions agree well with
the expected size of a disaccharide molecule. More recently, complex oligosaccharides were
studied by Wu et al. [104] in 2020, and it was found that STM was able to visualize the
connectivity in glycans and the resolution allowed the discrimination between regioisomers.
In this study, ES-IBD was also used to deposit the samples. The same group extended
Figure 5. STM
their analysis images
and, of monomers,
employing dimers, andcollision,
soft molecule–surface oligomers of Aβ1–40
analyzed on atomically flat g
oligosaccharides’
faces. Monomers
conformation contain
states; mainly,three
they or fourthat
found parallel strandscan
cellohexaose (arrows) (a).inDimers
be found diverse are constituted
confor-
monomers
mation states:(b). Oligomers
extended resulted from
conformation, partlythe fusion
coiled of monomers
conformation, or dimers
or a coiled (c), the arrows a
conformation
(Figure 9). Additionally,
cating the they(d).
parallel strands found that the with
Reprinted coiledpermission
populationfrom
was the [89]. Copyright
primary
Ref. gas-phase (2006), E
conformer, and their presence can be controlled by tuning the collision energy [105].

Figure
Figure 6.6.STM
STM images
images of unfixed
of unfixed microtubules
microtubules depositeddeposited on ITO
on ITO surface. surface.
Typical Typical
images images o
of micro-
tubules (a,b).
tubules (a,b). The
The images
images represent
represent a portion
a portion of a microtubule,
of a microtubule, in which thein which theofarrangemen
arrangement the
tubulin proteins is slightly revealed. Reprinted with permission from Ref. [12]. Copyright (1994), The
Company of Biologists.

Very little work was done for the analysis of carbohydrates. The group of Klaus
Kern [102,103,105,106] started to explore a new method for sample deposition, opening the
possibility to analyze complex carbohydrates (e.g., glycosaminoglycans, glycolipids, and
glycoproteins). The STM can be valuable to carbohydrates’ inherent polydispersity and
microheterogeneity.
 strates controlling the pH of the electrospray solution and filtering the charge states (un-
folded proteins were found mainly as high-charge state ions (z > +10). In contrast, folded
proteins were found as low-charge states (z < +8)). Notably, STM images of folded proteins
displayed a globular structure (Figure 7). Meanwhile, unfolded proteins were observed
as extended polymer strands. STM images of high resolution revealed internal lobes struc-
Nanomaterials 2022, 12, 3013 tures inferred as groups of amino acids. The reports indicated can give direct information 14 of 22
for the structure and organization of these types of assemblies and complement the clas-
sical methods of electron microscopy and X-ray diffraction.

Figure 7.7.STM
Figure STMimages
imagesof of
CytC deposited
CytC depositedby the
by ES-IBD method
the ES-IBD onto copper
method (a,c,d) (a,c,d)
onto copper and gold
andsur-
gold surfaces
facesFolded
(b). (b). Folded proteins
proteins displayeda aglobular
displayed globular structure
structure and
andunfolded
unfoldedproteins displayed
proteins an ex-an extended
displayed
tended conformation, and the observed lobes on extended proteins were interpreted as amino acids
conformation, and the observed lobes on extended proteins were interpreted as amino acids (d,e),
(d,e), BSA was used as reference (e). Reprinted with permission from Ref. [93]. Copyright (2020),
Nanomaterials 2022, 11, x FOR PEER BSA
REVIEWwas used as reference 20 of 23
American Chemical Society. (e). Reprinted with permission from Ref. [93]. Copyright (2020), American
Chemical Society.
3.4. Carbohydrates
Carbohydrates play various biological functions, including a source of chemical en-
ergy, energy storage, communication messenger, a structural component of cell mem-
branes, and functions as the key structural component in plants [94]. Carbohydrates can

Figure 8. STM images of molecular necklaces, where (a) is a necklace of 22 α-CyDs and (b) one of
Figure 8. STM images of molecular necklaces, where (a) is a necklace of 22 α-CyDs and (b) one of
15 α-CyDs. Reprinted with permission from Ref. [100]. Copyright (2003), American Chemical Soci-
ety.α-CyDs. Reprinted with permission from Ref. [100]. Copyright (2003), American Chemical Society.
15
 Figure 8. STM images of molecular necklaces, where (a) is a necklace of 22 α-CyDs and (b) one of
Nanomaterials 2022, 12, 3013 15 α-CyDs. Reprinted with permission from Ref. [100]. Copyright (2003), American Chemical15Soci-
of 22
ety.

Figure
Figure 9.
9. STM
STM image
image and
and STM
STM simulation
simulation of
of cellohexaose
cellohexaose on
on Cu(100).
Cu(100). The
The deposition
deposition parameters’
parameters’
control permitted the transition conformers between “extended” and “coiled”
control permitted the transition conformers between “extended” and “coiled” forms forms of cellohex-
of cellohexaose.
aose. Reprinted
Reprinted with permission
with permission from[105].
from Ref. Ref. [105] . Copyright
Copyright (2020),(2020), American
American Chemical
Chemical Society.
Society.

4.
4. Relevance
Relevance of of STM
STM Imaging
Imaging in in Biological
Biological Applications
Applications
Imaging
Imaging single
single biomolecules
biomolecules have
have been
been aa long-standing
long-standing ambition
ambition toto advance
advance various
various
fields, such as
as structural
structuralbiology,
biology,biophysics,
biophysics,nanotechnology,
nanotechnology, andand medicine.
medicine. Exception-
Exceptionally,
ally,
directdirect
STMSTM imaging
imaging of single
of single biomolecules
biomolecules canhelpful
can be be helpful to address
to address medical
medical prob-
problems
lems that require structural and dynamic information of biomolecules, mainly those
that require structural and dynamic information of biomolecules, mainly those that that
adopt
adopt heterogeneous
heterogeneous and non-crystalline
and non-crystalline structures
structures or for
or for those those
which whichcannot
crystals crystals
becannot be
obtained.
obtained. For amyloid
For example, example,fibrils
amyloid
are afibrils
centralare a central pathological
pathological feature of Alzheimer’s
feature of Alzheimer’s disease and
disease and other neurodegenerative
other neurodegenerative diseases [107]. diseases [107]. The characterization
The characterization of oligomers
of oligomers preceding the
preceding the formation of well-defined fibrils and the fibrils themselves are of particular
formation of well-defined fibrils and the fibrils themselves are of particular interest because
the understanding of the structure and dynamic assembly of fibrils can help to understand
the abnormal assembly and could be used for the rational design of therapeutic agents
for their prevention or disaggregation. Structural studies have shown it is generally not
possible to use conventional methods such as crystallography or solution nuclear magnetic
resonance since the fibers are insoluble and the oligomers are polymorphic. In this regard,
STM has been used to analyze oligomers and provided high-resolution STM images of Aβ
monomers, dimers, and oligomers [89]. STM has proved its utility in understanding the
processes of fibril formation.
STM also offers advantages over conventional techniques such as X-ray and electron
microscope. STM can be used to study the electronic properties of proteins, particularly
those involved in the conversion of energy in organism autotrophs and heterotrophs. As
mentioned in Section 3.3, metalloproteins are enzymes with metal ions that play a crucial
role in photosynthesis, respiration, and other biological processes where the production of
glucose and ATP is vital. Metalloproteins are very efficiently shuttling single electrons over
long distances and quickly among the active sites of biological partners [108]. STM has been
used not just for imaging of metalloproteins but also for analyzing electron transport across
these proteins in air and under aqueous conditions with a high spatial resolution. EC-STM
has been used to study small proteins, cytb562 , cyt c, and azurin, multiprotein complexes
as photosystems, and various metalloproteins [109]. Remarkably, STM has shown to be
valuable for analyzing proteins at a single-biomolecule level.
STM in biological applications can be related to nanotechnology. Nanoscale struc-
tures and materials (nanoparticles, nanowires, nanofibers, nanotubes) have been explored
in many biological applications (biosensing, biological separation, molecular imaging,
anticancer therapy) because their novel properties and functions differ drastically from
their bulk counterparts [110,111]. This is the field of nanobiotechnology, which combines
engineering and molecular biology, leading to novel multifunctional devices and systems
for biological analysis with better sensitivity, specificity, and a higher recognition rate.
Nanomaterials 2022, 12, 3013 16 of 22

The high demand and production of that new class of devices and systems will require
characterization techniques to be versatile and low cost.
An exciting application of biological nanoparticles is using RNA particles for tumor
diagnosis. Shu et al. [112] prepared RNA nanoparticles to target cancer exclusively in vivo
without accumulating normal organs and tissues. The advantage of this application is
that these miniaturized fluorescent nanoparticles can be quickly taken up by cells through
endocytosis and subsequently used for site-specific intracellular measurements. The use of
STM for imaging those particles can help analyze large biomolecules in real-time. How-
ever, breakthroughs in sample preparation techniques or STM instrumentation may be a
conditional factor for this application.
Dumont and Prakash [1] examined the hierarchical thread in which single molecules
give rise to a complex and dynamic structure at the cellular scale. Probing the mechanics of
complex macromolecular assemblies inside cells has been challenging for a long time. Such
mechanics require quantifying temporal dynamics, precise architectural parameters, active
force generation processes, and their relationship [113]. In this regard, STM/STS may also
be essential to measure electronic states and thus characterize individual molecules as a
response to modifications to their chemical structure.
Recently, Xia and Kanchanawong reviewed open questions about the cell adhesions
at the nanoscale level to grasp the inner logic of cellular decision-making [114]. The
need to characterize forces and imaging cellular ultrastructure is only reachable through
advanced techniques such as super-resolution fluorescence microscopy coupled to electron
microscopes [115]. STM/STS might evolve in advanced techniques to provide a three-
dimensional profile of the surface and local information at the contact point between
two different cell regions in a similar fashion to those measurements for semiconductor
heterojunctions [116].
Integrating biomolecules with synthetic materials can direct self-assembly, catalysis,
and other specific properties such as biorecognition. This can be exploited when using
biohybrid systems as electronic devices for diagnostics, health care, drug screening, or envi-
ronmental monitoring applications [117]. For those applications, integrating biomolecules
into inorganic surfaces is of great importance since the distribution and orientation of
biomolecules on surfaces can affect their functionality. Surface plasmon resonance (SPR)
sensors [118] are thin-film refractometers that measure changes in the refractive index
occurring at the surface of a metal film supporting a surface plasmon. Various types of
biorecognition elements have been employed in affinity SPR biosensors. Single-chain anti-
bodies fragments remain by far the most frequently used biorecognition element. Another
type of biorecognition element that has been employed in SPR sensors is peptides and
aptamers. These biorecognition elements are commonly immobilized on the sensor surface.
The surface chemistry must be designed to enable immobilization of a sufficient number
without affecting their biological activity [119]. Therefore, SPR sensors still require high-
cost investment, components, and operation, leading to unaffordability for implementation
in a low-cost point of care or laboratories [120]. In this regard, STM can help to analyze
and characterize the SPR sensors since STM has proven to be a powerful tool for analyzing
biomolecules adsorbed on surfaces [68].

5. Perspective
STM has proven to be an invaluable tool for studying conductive surfaces throughout
the last four decades. The incursion of novel substrates and new deposition techniques
helped to improve the capability to resolve structures at the atomic level, allowing the study
of complex phenomena such as molecular bonding and hybridization states [121], in-situ
chemical reactions for molecular engineering [122], or single-molecule junctions [123], to
mention a few applications. These advances drive STM expansion in all directions and help
conceptualize other instruments such as the AFM [124] or the scanning ion conductance
microscope [125] that also allow the observation of biological samples.
Nanomaterials 2022, 12, 3013 17 of 22

The most cited article featuring the capabilities of STM to resolve sub-nanometer struc-
tures was focused on the analysis of carbon nanotubes (CNTs). In 1998, Odom et al., [126]
were able to locate and resolve the hexagonal-ring structure of the CNTs walls. These re-
sults represented a significant implication to determine the structure of such nanosystems,
which were promptly compared with theoretical models. At that time when STM imagined
CNTs, this technique was still a young child [127] that required the accompaniment of other
mature techniques such as ultra-high vacuum systems, controlled atmospheres, sputtering,
annealing, and cooling systems (<100 K). Hence, STM owned fundamental implications in
the physics and surface-science communities.
STM grew up in the following years but was accompanied by scanning tunneling
spectroscopy (STS). The couple STM and STS correlate well the structure and the den-
sity of electric states that are of paramount importance for electronic applications. This
fact fits with the original prospect from their STM inventors, Binning and Roher [128],
which was to use STM as a Feynman molecular engine [129] to handle atoms and carry
out small force measurements down to those between individual atoms. High-resolution
STM/STS experiments are probably the proof for that expectation, as shown in the observa-
tional measurements of different types of nanostructures with variable composition and
bandgap [130].
In the last decade, the emphasis in STM studies was shifted from the research of
surface topography to surface chemistry [122] and surface dynamics [131]. In the case of
STM microscopists, a social differentiation within the SPM community made it an attractive
opportunity to focus on nanoscience as the most attractive proposition for research. There-
fore, the atomic resolution capability in STM was taken as what makes it unique to use as a
tool in nanotechnology. The STM research was pushed forward with the participation of
surface scientists and nanotechnologists while it was moving away from bio-applications.
In this way, biologists found the whole language of electron tunneling obscure and un-
helpful in interpreting these strange images. At the same time, surface scientists identified
biological systems as “dirty” and ill-defined [132]. Today, the complexity in STM imaging
interpretation has motivated experimental and theoretical groups to understand the origins
of image contrast [16] as the basis for interpreting images. In addition, STM is a technique
requiring specialized operators to optimize imaging parameters during sample analysis.
Their ability to recognize and identify structures may help spread the interest in using STM
in other applications than surface phenomena. The current STM experiments in biological
systems may require feedback from atomistic simulations to reproduce the key features of
the imagined structures [133]. Therefore, STM for imaging biological structures can be an
opportunity to train researchers who exceptionally can endorse the instrument attending
the sample preparation and imaging interpretation.
In perspective, STM applications for biomedicine are still an elusive goal, albeit not
necessarily unreachable. Our experience says that the electron microscope is preferable in
nanoscale research. STM can require several fiddling days to obtain meaningful images;
in comparison, a few hours are needed when working with electron microscopes [79].
However, our first option is to use STM when studying biological structures attached
to nanostructures. In general, the recent STM studies are clear evidence that the era
of this tool is not at its end but heading towards new ways other than applications by
which molecular systems can be manipulated and studied. STM imaging, computational
chemistry methods, sample deposition, and UHV techniques are pivotal in characterizing
biological structures. A continuous improvement in the physical features of substrates
is also profitable in both academic and commercial fields [134]. The STM research is still
an entire science research ecosystem; it is open for practitioners from various disciplines
interested in biology, technology, and instrumentation. History tells us that microscopy
played a crucial role in the growth of nanotechnology in which nanostructures are frozen
in space and time. STM can assist studies about dynamic biological systems with relevance
for nanobiotechnology applications.
Nanomaterials 2022, 12, 3013 18 of 22

Author Contributions: A.R.-G. and F.F.C.-T. wrote the first draft of the manuscript and discussed its
final version. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the National Autonomous University of Mexico (UNAM;
Grant DGAPA- IA204521) and the National Council on Science and Technology (CONACyT, Grant
No. 269399).
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Dumont, S.; Prakash, M. Emergent Mechanics of Biological Structures. Mol. Biol. Cell 2014, 25, 3461–3465. [CrossRef] [PubMed]
2. Chapman, H.N.; Fromme, P.; Barty, A.; White, T.A.; Kirian, R.A.; Aquila, A.; Hunter, M.S.; Schulz, J.; DePonte, D.P.; Weierstall, U.; et al.
Femtosecond X-ray Protein Nanocrystallography. Nature 2011, 470, 73–77. [CrossRef] [PubMed]
3. Selenko, P. Quo Vadis Biomolecular NMR Spectroscopy? Int. J. Mol. Sci. 2019, 20, 1278. [CrossRef] [PubMed]
4. Marini, M.; Falqui, A.; Moretti, M.; Limongi, T.; Allione, M.; Genovese, A.; Lopatin, S.; Tirinato, L.; Das, G.; Torre, B.; et al. The
Structure of DNA by Direct Imaging. Sci. Adv. 2015, 1, e1500734. [CrossRef] [PubMed]
5. Kermani, A.A. A Guide to Membrane Protein X-ray Crystallography. FEBS J. 2021, 288, 5788–5804. [CrossRef]
6. Frueh, D.P.; Goodrich, A.C.; Mishra, S.H.; Nichols, S.R. NMR methods for structural studies of large monomeric and multimeric
proteins. Curr. Opin. Struct. Biol. 2013, 23, 734–739. [CrossRef]
7. Longchamp, J.-N.; Rauschenbach, S.; Abb, S.; Escher, C.; Latychevskaia, T.; Kern, K.; Fink, H.-W. Imaging Proteins at the
Single-Molecule Level. Proc. Natl. Acad. Sci. USA 2017, 114, 1474–1479. [CrossRef]
8. Bian, K.; Gerber, C.; Heinrich, A.J.; Müller, D.J.; Scheuring, S.; Jiang, Y. Scanning Probe Microscopy. Nat. Rev. Methods Primer 2021, 1, 36.
[CrossRef]
9. Hansma, P.K.; Elings, V.B.; Marti, O.; Bracker, C.E. Scanning Tunneling Microscopy and Atomic Force Microscopy: Application to
Biology and Technology. Science 1988, 242, 209–216. [CrossRef]
10. Sonnenfeld, R.; Hansma, P.K. Atomic-Resolution Microscopy in Water. Science 1986, 232, 211–213. [CrossRef]
11. Rodríguez-Galván, A.; Heredia, A.; Plascencia-Villa, G.; Ramírez, O.T.; Palomares, L.A.; Basiuk, V.A. Scanning Tunneling
Microscopy of Rotavirus VP6 Protein Self-Assembled into Nanotubes and Nanospheres. J. Scanning Probe Microsc. 2008, 3, 25–31.
[CrossRef]
12. Maaloum, M.; Chretien, D.; Karsenti, E.; Horber, J.K. Approaching Microtubule Structure with the Scanning Tunneling Microscope
(STM). J. Cell Sci. 1994, 107, 3127–3131. [CrossRef]
13. Engel, A.; Müller, D.J. Observing Single Biomolecules at Work with the Atomic Force Microscope. Nat. Struct. Biol. 2000, 7,
715–718. [CrossRef] [PubMed]
14. Miranda, A.; Gómez-Varela, A.I.; Stylianou, A.; Hirvonen, L.M.; Sánchez, H.; Beule, P.A.A.D. How Did Correlative Atomic Force
Microscopy and Super-Resolution Microscopy Evolve in the Quest for Unravelling Enigmas in Biology? Nanoscale 2021, 13,
2082–2099. [CrossRef] [PubMed]
15. Davis, J.J.; Hill, H.A.O. The Scanning Probe Microscopy of Metalloproteins and Metalloenzymes. Chem. Commun. 2002, 393–401.
[CrossRef] [PubMed]
16. Hofer, W.A. Challenges and Errors: Interpreting High Resolution Images in Scanning Tunneling Microscopy. Prog. Surf. Sci. 2003,
71, 147–183. [CrossRef]
17. Spong, J.K.; Mizes, H.A.; LaComb Jr, L.J.; Dovek, M.M.; Frommer, J.E.; Foster, J.S. Contrast Mechanism for Resolving Organic
Molecules with Tunnelling Microscopy. Nature 1989, 338, 137–139. [CrossRef]
18. Miles, M.J.; McMaster, T.; Carr, H.J.; Tatham, A.S.; Shewry, P.R.; Field, J.M.; Belton, P.S.; Jeenes, D.; Hanley, B.; Whittam, M.; et al.
Scanning Tunneling Microscopy of Biomolecules. J. Vac. Sci. Technol. A 1990, 8, 698–702. [CrossRef]
19. Guckenberger, R.; Heim, M.; Cevc, G.; Knapp, H.; Wiegrabe, W.; Hillebrand, A. Scanning Tunneling Microscopy of Insulators and
Biological Specimens Based on Lateral Conductivity of Ultrathin Water Films. Science 1994, 266, 1538–1540. [CrossRef]
20. Baxter, S. Electrical Conduction of Textiles. Trans. Faraday Soc. 1943, 39, 207–214. [CrossRef]
21. Rosenberg, B. Electrical Conductivity of Proteins. II. Semiconduction in Crystalline Bovine Hemoglobin. J. Chem. Phys. 1962, 36,
816–823. [CrossRef]
22. Yuan, J.-Y.; Shao, Z.; Gao, C. Alternative Method of Imaging Surface Topologies of Nonconducting Bulk Specimens by Scanning
Tunneling Microscopy. Phys. Rev. Lett. 1991, 67, 863–866. [CrossRef]
23. Cross, G.; Schirmeisen, A.; Stalder, A.; Grütter, P.; Tschudy, M.; Dürig, U. Adhesion Interaction between Atomically Defined Tip
and Sample. Phys. Rev. Lett. 1998, 80, 4685–4688. [CrossRef]
24. Dunlap, D.D.; García, R.; Schabtach, E.; Bustamante, C. Masking Generates Contiguous Segments of Metal-Coated and Bare DNA
for Scanning Tunneling Microscope Imaging. Proc. Natl. Acad. Sci. USA 1993, 90, 7652–7655. [CrossRef]
25. Chaika, A.N.; Orlova, N.N.; Semenov, V.N.; Postnova, E.Y.; Krasnikov, S.A.; Lazarev, M.G.; Chekmazov, S.V.; Aristov, V.Y.;
Glebovsky, V.G.; Bozhko, S.I.; et al. Fabrication of [001]-Oriented Tungsten Tips for High Resolution Scanning Tunneling
Microscopy. Sci. Rep. 2014, 4, 3742. [CrossRef] [PubMed]
26. Barinov, N.A.; Prokhorov, V.V.; Dubrovin, E.V.; Klinov, D.V. AFM Visualization at a Single-Molecule Level of Denaturated States
of Proteins on Graphite. Colloids Surf. B Biointerfaces 2016, 146, 777–784. [CrossRef] [PubMed]
Nanomaterials 2022, 12, 3013 19 of 22

27. Kaliyaraj Selva Kumar, A.; Zhang, Y.; Li, D.; Compton, R.G. A Mini-Review: How Reliable Is the Drop Casting Technique?
Electrochem. Commun. 2020, 121, 106867. [CrossRef]
28. Frommer, J. Scanning Tunneling Microscopy and Atomic Force Microscopy in Organic Chemistry. Angew. Chem. Int. Ed. 1992, 31,
1298–1328. [CrossRef]
29. Rauschenbach, S.; Ternes, M.; Harnau, L.; Kern, K. Mass Spectrometry as a Preparative Tool for the Surface Science of Large
Molecules. Annu. Rev. Anal. Chem. 2016, 9, 473–498. [CrossRef]
30. Dürig, U.; Züger, O.; Pohl, D.W. Force Sensing in Scanning Tunnelling Microscopy: Observation of Adhesion Forces on Clean
Metal Surfaces. J. Microsc. 1988, 152, 259–267. [CrossRef]
31. Dürig, U.; Züger, O.; Pohl, D.W. Observation of Metallic Adhesion Using the Scanning Tunneling Microscope. Phys. Rev. Lett.
1990, 65, 349–352. [CrossRef]
32. Chen, C.J. Introduction to Scanning Tunneling Microscopy, 2nd ed.; Monographs on the Physics and Chemistry of Materials; Oxford
University Press: Oxford, UK, 2007.
33. Loppacher, C.; Bammerlin, M.; Guggisberg, M.; Schär, S.; Bennewitz, R.; Baratoff, A.; Meyer, E.; Güntherodt, H.-J. Dynamic Force
Microscopy of Copper Surfaces: Atomic Resolution and Distance Dependence of Tip-Sample Interaction and Tunneling Current.
Phys. Rev. B 2000, 62, 16944–16949. [CrossRef]
34. Schirmeisen, A.; Cross, G.; Stalder, A.; Grütter, P.; Dürig, U. Metallic Adhesion and Tunnelling at the Atomic Scale. New J. Phys.
2000, 2, 329. [CrossRef]
35. Sugimoto, Y.; Ondráček, M.; Abe, M.; Pou, P.; Morita, S.; Perez, R.; Flores, F.; Jelínek, P. Quantum Degeneracy in Atomic Point
Contacts Revealed by Chemical Force and Conductance. Phys. Rev. Lett. 2013, 111, 106803. [CrossRef]
36. Ashino, M.; Obergfell, D.; Haluška, M.; Yang, S.; Khlobystov, A.N.; Roth, S.; Wiesendanger, R. Atomically Resolved Mechanical
Response of Individual Metallofullerene Molecules Confined inside Carbon Nanotubes. Nat. Nanotechnol. 2008, 3, 337–341.
[CrossRef]
37. Gross, L.; Mohn, F.; Moll, N.; Liljeroth, P.; Meyer, G. The Chemical Structure of a Molecule Resolved by Atomic Force Microscopy.
Science 2009, 325, 1110–1114. [CrossRef]
38. Church, G.M.; Gao, Y.; Kosuri, S. Next-Generation Digital Information Storage in DNA. Science 2012, 337, 1628. [CrossRef]
39. Travaglini, G.; Rohrer, H.; Amrein, M. Scanning Tunneling Microscopy on Biological Matter. Surf. Sci. 1987, 181, 380–390.
[CrossRef]
40. Driscoll, R.J.; Youngquist, M.G.; Baldeschwieler, J.D. Atomic-Scale Imaging of DNA Using Scanning Tunnelling Microscopy.
Nature 1990, 346, 294–296. [CrossRef] [PubMed]
41. Chang, H.; Bard, A.J. Observation and Characterization by Scanning Tunneling Microscopy of Structures Generated by Cleaving
Highly Oriented Pyrolytic Graphite. Langmuir 1991, 7, 1143–1153. [CrossRef]
42. Tanaka, H.; Hamai, C.; Kanno, T.; Kawai, T. High-Resolution Scanning Tunneling Microscopy Imaging of DNA Molecules on
Cu(111) Surfaces. Surf. Sci. 1999, 432, L611–L616. [CrossRef]
43. Tanaka, H.; Kawai, T. Visualization of Detailed Structures within DNA. Surf. Sci. 2003, 539, L531–L536. [CrossRef]
44. Jing, T.W.; Jeffrey, A.M.; DeRose, J.A.; Lyubchenko, Y.L.; Shlyakhtenko, L.S.; Harrington, R.E.; Appella, E.; Larsen, J.; Vaught, A.;
Rekesh, D. Structure of Hydrated Oligonucleotides Studied by in Situ Scanning Tunneling Microscopy. Proc. Natl. Acad. Sci. USA
1993, 90, 8934–8938. [CrossRef]
45. Wang, H.; Tang, Z.; Li, Z.; Wang, E. Self-Assembled Monolayer of SsDNA on Au(111) Substrate. Surf. Sci. 2001, 480, L389–L394.
[CrossRef]
46. Shapir, E.; Cohen, H.; Borovok, N.; Kotlyar, A.B.; Porath, D. High-Resolution STM Imaging of Novel Poly(G)−Poly(C) DNA
Molecules. J. Phys. Chem. B 2006, 110, 4430–4433. [CrossRef]
47. Shapir, E.; Cohen, H.; Calzolari, A.; Cavazzoni, C.; Ryndyk, D.A.; Cuniberti, G.; Kotlyar, A.; Di Felice, R.; Porath, D. Electronic
Structure of Single DNA Molecules Resolved by Transverse Scanning Tunnelling Spectroscopy. Nat. Mater. 2008, 7, 68–74.
[CrossRef] [PubMed]
48. Tanaka, H.; Mielke, S.P.; Benham, C.J.; Kawai, T. Visualization of the Detailed Structure of Plasmid DNA. J. Phys. Chem. B 2008,
112, 16788–16792. [CrossRef]
49. Tanaka, H.; Kawai, T. Partial Sequencing of a Single DNA Molecule with a Scanning Tunnelling Microscope. Nat. Nanotechnol.
2009, 4, 518–522. [CrossRef]
50. Abel, G.R.; Korshoj, L.E.; Otoupal, P.B.; Khan, S.; Chatterjee, A.; Nagpal, P. Nucleotide and Structural Label Identification in
Single RNA Molecules with Quantum Tunneling Spectroscopy. Chem. Sci. 2019, 10, 1052–1063. [CrossRef] [PubMed]
51. Fardian-Melamed, N.; Eidelshtein, G.; Rotem, D.; Kotlyar, A.; Porath, D. Scanning Tunneling Microscopy and Spectroscopy of
Novel Silver–Containing DNA Molecules. Adv. Mater. 2019, 31, 1902816. [CrossRef]
52. Fardian-Melamed, N.; Eidelshtein, G.; Rotem, D.; Kotlyar, A.; Porath, D. Temperature Dependence of the STM Morphology and
Electronic Level Structure of Silver-Containing DNA. Small 2020, 16, 1905901. [CrossRef]
53. Chen, Z.; Liu, C.; Cao, F.; Ren, J.; Qu, X. DNA Metallization: Principles, Methods, Structures, and Applications. Chem. Soc. Rev.
2018, 47, 4017–4072. [CrossRef]
54. Van Meer, G.; Voelker, D.R.; Feigenson, G.W. Membrane Lipids: Where They Are and How They Behave. Nat. Rev. Mol. Cell Biol.
2008, 9, 112–124. [CrossRef]
Nanomaterials 2022, 12, 3013 20 of 22

55. Smith, D.P.; Bryant, A.; Quate, C.F.; Rabe, J.P.; Gerber, C.; Swalen, J.D. Images of a Lipid Bilayer at Molecular Resolution by
Scanning Tunneling Microscopy. Proc. Natl. Acad. Sci. USA 1987, 84, 969–972. [CrossRef]
56. Hörber, J.K.H.; Lang, C.A.; Hänsch, T.W.; Heckl, W.M.; Möhwald, H. Scanning Tunneling Microscopy of Lipid Films and
Embedded Biomolecules. Chem. Phys. Lett. 1988, 145, 151–158. [CrossRef]
57. Matsuda, H.; Kishi, E.; Kuroda, R.; Albrecht, O.; Eguchi, K.; Hatanaka, K.; Nakagiri, T. Parallel Arrangement of Fatty Acid
Molecules in Films Deposited at a Lower Surface Pressure. Thin Solid Films 1993, 224, 248–252. [CrossRef]
58. Woodward IV, J.T.; Zasadzinski, J.A. High-Resolution Scanning Tunneling Microscopy of Fully Hydrated Ripple- Phase Bilayers.
Biophys. J. 1997, 72, 964–976. [CrossRef]
59. Zareýe, M.H.; Mozafarý, M.R.; Hasirci, V.; Pýkýn, E. Scanning Tunnelling Microscopy Investigation of Liposome-DNA-Ca2+
Complexes. J. Liposome Res. 1997, 7, 491–502. [CrossRef]
60. Xu, S.; Szymanski, G.; Lipkowski, J. Self-Assembly of Phospholipid Molecules at a Au(111) Electrode Surface. J. Am. Chem. Soc.
2004, 126, 12276–12277. [CrossRef] [PubMed]
61. Sek, S.; Xu, S.; Chen, M.; Szymanski, G.; Lipkowski, J. STM Studies of Fusion of Cholesterol Suspensions and Mixed 1,2-
Dimyristoyl-Sn-Glycero-3-Phosphocholine (DMPC)/Cholesterol Vesicles onto a Au(111) Electrode Surface. J. Am. Chem. Soc.
2008, 130, 5736–5743. [CrossRef]
62. Pawłowski, J.; Juhaniewicz, J.; Güzeloğlu, A.; S˛ek, S. Mechanism of Lipid Vesicles Spreading and Bilayer Formation on a Au(111)
Surface. Langmuir 2015, 31, 11012–11019. [CrossRef] [PubMed]
63. Sek, S.; Laredo, T.; Dutcher, J.R.; Lipkowski, J. Molecular Resolution Imaging of an Antibiotic Peptide in a Lipid Matrix. J. Am.
Chem. Soc. 2009, 131, 6439–6444. [CrossRef]
64. Pieta, P.; Mirza, J.; Lipkowski, J. Direct Visualization of the Alamethicin Pore Formed in a Planar Phospholipid Matrix. Proc. Natl.
Acad. Sci. USA 2012, 109, 21223–21227. [CrossRef]
65. Smetanin, M.; Sek, S.; Maran, F.; Lipkowski, J. Molecular Resolution Visualization of a Pore Formed by Trichogin, an Antimicrobial
Peptide, in a Phospholipid Matrix. Biochim. Biophys. Acta BBA-Biomembr. 2014, 1838, 3130–3136. [CrossRef]
66. Matsunaga, S.; Shimizu, H.; Yamada, T.; Kobayashi, T.; Kawai, M. In Situ STM and Vibrational Study of Nanometer-Scale
Reorganization of a Phospholipid Monolayer Accompanied by Potential-Driven Headgroup Digestion. Langmuir 2017, 33,
13157–13167. [CrossRef] [PubMed]
67. Doerr, A. High-Speed Protein Crystallography. Nat. Methods 2018, 15, 855. [CrossRef]
68. Bonanni, B.; Andolfi, L.; Bizzarri, A.R.; Cannistraro, S. Functional Metalloproteins Integrated with Conductive Substrates:
Detecting Single Molecules and Sensing Individual Recognition Events. J. Phys. Chem. B 2007, 111, 5062–5075. [CrossRef]
69. Edstrom, R.D.; Meinke, M.H.; Yang, X.; Yang, R.; Evans, D.F. Direct Observation of Phosphorylase Kinase and Phosphorylase b by
Scanning Tunneling Microscopy. Biochemistry 1989, 28, 4939–4942. [CrossRef] [PubMed]
70. Miles, M.J.; Carr, H.J.; McMaster, T.C.; I’Anson, K.J.; Belton, P.S.; Morris, V.J.; Field, J.M.; Shewry, P.R.; Tatham, A.S. Scanning
Tunneling Microscopy of a Wheat Seed Storage Protein Reveals Details of an Unusual Supersecondary Structure. Proc. Natl. Acad.
Sci. USA 1991, 88, 68–71. [CrossRef]
71. Arakawa, H.; Umemura, K.; Ikai, A. Protein Images Obtained by STM, AFM and TEM. Nature 1992, 358, 171–173. [CrossRef]
72. Liu, Z.F.; Manivannan, A.; Yanagi, H.; Ashida, M.; Fujishima, A.; Inokuchi, H. Direct Observation of the Secondary Structure of
Unfolded Pseudomonas-Cytochrome C551 by Scanning Tunneling Microscopy. Surf. Sci. 1993, 284, L411–L415. [CrossRef]
73. Chi, Q.; Zhang, J.; Dong, S.; Wang, E. Direct Observation of Native and Unfolded Glucose Oxidase Structures by Scanning
Tunnelling Microscopy. J. Chem. Soc. Faraday Trans. 1994, 90, 2057–2060. [CrossRef]
74. Zhang, J.-D.; Chi, Q.; Dong, S.-J.; Wang, E. High Resolution Imaging of Native Hemoglobin by Scanning Tunneling Microscopy.
J. Electroanal. Chem. 1994, 379, 535–539. [CrossRef]
75. Zhang, B.; Wang, E. Tip-Induced Dynamic Characterization of Multilayers Andvariable Morphologies of Cytochrome c Molecules
on Highlyoriented Pyrolytic Graphite Revealed by in-Situscanning Tunnelling Microscopy. J. Chem. Soc. Faraday Trans. 1997, 93,
327–331. [CrossRef]
76. Davis, J.J.; Halliwell, C.M.; Hill, H.A.O.; Canters, G.W.; van Amsterdam, M.C.; Verbeet, M.P. Protein Adsorption at a Gold
Electrode Studied by Insitu Scanning Tunnelling Microscopy. New J. Chem. 1998, 22, 1119–1123. [CrossRef]
77. Friis, E.P.; Andersen, J.E.T.; Kharkats, Y.I.; Kuznetsov, A.M.; Nichols, R.J.; Zhang, J.-D.; Ulstrup, J. An Approach to Long-Range
Electron Transfer Mechanisms in Metalloproteins: In Situ Scanning Tunneling Microscopy with Submolecular Resolution.
Proc. Natl. Acad. Sci. USA 1999, 96, 1379–1384. [CrossRef] [PubMed]
78. Mukhopadhyay, R.; Davis, J.J.; Kyritsis, P.; Hill, H.A.O.; Meyer, J. A Scanning Tunnelling Microscopy Study of Clostridium
Pasteurianum Rubredoxin. J. Inorg. Biochem. 2000, 78, 251–254. [CrossRef]
79. Rodríguez-Galván, A.; Contreras-Torres, F.F.; Basiuk, E.V.; Alvarez-Zauco, E.; Heredia, A.; Basiuk, V.A. Aggregation of Human
Serum Albumin on Graphite and Single-Walled Carbon Nanotubes as Studied by Scanning Probe Microscopies. J. Nanosci.
Nanotechnol. 2011, 11, 5491–5498. [CrossRef]
80. Riordan, J.F. The Role of Metals in Enzyme Activity. Ann. Clin. Lab. Sci. 1977, 7, 119–129.
81. Contera, S.A.; Iwasaki, H.; Suzuki, S. Ambient STM and in Situ AFM Study of Nitrite Reductase Proteins Adsorbed on Gold and
Graphite: Influence of the Substrate on Protein Interactions. Ultramicroscopy 2003, 97, 65–72. [CrossRef]
82. Contera, S.A.; Iwasaki, H. Imaging the Proteins Pseudoazurin and Apo-Pseudoazurin on Gold by STM in Air: Effect of the Bias
Voltage. Ultramicroscopy 2002, 91, 231–243. [CrossRef]
Nanomaterials 2022, 12, 3013 21 of 22

83. Contera, S.A.; Okajima, T.; Iwasaki, H. Scanning Tunnelling Microscopy Images of the Copper-Containing Amine Oxidase from
Arthrobacter Globiformis in the Holo and Apo Forms Adsorbed on Gold under Ambient Conditions. Jpn. J. Appl. Phys. 2002, 41, 3916.
[CrossRef]
84. Parker, M.; Davies, M.C.; Tendler, S.J.B. Hydrogen Bonding Molecules and Their Effect on Scanning Tunneling Microscope Image
Contrast of Covalently Immobilized Protein Molecules. J. Vac. Sci. Technol. B Microelectron. Nanometer Struct. Process. Meas.
Phenom. 1996, 14, 1432–1437. [CrossRef]
85. Wang, J.; Zhang, L.; Hu, C.; Liu, Q.; Hou, Y.; Zhang, X.; Lu, Q. Sub-Molecular Features of Single Proteins in Solution Resolved
with Scanning Tunneling Microscopy. Nano Res. 2016, 9, 2551–2560. [CrossRef]
86. Zhang, L.; Wang, J.; Wang, H.; Wang, W.; Li, Z.; Liu, J.; Yang, X.; Ji, X.; Luo, Y.; Hu, C.; et al. Moderate and Strong Static Magnetic
Fields Directly Affect EGFR Kinase Domain Orientation to Inhibit Cancer Cell Proliferation. Oncotarget 2016, 7, 41527–41539.
[CrossRef]
87. Shivji, A.P.; Brown, F.; Davies, M.C.; Jennings, K.H.; Roberts, C.J.; Tendler, S.J.B.; Wilkinson, M.J.; Williams, P.M. Scanning
Tunnelling Microscopy Studies of β-Amyloid Fibril Structure and Assembly. FEBS Lett. 1995, 371, 25–28. [CrossRef]
88. Wang, Z.; Zhou, C.; Wang, C.; Wan, L.; Fang, X.; Bai, C. AFM and STM Study of β-Amyloid Aggregation on Graphite.
Ultramicroscopy 2003, 97, 73–79. [CrossRef]
89. Losic, D.; Martin, L.L.; Mechler, A.; Aguilar, M.-I.; Small, D.H. High Resolution Scanning Tunnelling Microscopy of the β-Amyloid
Protein (Aβ1–40) of Alzheimer’s Disease Suggests a Novel Mechanism of Oligomer Assembly. J. Struct. Biol. 2006, 155, 104–110.
[CrossRef]
90. Naruse, N.; Satooka, H.; Todo, K.; Nakanishi, A.; Taguchi, H.; Mera, Y. Oligomers Imaging of Amyloid-B1-42 by Scanning
Tunneling Microscopy. Jpn. J. Appl. Phys. 2019, 58, SIIB30. [CrossRef]
91. Forman, C.J.; Wang, N.; Yang, Z.Y.; Mowat, C.G.; Jarvis, S.; Durkan, C.; Barker, P.D. Probing the Location of Displayed Cytochrome
B562on Amyloid by Scanning Tunnelling Microscopy. Nanotechnology 2013, 24, 175102. [CrossRef]
92. Gathercole, L.J.; Miles, M.J.; McMaster, T.J.; Holmes, D.F. Scanning Probe Microscopy of Collagen I and PN-Collagen I Assemblies
and the Relevance to Scanning Tunnelling Microscopy Contrast Generation in Proteins. J. Chem. Soc. Faraday Trans. 1993, 89,
2589–2594. [CrossRef]
93. Deng, Z.; Thontasen, N.; Malinowski, N.; Rinke, G.; Harnau, L.; Rauschenbach, S.; Kern, K. A Close Look at Proteins: Submolecular
Resolution of Two- and Three-Dimensionally Folded Cytochrome c at Surfaces. Nano Lett. 2012, 12, 2452–2458. [CrossRef]
[PubMed]
94. Perez, S. X-ray Diffraction and Crystallography of Oligosaccharides and Polysaccharides. In Encyclopedia of Biophysics; Roberts,
G.C.K., Ed.; Springer: Berlin/Heidelberg, Germany, 2013; pp. 2767–2777.
95. Cummings, J.H.; Stephen, A.M. Carbohydrate Terminology and Classification. Eur. J. Clin. Nutr. 2007, 61, S5–S18. [CrossRef]
96. Davis, M.E.; Brewster, M.E. Cyclodextrin-Based Pharmaceutics: Past, Present and Future. Nat. Rev. Drug Discov. 2004, 3,
1023–1035. [CrossRef] [PubMed]
97. Rabe, J.P.; Buchholz, S.; Ritcey, A.M. Reactive Graphite Etch and the Structure of an Adsorbed Organic Monolayer—A Scanning
Tunneling Microscopy Study. J. Vac. Sci. Technol. A 1990, 8, 679–683. [CrossRef]
98. Yang, X.; Miller, M.A.; Yang, R.; Evans, D.F.; Edstrom, R.D. Scanning Tunneling Microscopic Images Show a Laminated Structure
for Glycogen Molecules. FASEB J. 1990, 4, 3140–3143. [CrossRef]
99. Lee, I.; Atkins, E.D.T.; Miles, M.J. Visualization of the Algal Polysaccharide Carrageenan by Scanning Tunnelling Microscopy.
Ultramicroscopy 1992, 42–44, 1107–1112. [CrossRef]
100. Miyake, K.; Yasuda, S.; Harada, A.; Sumaoka, J.; Komiyama, M.; Shigekawa, H. Formation Process of Cyclodextrin
Necklace−Analysis of Hydrogen Bonding on a Molecular Level. J. Am. Chem. Soc. 2003, 125, 5080–5085. [CrossRef]
101. Jia, Y.-G.; Malveau, C.; Mezour, M.A.; Perepichka, D.F.; Zhu, X.X. A Molecular Necklace: Threading β-Cyclodextrins onto
Polymers Derived from Bile Acids. Angew. Chem. Int. Ed. 2016, 55, 11979–11983. [CrossRef]
102. Abb, S.; Tarrat, N.; Cortés, J.; Andriyevsky, B.; Harnau, L.; Schön, J.C.; Rauschenbach, S.; Kern, K. Carbohydrate Self-Assembly at
Surfaces: STM Imaging of Sucrose Conformation and Ordering on Cu(100). Angew. Chem. Int. Ed. 2019, 58, 8336–8340. [CrossRef]
103. Abb, S.; Tarrat, N.; Cortés, J.; Andriyevsky, B.; Harnau, L.; Schön, J.C.; Rauschenbach, S.; Kern, K. Polymorphism in Carbohydrate
Self-Assembly at Surfaces: STM Imaging and Theoretical Modelling of Trehalose on Cu(100). RSC Adv. 2019, 9, 35813–35819.
[CrossRef]
104. Wu, X.; Delbianco, M.; Anggara, K.; Michnowicz, T.; Pardo-Vargas, A.; Bharate, P.; Sen, S.; Pristl, M.; Rauschenbach, S.; Schlickum,
U.; et al. Imaging Single Glycans. Nature 2020, 582, 375–378. [CrossRef] [PubMed]
105. Anggara, K.; Zhu, Y.; Delbianco, M.; Rauschenbach, S.; Abb, S.; Seeberger, P.H.; Kern, K. Exploring the Molecular Conformation
Space by Soft Molecule–Surface Collision. J. Am. Chem. Soc. 2020, 142, 21420–21427. [CrossRef] [PubMed]
106. Singh, A. Imaging Single Glycan Molecules. Nat. Methods 2020, 17, 757. [CrossRef] [PubMed]
107. Selkoe, D.J.; Hardy, J. The Amyloid Hypothesis of Alzheimer’s Disease at 25 Years. EMBO Mol. Med. 2016, 8, 595–608. [CrossRef]
[PubMed]
108. Baldacchini, C.; Bizzarri, A.R.; Cannistraro, S. Electron Transfer, Conduction and Biorecognition Properties of the Redox
Metalloprotein Azurin Assembled onto Inorganic Substrates. Eur. Polym. J. 2016, 83, 407–427. [CrossRef]
109. Elliott, M.; Jones, D.D. Approaches to Single-Molecule Studies of Metalloprotein Electron Transfer Using Scanning Probe-Based
Techniques. Biochem. Soc. Trans. 2017, 46, 1–9. [CrossRef]
Nanomaterials 2022, 12, 3013 22 of 22

110. De Morais, M.G.; Martins, V.G.; Steffens, D.; Pranke, P.; da Costa, J.A.V. Biological Applications of Nanobiotechnology. J. Nanosci.
Nanotechnol. 2014, 14, 1007–1017. [CrossRef]
111. Rodríguez-Galván, A.; Heredia, A.; Amelines-Sarria, O.; Rivera, M.; Medina, L.A.; Basiuk, V.A. Non-Covalent Attachment of
Silver Nanoclusters onto Single-Walled Carbon Nanotubes with Human Serum Albumin as Linking Molecule. Appl. Surf. Sci.
2015, 331, 271–277. [CrossRef]
112. Shu, Y.; Cinier, M.; Shu, D.; Guo, P. Assembly of Multifunctional Phi29 PRNA Nanoparticles for Specific Delivery of SiRNA and
Other Therapeutics to Targeted Cells. Methods 2011, 54, 204–214. [CrossRef]
113. Cai, Y.; Sheetz, M.P. Force Propagation across Cells: Mechanical Coherence of Dynamic Cytoskeletons. Curr. Opin. Cell Biol. 2009,
21, 47–50. [CrossRef] [PubMed]
114. Xia, S.; Kanchanawong, P. Nanoscale Mechanobiology of Cell Adhesions. Semin. Cell Dev. Biol. 2017, 71, 53–67. [CrossRef]
[PubMed]
115. Shtengel, G.; Wang, Y.; Zhang, Z.; Goh, W.I.; Hess, H.F.; Kanchanawong, P. Chapter 15—Imaging Cellular Ultrastructure by
PALM, IPALM, and Correlative IPALM-EM. In Methods in Cell Biology; Waters, J.C., Wittman, T., Eds.; Quantitative Imaging in
Cell Biology; Academic Press: Cambridge, MA, USA, 2014; Volume 123, pp. 273–294.
116. Peng, W.; Wang, H.; Lu, H.; Yin, L.; Wang, Y.; Grandidier, B.; Yang, D.; Pi, X. Recent Progress on the Scanning Tunneling
Microscopy and Spectroscopy Study of Semiconductor Heterojunctions. Small 2021, 17, 2100655. [CrossRef] [PubMed]
117. Willner, I. Biomaterials for Sensors, Fuel Cells, and Circuitry. Science 2002, 298, 2407–2408. [CrossRef]
118. Homola, J. Surface Plasmon Resonance Sensors for Detection of Chemical and Biological Species. Chem. Rev. 2008, 108, 462–493.
[CrossRef]
119. Rusmini, F.; Zhong, Z.; Feijen, J. Protein Immobilization Strategies for Protein Biochips. Biomacromolecules 2007, 8, 1775–1789.
[CrossRef]
120. Prabowo, B.A.; Purwidyantri, A.; Liu, K.-C. Surface Plasmon Resonance Optical Sensor: A Review on Light Source Technology.
Biosensors 2018, 8, 80. [CrossRef]
121. Rabia, A.; Tumino, F.; Milani, A.; Russo, V.; Bassi, A.L.; Achilli, S.; Fratesi, G.; Onida, G.; Manini, N.; Sun, Q.; et al. Scanning
Tunneling Microscopy and Raman Spectroscopy of Polymeric Sp–Sp2 Carbon Atomic Wires Synthesized on the Au(111) Surface.
Nanoscale 2019, 11, 18191–18200. [CrossRef]
122. Hla, S.-W.; Bartels, L.; Meyer, G.; Rieder, K.-H. Inducing All Steps of a Chemical Reaction with the Scanning Tunneling Microscope
Tip: Towards Single Molecule Engineering. Phys. Rev. Lett. 2000, 85, 2777–2780. [CrossRef]
123. Reimers, J.R.; Yang, J.; Darwish, N.; Kosov, D.S. Silicon-Single Molecule-Silicon Circuits. Chem. Sci. 2021, 12, 15870–15881.
[CrossRef]
124. Deng, X.; Xiong, F.; Li, X.; Xiang, B.; Li, Z.; Wu, X.; Guo, C.; Li, X.; Li, Y.; Li, G.; et al. Application of Atomic Force Microscopy in
Cancer Research. J. Nanobiotechnol. 2018, 16, 102. [CrossRef] [PubMed]
125. Chen, C.-C.; Zhou, Y.; Baker, L.A. Scanning Ion Conductance Microscopy. Annu. Rev. Anal. Chem. 2012, 5, 207–228. [CrossRef]
[PubMed]
126. Odom, T.W.; Huang, J.-L.; Kim, P.; Lieber, C.M. Atomic Structure and Electronic Properties of Single-Walled Carbon Nanotubes.
Nature 1998, 391, 62–64. [CrossRef]
127. Binnig, G.; Rohrer, H. Scanning Tunneling Microscopy—From Birth to Adolescence. Rev. Mod. Phys. 1987, 59, 615–625. [CrossRef]
128. Binnig, G.; Rohrer, H. Scanning Tunneling Microscopy. Surf. Sci. 1985, 152–153, 17–26. [CrossRef]
129. Drexler, K.E. Molecular Engineering: An Approach to the Development of General Capabilities for Molecular Manipulation.
Proc. Natl. Acad. Sci. USA 1981, 78, 5275–5278. [CrossRef]
130. Potorochin, D.V.; Chaika, A.N.; Molodtsova, O.V.; Aristov, V.Y.; Marchenko, D.E.; Smirnov, D.A.; Makarova, A.A.; Walls, B.;
Zhussupbekov, K.; Walshe, K.; et al. Surface Functionalization of Few-Layer Graphene on β-SiC(001) by Neutral Red Dye.
Appl. Surf. Sci. 2022, 585, 152542. [CrossRef]
131. Jäckel, F.; Perera, U.G.E.; Iancu, V.; Braun, K.-F.; Koch, N.; Rabe, J.P.; Hla, S.-W. Investigating Molecular Charge Transfer Complexes
with a Low Temperature Scanning Tunneling Microscope. Phys. Rev. Lett. 2008, 100, 126102. [CrossRef]
132. Baird, D.; Nordmann, A.; Schummer, J. Discovering the Nanoscale, 3rd ed.; IOS Press: Amsterdam, The Netherlands, 2004.
133. Abb, S.; Harnau, L.; Gutzler, R.; Rauschenbach, S.; Kern, K. Two-Dimensional Honeycomb Network through Sequence-Controlled
Self-Assembly of Oligopeptides. Nat. Commun. 2016, 7, 10335. [CrossRef]
134. Anand, G.; Sharma, S.; Dutta, A.K.; Kumar, S.K.; Belfort, G. Conformational Transitions of Adsorbed Proteins on Surfaces of
Varying Polarity. Langmuir 2010, 26, 10803–10811. [CrossRef]