Noncontact Atomic Force Microscopy Images Research Articles

Determination of in-plane surface directions in scanning probe microscopy images.

We describe an approach to determine the in-plane crystallographic surface directions in scanning probe microscopy (SPM) images. This method is based on a one-time characterization of the SPM instrument with an appropriate test sample and is exemplified by the analysis of non-contact atomic force microscopy (NC-AFM) images on surfaces whose natural cleavage occurs along {111} planes. We introduce a two-dimensional rotation matrix relating the crystallographic surface directions known from an analysis of the macroscopic crystal to the directions in the NC-AFM images. The procedure takes into account rotations and mirror axes resulting from sample mounting, the SPM scanner rotation, the choice of scan direction, as well as data processing, storage, and display. We demonstrate the practicability of the approach by determining the [112̄] direction in topographic images of a CeO2(111) film grown on a Si(111) wafer and atomic resolution images of CaF2(111) with an instrument based on the beetle-type scanner.

Development of a cryogen-free sub-3K low-temperature scanning probe microscope by remote liquefaction scheme.

We developed a new scheme for cryogen-free cooling down to sub-3K temperature range and ultra-low vibration level. An ultra-high-vacuum cryogen-free scanning probe microscope (SPM) system was built based on the new scheme. Instead of mounting a below-decoupled cryocooler directly onto the system, the new design was realized by integrating a Gifford-McMahon cryocooler into a separate liquefying chamber, providing two-stage heat exchangers in a remote way. About 10 L of helium gas inside the gas handling system was cooled, liquefied in the liquefying chamber, and then transferred to a continuous-flow cryostat on the SPM chamber through an ∼2m flexible helium transfer line. The exhausted helium gas from the continuous-flow cryostat was then returned to the liquefying chamber for reliquefaction. A base temperature of ∼2.84K at the scanner sample stage and a temperature fluctuation of almost within ±0.1 mK at 4K were achieved. The cooling curves, tunneling current noise, variable-temperature test, scanning tunneling microscopy and non-contact atomic force microscopy imaging, and first and second derivatives of I(V) spectra are characterized to verify that the performance of our cryogen-free SPM system is comparable to the bath cryostat-based low-temperature SPM system. This remote liquefaction close-cycle scheme shows conveniency to upgrade the existing bath cryostat-based SPM system, upgradeability of realizing even lower temperature down to sub-1K range, and great compatibility of other physical environments, such as high magnetic field and optical accesses. We believe that the new scheme could also pave a way for other cryogenic applications requiring low temperature but sensitive to vibration.

Identification of OH groups on SrTiO3(100)-(13×13)-R33.7° reconstructed surface by non-contact atomic force microscopy and scanning tunneling microscopy

Atomic resolution imaging of a SrTiO3(100)-(13×13)-R33.7° reconstructed surface using non-contact atomic force microscopy (NC-AFM) and its simultaneous measurement with scanning tunneling microscopy (STM) is presented. Simultaneous STM and NC-AFM imaging reveals three patterns of image contrast depending on the tip apex condition and the relationship between the SrTiO3(100)-(13×13)-R33.7° surface reconstructed structure and the NC-AFM image contrast. The NC-AFM image contrast variation is deduced from the tip apex polarity on the basis of an analysis of two images with opposite contrast. This interpretation is consistent with the results of simultaneous imaging of the SrTiO3(100)-(13×13)-R33.7° surface. Furthermore, the results and interpretation identified an OH group, which is one of the surface defects, and this adsorption site.

Automated real-space lattice extraction for atomic force microscopy images

Analyzing atomically resolved images is a time-consuming process requiring solid experience and substantial human intervention. In addition, the acquired images contain a large amount of information such as crystal structure, presence and distribution of defects, and formation of domains, which need to be resolved to understand a material’s surface structure. Therefore, machine learning techniques have been applied in scanning probe and electron microscopies during the last years, aiming for automatized and efficient image analysis. This work introduces a free and open source tool (AiSurf: Automated Identification of Surface Images) developed to inspect atomically resolved images via scale-invariant feature transform and clustering algorithms. AiSurf extracts primitive lattice vectors, unit cells, and structural distortions from the original image, with no pre-assumption on the lattice and minimal user intervention. The method is applied to various atomically resolved non-contact atomic force microscopy images of selected surfaces with different levels of complexity: anatase TiO2(101), oxygen deficient rutile TiO2(110) with and without CO adsorbates, SrTiO3(001) with Sr vacancies and graphene with C vacancies. The code delivers excellent results and is tested against atom misclassification and artifacts, thereby facilitating the interpretation of scanning probe microscopy images.

On-Surface Synthesis of C144 Hexagonal Coronoid with Zigzag Edges.

Coronoids as polycyclic aromatic macrocycles enclosing a cavity have attracted a lot of attention due to their distinctive molecular and electronic structures. They can be also regarded as nanoporous graphene molecules whose electronic properties are critically dependent on the size and topology of their outer and inner peripheries. However, because of their synthetic challenges, the extended hexagonal coronoids with zigzag outer edges have not been reported yet. Here, we report the on-surface synthesis of C144 hexagonal coronoid with outer zigzag edges on a designed precursor undergoing hierarchical Ullmann coupling and cyclodehydrogenation on the Au(111) surface. The molecular structure is unambiguously characterized by bond-resolved noncontact atomic force microscopy imaging. The electronic properties are further investigated by scanning tunneling spectroscopy measurements, in combination with the density functional theory calculations. Moreover, the values of the harmonic oscillator model of aromaticity are derived from calculations that suggest that the molecular structure is ideally represented by Clar's model. Our results provide approaches toward realizing a hexagonal coronoid with zigzag edges, potentially inspiring fabrication of hexagonal zigzag coronoids with multiple radical characters in the future.

Development of a near-5-Kelvin, cryogen-free, pulse-tube refrigerator-based scanning probe microscope.

We report the design and performance of a cryogen-free, pulse-tube refrigerator (PTR)-based scanning probe microscopy (SPM) system capable of operating at a base temperature of near 5 K. We achieve this by combining a home-made interface design between the PTR cold head and the SPM head, with an automatic gas-handling system. The interface design isolates the PTR vibrations by a combination of polytetrafluoroethylene and stainless-steel bellows and by placing the SPM head on a passive vibration isolation table via two cold stages that are connected to thermal radiation shields using copper heat links. The gas-handling system regulates the helium heat-exchange gas pressures, facilitating both the cooldown to and maintenance of the base temperature. We discuss the effects of each component using measured vibration, current-noise, temperature, and pressure data. We demonstrate that our SPM system performance is comparable to known liquid-helium-based systems with the measurements of the superconducting gap spectrum of Pb, atomic-resolution scanning tunneling microscopy image and quasiparticle interference pattern of Au(111) surface, and non-contact atomic force microscopy image of NaCl(100) surface. Without the need for cryogen refills, the present SPM system enables uninterrupted low-temperature measurements.

NBN-doped nanographene embedded with five- and seven-membered rings on Au(111) surface* *Project supported by the National Natural Science Foundation of China (Grant Nos. 51761135130 and 61888102), the National Key R&D Program of China (Grant Nos. 2018YFA0305800 and 2019YFA0308500), DFG EnhanceNano (Grant No. 391979941), the Strategic Priority Research Program of Chinese Academy of Sciences (Grant No. XDB30000000), the International Partnership Program

Nanographenes (NGs) can be embedded with predesigned dopants or nonhexagonal rings to tailor the electronic properties and provide ideal platforms to study the unique physical and chemical properties. Here, we report the on-surface synthesis of NBN-doped NG embedded with five- and seven-membered rings (NBN-575-NG) on Au(111) from a oligophenylene precursor preinstalled with a NBN unit and a heptagonal ring. Scanning tunneling microscopy and non-contact atomic force microscopy images elucidate the intramolecular cyclodehydrogenation and the existence of the five- and seven-membered rings. Scanning tunneling spectroscopy spectra reveal that the NBN-575-NG is a semiconductor, which agrees with the density functional theory calculation results on a freestanding NBN-575-NG with the same structure. This work provides a feasible approach for the on-surface synthesis of novel NGs containing non-hexagonal rings.

Synthesis and characterization of Mg0.4Ti0.6O2 alloy thin film

The present work includes the fabrication of Mg0.4Ti0.6O2 thin film (TF) based Metal-oxide–semiconductor (MOS) capacitor on (100) n-type GaAs substrates by electron beam evaporation technique. The thickness of the thin film alloy is found to be about 300 nm measured from Field emission gun-scanning electron microscope (FEG-SEM) data. The pore volume, surface roughness and grain size of the thin film samples are calculated from non-contact atomic force microscopy (NC-AFM) images. Experimental as well as analytical studies have been carried out to determine the bandgap and defect level transition of the material. The band-to-band transition of the Mg0.4Ti0.6O2 sample is calculated to be at ~ 5.2 eV. The as prepared sample shows tetragonal crystal structure. Presence of anatase phase of TiO2 in the TF material is confirmed through XRD (X-ray diffraction) investigation. Current (I) – Voltage (V) characteristics of the Mg0.4Ti0.6O2 Schottky devices reveal that the leakage current at −1 V is 8.1 × 10-4 A.

Surface and electronic structure of antiferromagnetic axion insulator candidate EuIn<sub>2</sub>As<sub>2</sub>

The interplay between non-trivial band topology and magnetic order can induce exotic quantum phenomena, such as the quantum anomalous Hall effect and axion insulator state. A prevalent approach to realizing such topological states is either by magnetic doping or through heterostructure engineering, while the former will bring in inhomogeneity and the latter requires complex procedures. Intrinsic magnetic topological insulators are expected to avoid the aforementioned disadvantages, which is of great significance in both studying and practically using these exotic quantum phenomena. Recently, a Zintl compound EuIn<sub>2</sub>As<sub>2</sub> is predicted to be an intrinsic antiferromagnetic axion insulator. The bulk magnetic order of EuIn<sub>2</sub>As<sub>2</sub> has been reported in a lot of experiments, while the topological nature has not yet been confirmed. The surface properties of intrinsic magnetic topological insulators play an important role in the interplay between magnetic order and non-trivial surface state. Here in this work, we study the surface structure and electronic property of EuIn<sub>2</sub>As<sub>2</sub> single crystal by using scanning tunneling microscopy/spectroscopy (STM/S) and non-contact atomic force microscopy (NC-AFM). Considering the strength of bonds, the easy cleavage plane of the crystals possibly lies between In-In layers or between Eu-As layers. The STM topographies show that the cleaved surface is dominated by a striped pattern. And the dominated step height is an integer multiple of <i>c</i>/2, which implies that only one kind of cleavage plane is preferred. Atomic-resolved surface topographies show that the striped pattern is the <inline-formula><tex-math id="M2">\begin{document}$ 1\times 2 $\end{document}</tex-math><alternatives><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="18-20210783_M2.jpg"/><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="18-20210783_M2.png"/></alternatives></inline-formula> surface reconstruction with 50% coverage. Hence an In-terminated surface which will be 100% coverage is ruled out. The spatial evolution of STS near vacancies on the striped pattern shows a hole-doping feature. All of these results reveal that the striped pattern is the <inline-formula><tex-math id="M3">\begin{document}$ 1\times 2 $\end{document}</tex-math><alternatives><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="18-20210783_M3.jpg"/><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="18-20210783_M3.png"/></alternatives></inline-formula> surface reconstruction of the Eu terminated surface with 50% coverage. Using the STS, we measure the local densities of states on the striped surface at various temperatures. We find that there is an asymmetric valley-peak feature in the density of states near the Fermi energy at 4 K, which is gradually weakened with increasing temperature, and disappears above the antiferromagnetic Néel temperature, indicating that the asymmetric valley-peak feature is closely related to the antiferromagnetic order. Besides, a maze-like pattern is observed occasionally near some step edges. The STM topographies show atoms both on bright and dark stripes of the maze-like pattern, which form a whole hexagonal lattice. And the NC-AFM images show that the maze-like pattern is about 1 Å higher than the Eu terminated striped pattern. Based on these results, the maze-like pattern can be explained as the buckled Eu surface with 100% coverage. These results provide important information for understanding the surface electronic band structure and topological nature of EuIn<sub>2</sub>As<sub>2</sub>.

Protruding hydrogen atoms as markers for the molecular orientation of a metallocene.

A distinct dumbbell shape is observed as the dominant contrast feature in the experimental data when imaging 1,1’-ferrocene dicarboxylic acid (FDCA) molecules on bulk and thin film CaF2(111) surfaces with non-contact atomic force microscopy (NC-AFM). We use NC-AFM image calculations with the probe particle model to interpret this distinct shape by repulsive interactions between the NC-AFM tip and the top hydrogen atoms of the cyclopentadienyl (Cp) rings. Simulated NC-AFM images show an excellent agreement with experimental constant-height NC-AFM data of FDCA molecules at several tip–sample distances. By measuring this distinct dumbbell shape together with the molecular orientation, a strategy is proposed to determine the conformation of the ferrocene moiety, herein on CaF2(111) surfaces, by using the protruding hydrogen atoms as markers.

Chemical and steric effects in simulating noncontact atomic force microscopy images of organic molecules on a Cu (111) substrate

Noncontact mode of atomic force microscopy (nc-AFM) employing a CO-functionalized tip is a very powerful tool for studying molecular structures. However, interpreting nc-AFM images for nonplanar molecules can sometimes be problematic. To illustrate and resolve the nature of such problematic systems, we employ real-space pseudopotentials constructed within density functional theory to simulate nc-AFM images. We focus on several representative nonplanar organic molecules (pentacene, naphthanthrone, olympicene, and 6-phenylhexa-1,3,5-triynybenzene (PHTB)) on a typical substrate: the Cu (111) surface. This substrate results in significant distortions in the molecular geometries of pentacene and naphthanthrone. Including these distortions in simulated nc-AFM imaging notably improves the agreement between the simulated and measured images. In naphthanthrone, the relatively large interaction between the O atom and the Cu substrate offers a straightforward explanation for the absence of the $\mathrm{C}=\mathrm{O}$ bond in the measured image. Nonplanar features such as tilting or twisting are also apparent in olympicene and PHTB. A ``triangular'' bright feature associated with the $\ensuremath{-}\mathrm{C}{\mathrm{H}}_{2}$ group in olympicene appears in simulated and measured nc-AFM images. This feature is directly related to the tilting angle of the molecule with respect to the Cu substrate. The ``defective'' benzene ring feature and the faint ellipsoidal $\mathrm{C}\ensuremath{\equiv}\mathrm{C}$ feature in PHTB can be ascribed to its twisted nature. The ability to simulate such subatomic images in nc-AFM reflects the accuracy and efficiency of calculating quantum forces in real space.

High resolution noncontact atomic force microscopy imaging with oxygen-terminated copper tips at 78 K.

Functionalizing atomic force microscopy (AFM) tips by picking up single inert probe particles like CO or Xe from the surface drastically increase the resolution. In particular, this approach allows imaging organic molecules with submolecular resolution revealing their internal bonding structure. However, due to the weak coupling of these probe particles to both, the surface they are picked up from and the tip apex, these experiments require liquid helium temperatures (i.e.≈5 K). In the present study we demonstrate that functionalizing an AFM tip with an atomically defined O-terminated copper tip (CuOx tip) allows performing such experiments at liquid nitrogen temperatures (i.e.≈78 K) with outstanding quality. We show that it is possible to utilize CuOx tips for chemically selective imaging of a copper oxide nanodomain on a partially oxidized Cu(110) surface in the repulsive force regime at elevated temperatures. Moreover, the high structural and chemical stability of CuOx tips allow even ex situ investigations where these tips are used to perform experiments on other, non-Cu, non-oxidized, substrates. In particular, we present results obtained from a dicoronylene (DCLN) molecule with submolecular resolution. An analysis of inner and peripheral bond lengths of the DCLN molecule shows excellent agreement with theoretical gas phase simulations emphasizing the exceptional imaging properties of CuOx tips also at elevated temperatures.

Strain-Induced Isomerization in One-Dimensional Metal-Organic Chains.

The ability to use mechanical strain to steer chemical reactions creates completely new opportunities for solution- and solid-phase synthesis of functional molecules and materials. However, this strategy is not readily applied in the bottom-up on-surface synthesis of well-defined nanostructures. We report an internal strain-induced skeletal rearrangement of one-dimensional (1D) metal-organic chains (MOCs) via a concurrent atom shift and bond cleavage on Cu(111) at room temperature. The process involves Cu-catalyzed debromination of organic monomers to generate 1,5-dimethylnaphthalene diradicals that coordinate to Cu adatoms, forming MOCs with both homochiral and heterochiral naphthalene backbone arrangements. Bond-resolved non-contact atomic force microscopy imaging combined with density functional theory calculations showed that the relief of substrate-induced internal strain drives the skeletal rearrangement of MOCs via 1,3-H shifts and shift of Cu adatoms that enable migration of the monomer backbone toward an energetically favorable registry with the Cu(111) substrate. Our findings on this strain-induced structural rearrangement in 1D systems will enrich the toolbox for on-surface synthesis of novel functional materials and quantum nanostructures.

Advances in Atomic Force Microscopy: Weakly Perturbative Imaging of the Interfacial Water.

The structure and dynamics of interfacial water, determined by the water-interface interactions, are important for a wide range of applied fields and natural processes, such as water diffusion (Kim et al., 2013), electrochemistry (Markovic, 2013), heterogeneous catalysis (Over et al., 2000), and lubrication (Zilibotti et al., 2013). The precise understanding of water-interface interactions largely relies on the development of atomic-scale experimental techniques (Guo et al., 2014) and computational methods (Hapala et al., 2014b). Scanning probe microscopy has been extensively applied to probe interfacial water in many interdisciplinary fields (Ichii et al., 2012; Shiotari and Sugimoto, 2017; Peng et al., 2018a). In this perspective, we review the recent progress in the noncontact atomic force microscopy (nc-AFM) imaging and AFM simulation techniques and discuss how the newly developed techniques are applied to study the properties of interfacial water. The nc-AFM with the quadrupole-like CO-terminated tip can achieve ultrahigh-resolution imaging of the interfacial water on different surfaces, trace the reconstruction of H-bonding network and determine the intrinsic structures of the weakly bonded water clusters and even their metastable states. In the end, we present an outlook on the directions of future AFM studies of interfacial water as well as the challenges faced by this field.

On-Surface Synthesis and Characterization of Acene-Based Nanoribbons Incorporating Four-Membered Rings.

A bottom up method for the synthesis of unique tetracene-based nanoribbons, which incorporate cyclobutadiene moieties as linkers between the acene segments, is reported. These structures were achieved through the formal [2+2] cycloaddition reaction of ortho-functionalized tetracene precursor monomers. The formation mechanism and the electronic and magnetic properties of these nanoribbons were comprehensively studied by means of a multitechnique approach. Ultra-high vacuum scanning tunneling microscopy showed the occurrence of metal-coordinated nanostructures at room temperature and their evolution into nanoribbons through formal [2+2] cycloaddition at 475 K. Frequency-shift non-contact atomic force microscopy images clearly proved the presence of bridging cyclobutadiene moieties upon covalent coupling of activated tetracene molecules. Insight into the electronic and vibrational properties of the so-formed ribbons was obtained by scanning tunneling microscopy, Raman spectroscopy, and theoretical calculations. Magnetic properties were addressed from a computational point of view, allowing us to propose promising candidates to magnetic acene-based ribbons incorporating four-membered rings. The reported findings will increase the understanding and availability of new graphene-based nanoribbons with high potential in future spintronics.

Imaging patterns of anatase TiO2(001) with non-contact atomic force microscopy

Image patterns of anatase TiO2(001) with non-contact atomic force microscopy (NC-AFM) are presented. A combined system of NC-AFM with pulsed laser deposition, scanning tunneling microscopy (STM), and low energy electron diffraction (LEED) enables us to prepare clean surfaces of anatase TiO2 thin films and to perform measurements on them without breaking the vacuum or the need of cleaning the surface with sputtering and annealing cycles. Results from STM and LEED show that the anatase TiO2(001) films display a (1 × 4) surface pattern of row structures. At far distances, in which the frequency shift was relatively small, NC-AFM images show three different patterns (labeled as protrusion, hole, and neutral contrast, respectively) similar to the ones that have been reported before in the studies of the rutile TiO2 surface. At closer tip-sample distances, a periodic pattern consistent with the TiO2 lattice constant was observed along the higher-positioned TiO3 rows. We have also observed that energy dissipation is induced only with platinum tips, and not with other tip apexes.

Micrometre-long covalent organic fibres by photoinitiated chain-growth radical polymerization on an alkali-halide surface.

On-surface polymerization is a promising technique to prepare organic functional nanomaterials that are challenging to synthesize in solution, but it is typically used on metal substrates, which play a catalytic role. Previous examples on insulating surfaces have involved intermediate self-assembled structures, which face high barriers to diffusion, or annealing to higher temperatures, which generally causes rapid dewetting and desorption of the monomers. Here we report the photoinitiated radical polymerization, initiated from a two-dimensional gas phase, of a dimaleimide monomer on an insulating KCl surface. Polymer fibres up to 1 μm long are formed through chain-like rather than step-like growth. Interactions between potassium cations and the dimaleimide's oxygen atoms facilitate the propagation of the polymer fibres along a preferred axis of the substrate over long distances. Density functional theory calculations, non-contact atomic force microscopy imaging and manipulations at room temperature were used to explore the initiation and propagation processes, as well as the structure and stability of the resulting one-dimensional polymer fibres.

Combined pulsed laser deposition and non-contact atomic force microscopy system for studies of insulator metal oxide thin films.

We have designed and developed a combined system of pulsed laser deposition (PLD) and non-contact atomic force microscopy (NC-AFM) for observations of insulator metal oxide surfaces. With this system, the long-period iterations of sputtering and annealing used in conventional methods for preparing a metal oxide film surface are not required. The performance of the combined system is demonstrated for the preparation and high-resolution NC-AFM imaging of atomically flat thin films of anatase TiO2(001) and LaAlO3(100).

Atomic White-Out: Enabling Atomic Circuitry through Mechanically Induced Bonding of Single Hydrogen Atoms to a Silicon Surface

We report the mechanically induced formation of a silicon-hydrogen covalent bond and its application in engineering nanoelectronic devices. We show that using the tip of a noncontact atomic force microscope (NC-AFM), a single hydrogen atom could be vertically manipulated. When applying a localized electronic excitation, a single hydrogen atom is desorbed from the hydrogen-passivated surface and can be transferred to the tip apex, as evidenced from a unique signature in frequency shift curves. In the absence of tunnel electrons and electric field in the scanning probe microscope junction at 0 V, the hydrogen atom at the tip apex is brought very close to a silicon dangling bond, inducing the mechanical formation of a silicon-hydrogen covalent bond and the passivation of the dangling bond. The functionalized tip was used to characterize silicon dangling bonds on the hydrogen-silicon surface, which was shown to enhance the scanning tunneling microscope contrast, and allowed NC-AFM imaging with atomic and chemical bond contrasts. Through examples, we show the importance of this atomic-scale mechanical manipulation technique in the engineering of the emerging technology of on-surface dangling bond based nanoelectronic devices.

Influence of antibody immobilization strategies on the analytical performance of a magneto-elastic immunosensor for Staphylococcus aureus detection

Magneto-elastic (ME) sensors have a great advantage in microbiology due to their ability to be queried wirelessly. Staphylococcus aureus is one of the most common bacteria widespread in the environment and a major human pathogen related to numerous illnesses. Immunosensors are affinity-based assays where the analyte is highly selective. The immobilization of antibodies (Ab) is an important step in the development of such devices. This study compared the effects of two antibody immobilization strategies on the analytical performance of a magneto-elastic immunosensor: (1) random antibody covalent immobilization (CysAb) and (2) specific-oriented antibody covalent immobilization (PrGAb). Immunosensors were exposed to solutions containing S. aureus at different concentrations (104 to 108CFU/ml) and sensor resonant frequencies were measured. In order to confirm that the frequency shifts were mainly caused by the binding of S. aureus to the sensor's surface, scanning electron microscope (SEM) and indirect immunofluorescence (IIF) images were taken after bacteria exposure at 108CFU/ml. Sensor surfaces were further monitored by non-contact topographic atomic force microscopy (AFM) images. In the covalent-oriented strategy, PrG was first bound covalently to the surface, which in turn, then binds the anti-S. aureus antibody in an oriented manner. Topographic AFM images showed different surface patterns between the two antibody immobilization strategies. Specific-oriented antibody covalent immobilization (PrGAb) strategy gave the highest anti-S. aureus antibody immobilization density. Therefore, the covalent-oriented strategy presented the best performance for S. aureus capture, detecting 104CFU/ml.