Sub-nanometer Resolution Research Articles

Improving the accuracy of SIF quantified from moderate spectral resolution airborne hyperspectral imager using SCOPE: assessment with sub-nanometer imagery

Hyperspectral imaging of solar-induced chlorophyll fluorescence (SIF) is required for plant phenotyping and stress detection. However, the most accurate instruments for SIF quantification, such as sub-nanometer (≤1-nm full-width at half-maximum, FWHM) airborne hyperspectral imagers, are expensive and uncommon. Previous studies have demonstrated that standard narrow-band hyperspectral imagers (i.e., 4–6-nm FWHM) are more cost-effective and can provide far-red SIF quantified at 760 nm (SIF760), which correlates strongly with precise sub-nanometer resolution measurements. Nevertheless, narrow-band SIF760 quantifications are subject to systematic overestimation owing to the influence of the spectral resolution (SR). In this study, we propose a modelling approach based on the Soil Canopy Observation, Photochemistry and Energy Fluxes (SCOPE) model with the objective of enhancing the accuracy of absolute SIF760 levels derived from standard airborne hyperspectral imagers in practical settings. The performance of the proposed method was evaluated using airborne imagery acquired from two airborne hyperspectral imagers (FWHM ≤ 0.2-nm and 5.8-nm) flown in tandem on board an aircraft that collected data from two different wheat and maize phenotyping trials. Leaf biophysical and biochemical traits were first estimated from airborne narrow-band reflectance imagery and subsequently used as SCOPE model inputs to simulate a range of top-of-canopy (TOC) radiance and SIF spectra at 1-nm FWHM. The SCOPE simulated radiance spectra were then convolved to match the spectral configuration of the narrow-band imager to compute the 5.8-nm FWHM SIF760. A site-specific model was constructed by employing the convolved 5.8-nm SR SIF760 as the independent variable and the 1-nm SR SIF760 directly simulated by SCOPE as the dependent variable. When applied to the airborne dataset, the estimated SIF760 at 1-nm SR from the standard narrow-band hyperspectral imager matched the reference sub-nanometer quantified SIF760 with root mean square error (RMSE) less than 0.5 mW/m2/nm/sr, yielding R2 = 0.93–0.95 from the two experiments. These results suggest that the proposed modelling approach enables the interpretation of SIF760 quantified using standard hyperspectral imagers of 4–6 nm FWHM for stress detection and plant physiological condition assessment.

Multiscale rheology from bulk to nano using a quartz tuning fork-atomic force microscope.

Rheological characteristics exhibit significant variations at nanoscale confinement or near interfaces, compared to bulk rheological properties. To bridge the gap between nano- and bulk-scale rheology, allowing for a better and holistic understanding of rheology, developing a single experimental platform that provides rheological measurements across different scales, from nano to bulk, is desirable. Here, we present the novel methodology for multiscale rheology using a highly sensitive atomic force microscope based on a quartz tuning fork (QTF) force sensor. We employ microscale and nanoscale shear probes attached to the QTF, oscillating parallel to a substrate surface for rheological measurements as a function of the tip-substrate distance with sub-nanometer resolution. Silicone oils with viscosities ranging from 5 cSt to 10 000 cSt are used as calibration samples, and we have successfully derived the bulk rheological moduli. Furthermore, an increase in modulus is observed within the regime of tribo-nanorheology at distances less than 50nm from the surface. Through such multiscale measurements, it is confirmed that this increase is due to the formation of a layered structure of silicone oil polymers on the solid surface. These results provide a comprehensive understanding of the tribo-rheological properties of complex fluids across different scales.

Low-dose cryo-electron ptychography of proteins at sub-nanometer resolution

Cryo-transmission electron microscopy (cryo-EM) of frozen hydrated specimens is an efficient method for the structural analysis of purified biological molecules. However, cryo-EM and cryo-electron tomography are limited by the low signal-to-noise ratio (SNR) of recorded images, making detection of smaller particles challenging. For dose-resilient samples often studied in the physical sciences, electron ptychography – a coherent diffractive imaging technique using 4D scanning transmission electron microscopy (4D-STEM) – has recently demonstrated excellent SNR and resolution down to tens of picometers for thin specimens imaged at room temperature. Here we apply 4D-STEM and ptychographic data analysis to frozen hydrated proteins, reaching sub-nanometer resolution 3D reconstructions. We employ low-dose cryo-EM with an aberration-corrected, convergent electron beam to collect 4D-STEM data for our reconstructions. The high frame rate of the electron detector allows us to record large datasets of electron diffraction patterns with substantial overlaps between the interaction volumes of adjacent scan positions, from which the scattering potentials of the samples are iteratively reconstructed. The reconstructed micrographs show strong SNR enabling the reconstruction of the structure of apoferritin protein at up to 5.8 Å resolution. We also show structural analysis of the Phi92 capsid and sheath, tobacco mosaic virus, and bacteriorhodopsin at slightly lower resolutions.

The Structure of Cilium Inner Junctions Revealed by Electron Cryo-tomography.

The cilium is a microtubule-based organelle critical for many cellular functions. Its assembly initiates at a basal body and continues as an axoneme that projects out of the cell to form a functional cilium. This assembly process is tightly regulated. However, our knowledge of the molecular architecture and the mechanism of assembly is limited. By applying electron cryotomography and subtomogram averaging, we obtained subnanometer resolution structures of the inner junction in three distinct regions of the cilium: the proximal region of the basal body, the central core of the basal body, and the flagellar axoneme. The structures allowed us to identify several basal body and axoneme components. While a few proteins are distributed throughout the entire length of the organelle, many are restricted to particular regions of the cilium, forming intricate local interaction networks and bolstering local structural stability. Finally, by knocking out a critical basal body inner junction component Poc1, we found the triplet MT was destabilized, resulting in a defective structure. Surprisingly, several axoneme-specific components were found to "infiltrate" into the mutant basal body. Our findings provide molecular insight into cilium assembly at its inner Junctions, underscoring its precise spatial regulation.

Fine-Tuning of Optical Resonance Wavelength of Surface-Micromachined Optical Ultrasound Transducer Arrays for Single-Wavelength Light Source Readout.

This article reports the fine-tuning of the optical resonance wavelength (ORW) of surface-micromachined optical ultrasound transducer (SMOUT) arrays to enable ultrasound data readout with non-tunable interrogation light sources for photoacoustic computed tomography (PACT). Permanent ORW tuning is achieved by material deposition onto or subtraction from the top diaphragm of each element with sub-nanometer resolution. For demonstration, a SMOUT array is first fabricated, and its ORW is tuned for readout with an 808 nm laser diode (LD). Experiments are conducted to characterize the optical and acoustic performances of the elements within the center region of the SMOUT array. Two-dimensional and three-dimensional PACT (photoacoustic computed tomography) is also performed to evaluate the imaging performance of the ORW-tuned SMOUT array. The results show that the ORW tuning does not degrade the optical, acoustic, and overall imaging performances of the SMOUT elements. As a result, the fine-tuning method enables new SMOUT-based PACT systems that are low cost, compact, powerful, and even higher speed, with parallel readout capability.

Theoretical study of tip-enhanced photoluminescence of single molecule in nanocavity formed by apex protrusion and substrate

Tip-enhanced photoluminescence techniques, characterized by ultra-high detection sensitivity and spatial resolution, have exhibited unprecedented capabilities in exploring optoelectronic phenomena and unraveling intrinsic physical mechanism at the single-molecule level. To suppress the quenching of molecular luminescence and achieve high-resolution optical imaging, a rational design of the tip-substrate configuration is required. In this paper, we systematically investigate the tip-enhanced photoluminescence characteristics of single molecules located within the plasmonic nanocavity formed by the tip protrusion and the substrate from a theoretical perspective. The impacts of electron tunneling and electron-hole pair creation on localized electric field and quantum yield are discussed using the quantum correction model and nonlocal dielectric theory, respectively. The crucial roles of atomic-scale protrusion at the tip apex and the decoupling layer on the substrate in enhancing vertical and horizontal electric fields as well as quantum yields have been emphasized. The results indicate that in smaller sub-nanometer cavities, electron tunneling and nonlocal dielectric effects greatly weaken the electric field intensity and fluorescence quantum yield. Meanwhile, due to the sharp lightning rod effect, the presence of atomic-level protrusion at the tip apex can provide a strong and highly localized plasmonic field. We find that the added NaCl layer not only blocks charge transfer between molecules and the substrate but also effectively prevents fluorescence quenching. The maximum fluorescence and Raman enhancement factors near the tip apex approach up to 5 and 12 orders of magnitude, respectively, achieving sub-nanometer spatial resolution. Finally, compared to vertical electric field and dipole, sharp protrusion can excite a stronger horizontal electric field and couple with horizontal dipoles to yield a higher quantum yield, resulting in a three orders of magnitude higher in the fluorescence enhancement of horizontal dipole compared to the tip without protrusion. Our theoretical research not only confirms the experimental results of spectral enhancement with the proximity of the tip to the molecule in tip-enhanced photoluminescence, but also provides effective guidance for a deeper understanding of the mechanism of tip-enhanced fluorescence and Raman spectroscopy, as well as optimizing the experimental system.

Laboratory-based 3D X-ray standing-wave analysis of nanometre-scale gratings.

The increasing structural complexity and downscaling of modern nanodevices require continuous development of structural characterization techniques that support R&D and manufacturing processes. This work explores the capability of laboratory characterization of periodic planar nanostructures using 3D X-ray standing waves as a promising method for reconstructing atomic profiles of planar nanostructures. The non-destructive nature of this metrology technique makes it highly versatile and particularly suitable for studying various types of samples. Moreover, it eliminates the need for additional sample preparation before use and can achieve sub-nanometre reconstruction resolution using widely available laboratory setups, as demonstrated on a diffractometer equipped with a microfocus X-ray tube with a copper anode.

Precise and Omnidirectional Opto-Thermo-Elastic Actuation in Van Der Waals Contacting Systems.

Actuation of micro-objects along unconstrained trajectories in van der Waals contacting systems-in the same capacity as optical tweezers to manipulate particles in fluidic environments-remains a formidable challenge due to the lack of effective methods to overcome and exploit surface friction. Herein, a technique that aims to resolve this difficulty is proposed. This study shows that, by utilizing a moderate power beam of light, micro-objects adhered on planar solid substrates can be precisely guided to move in arbitrary directions, realizing sub-nanometer resolution across extended surfaces. The underlying mechanism is the interplay between surface friction and pulsed opto-thermo-elastic deformations, and to render a biased motion with off-centroid light illumination. This technique enables high-precision assembly, separation control of nanogaps, regulation of rotation angles in various material-substrate systems, whose capability is further tested in reconfigurable construction of optoelectronic devices. With simple set-up and theoretical generality, opto-thermo-elastic actuation opens up an avenue for versatile optical manipulation in the solid domain.

Cryogenic Atom Probe Tomograph to Resolve Confined Electrochemical Interfaces

Atom Probe Tomography (APT) is an analysis technique capable of providing a 3-dimensional, element-sensitive map of needle-shaped samples (where the tip radius is about 50 nm) with sub-nanometre resolution [1]. APT analysis is carried out at temperatures in the range of 25-75 K to limit the thermal motion of surface atoms which improves the spatial and mass resolution. As a result, this enables the analysis of frozen liquids, and by extension solid-liquid interfaces, by so-called “cryo-APT”. While this was historically challenging, recent advancements in UHV-cryogenic transfer and sample preparation methods now make it possible for the atomic scale analysis of interfacial species in electrochemical systems [2-4].In this study, we will attempt to explore the effect of nanoscale geometrical confinement on the distribution of interfacial ions for the first time and to resolve the electric double layer, using cryo-APT. We will describe the workflow for cryo-APT sample preparation to study confined solid-liquid interfaces, using nanoporous gold as a template material with a frozen sodium iodide solution inside its pores. APT reconstructions will allow 3-dimensional mapping of aqueous interfacial species, helping to develop our understanding of the structure of the electric double layer under nanoscale geometrical confinement. The attached Figure shows an SEM image of the later stages of cryo-FIB milling of an APT tip of frozen deionised water and 50 mM sodium iodide and a segment of the related APT atom map reconstruction.

Single-cavity loss-enabled nanometrology.

Optical monitoring of the position and alignment of objects with a precision of only a few nanometres is key in applications such as smart manufacturing and force sensing. Traditional optical nanometrology requires precise nanostructure fabrication, multibeam interference or complex postprocessing algorithms, sometimes hampering wider adoption of this technology. Here we show a simplified, yet robust, approach to achieve nanometric metrology down to 2 nm resolution that eliminates the need for any reference signal for interferometric measurements. We insert an erbium-doped quartz crystal absorber into a single Fabry-Pérot cavity with a length of 3 cm and then induce exceptional points by matching the optical loss with the intercavity coupling. We experimentally achieve a displacement response enhancement of 86 times compared with lossless methods, and theoretically argue that an enhancement of over 450 times, corresponding to subnanometre resolution, may be achievable. We also show a fivefold enhancement in the signal-to-noise ratio, thus demonstrating that non-Hermitian sensors can lead to improved performances over the Hermitian counterpart.

An Ultrasensitive Perovskite Single‐Model Plasmonic Strain Sensor Based on Piezoelectric Effect

AbstractInterest in flexible photonics has been motivated by the development of artificial smart skins. In particular, coupling of photonics and mechanics can offer opportunities to realize ultrasensitive strain sensor, however, low‐cost fabrication of flexible sensing device with desired photonic functionality remains a challenge. Hereby, the study reports an ultrasensitive strain‐gauge sensor based on the poly(ethylenenaphthalate (PEN))/monocrystal Au/MgF2/CsPbBr3 nanorod/Al2O3/polyacrylonitrile (in short P/mAu/M/CPB@Al2O3@PAN), which are sensitive to nanoscale structure alterations of PEN substrate via the stress response of the single‐mode laser based on the piezoelectric‐effect. Wherein a low‐threshold single‐mode lasing (Pth ≈ 170 nJ cm−2) is achieved through coating Al2O3 on the CsPbBr3 nanorod, producing the higher quality factor (Q ≈ 1637) to guarantee a much higher sensitivity in sensing application. Reversible spectral regulating of ≈3 nm in single‐mode‐lasing wavelength, with a subnanometre scale resolution <0.4 nm and the wavelength sensitivity (Sλ) as high as 160 nm RIU−1, is validated in response to applied strain ranging from −1.31% to 1.31%. This work not only represents essential progress in construction of ultrasensitive and cost‐effective flexible photonic sensor, but also lays the foundation for the potential application in smart photonic skins.

Use of phase plate cryo-EM reveals conformation diversity of therapeutic IgG with 50 kDa Fab fragment resolved below 6 Å

While cryogenic electron microscopy (cryo-EM) is fruitfully used for harvesting high-resolution structures of sizable macromolecules, its application to small or flexible proteins composed of small domains like immunoglobulin (IgG) remain challenging. Here, we applied single particle cryo-EM to Rituximab, a therapeutic IgG mediating anti-tumor toxicity, to explore its solution conformations. We found Rituximab molecules exhibited aggregates in cryo-EM specimens contrary to its solution behavior, and utilized a non-ionic detergent to successfully disperse them as isolated particles amenable to single particle analysis. As the detergent adversely reduced the protein-to-solvent contrast, we employed phase plate contrast to mitigate the impaired protein visibility. Assisted by phase plate imaging, we obtained a canonical three-arm IgG structure with other structures displaying variable arm densities co-existing in solution, affirming high flexibility of arm-connecting linkers. Furthermore, we showed phase plate imaging enables reliable structure determination of Fab to sub-nanometer resolution from ab initio, yielding a characteristic two-lobe structure that could be unambiguously docked with crystal structure. Our findings revealed conformation diversity of IgG and demonstrated phase plate was viable for cryo-EM analysis of small proteins without symmetry. This work helps extend cryo-EM boundaries, providing a valuable imaging and structural analysis framework for macromolecules with similar challenging features.

Suppressed thermal transport in silicon nanoribbons by inhomogeneous strain.

Nanoscale structures can produce extreme strain that enables unprecedented material properties, such as tailored electronic bandgap1-5, elevated superconducting temperature6,7 and enhanced electrocatalytic activity8,9. While uniform strains are known to elicit limited effects on heat flow10-15, the impact of inhomogeneous strains has remained elusive owing to the coexistence of interfaces16-20 and defects21-23. Here we address this gap by introducing inhomogeneous strain through bending individual silicon nanoribbons on a custom-fabricated microdevice and measuring its effect on thermal transport while characterizing the strain-dependent vibrational spectra with sub-nanometre resolution. Our results show that a strain gradient of 0.112% per nanometre could lead to a drastic thermal conductivity reduction of 34 ± 5%, in clear contrast to the nearly constant values measured under uniform strains10,12,14,15. We further map the local lattice vibrational spectra using electron energy-loss spectroscopy, which reveals phonon peak shifts of several millielectron-volts along the strain gradient. This unique phonon spectra broadening effect intensifies phonon scattering and substantially impedes thermal transport, as evidenced by first-principles calculations. Our work uncovers a crucial piece of the long-standing puzzle of lattice dynamics under inhomogeneous strain, which is absent under uniform strain and eludes conventional understanding.

Miniature computational spectrometer with a plasmonic nanoparticles-in-cavity microfilter array

Optical spectrometers are essential tools for analysing light‒matter interactions, but conventional spectrometers can be complicated and bulky. Recently, efforts have been made to develop miniaturized spectrometers. However, it is challenging to overcome the trade-off between miniaturizing size and retaining performance. Here, we present a complementary metal oxide semiconductor image sensor-based miniature computational spectrometer using a plasmonic nanoparticles-in-cavity microfilter array. Size-controlled silver nanoparticles are directly printed into cavity-length-varying Fabry‒Pérot microcavities, which leverage strong coupling between the localized surface plasmon resonance of the silver nanoparticles and the Fabry‒Pérot microcavity to regulate the transmission spectra and realize large-scale arrayed spectrum-disparate microfilters. Supported by a machine learning-based training process, the miniature computational spectrometer uses artificial intelligence and was demonstrated to measure visible-light spectra at subnanometre resolution. The high scalability of the technological approaches shown here may facilitate the development of high-performance miniature optical spectrometers for extensive applications.

Design and experiment of multidimensional and subnanometer stage driven by spatially distributed piezoelectric ceramics.

Multidimensional microdriving stage is one of the key components to realize precision driving and high-precision positioning. To meet nanometer displacement and positioning in the fields of micro-/nano-machining and precision testing, a new six-degree-of-freedom microdriving stage (6-DOF-MDS) of multilayer spatially distributed piezoelectric ceramic actuators (PZTs) is proposed and designed. The interior of the 6-DOF-MDS is a hollow design. The flexure hinge is used as the transmission mechanism, and the series-parallel hybrid driving of the corresponding PZTs achieves the microtranslation in the X, Y, and Z directions and the microrotation around the three axes of the microdriving stage, forming a microdisplacement mechanism with high rigidity and simple structure, which can realize the microfeed of 6-DOF. The force-displacement theory and lug boss structure optimization of the 6-DOF-MDS are analyzed, while the strength checking and natural frequency of the 6-DOF-MDS are also simulated by the finite element method. In addition, the real-time motion control system of the 6-DOF-MDS is designed based on Advanced RISC Machines. Through a series of verification experiments, the stroke and resolution results of the 6-DOF-MDS are obtained, where the displacements in the X, Y, and Z directions are 20.72, 20.02, and 37.60μm, respectively. The resolution is better than 0.68nm. The rotation angles around X, Y, and Z are 38.96″, 33.80″, and 27.87″, respectively, with an angular resolution of 0.063″. Relevant coupling experiments were also performed in this paper; in the full stroke linear running of X-axis, the maximum coupling displacements of the Y- and Z-axes are 1.04 and 0.17μm, respectively, with the corresponding coupling rates of ∼5.0% and 0.8%. The maximum coupling angles for the X-, Y-, and Z-axes are 0.33″, 0.14″, and 2.30″, respectively. Considering the coupling of the 6-DOF-MDS, decoupling measures and specific mathematical models have also been proposed. The proposed multidimensional microdriving stage achieves subnanometer resolution and can be used for the precise positioning and attitude control of precision instruments at the nano-/subnanometer level.

The Contribution of Scanning Force Microscopy on Dental Research: A Narrative Review.

Scanning force microscopy (SFM) is one of the most widely used techniques in biomaterials research. In addition to imaging the materials of interest, SFM enables the mapping of mechanical properties and biological responses with sub-nanometer resolution and piconewton sensitivity. This review aims to give an overview of using the scanning force microscope (SFM) for investigations on dental materials. In particular, SFM-derived methods such as force-distance curves (scanning force spectroscopy), lateral force spectroscopy, and applications of the FluidFM® will be presented. In addition to the properties of dental materials, this paper reports the development of the pellicle by the interaction of biopolymers such as proteins and polysaccharides, as well as the interaction of bacteria with dental materials.

A soft touch with electron beams: Digging out structural information of nanomaterials with advanced scanning low energy electron microscopy coupled with deep learning

Nanostructured materials continue to find applications in various electronic and sensing devices, chromatography, separations, drug delivery, renewable energy, and catalysis. While major advancements on the synthesis and characterization of these materials have already been made, getting information about their structures at sub-nanometer resolution remains challenging. It is also unfortunate to find that many emerging or already available powerful analytical methods take time to be fully adopted for characterization of various nanomaterials. The scanning low energy electron microscopy (SLEEM) is a good example to this. In this report, we show how clearer structural and surface information at nanoscale can be obtained by SLEEM, coupled with deep learning. The method is demonstrated using Au nanoparticles-loaded mesoporous silica as a model system. Moreover, unlike conventional scanning electron microscopy (SEM), SLEEM does not require the samples to be coated with conductive films for analysis; thus, not only it is convenient to use but it also does not give artifacts. The results further reveal that SLEEM and deep learning can serve as great tools to analyze materials at nanoscale well. The biggest advantage of the presented method is its availability, as most modern SEMs are able to operate at low energies and deep learning methods are already being widely used in many fields.

Unravelling lithium distribution in carbon fibre electrodes for structural batteries with atom probe tomography

Carbon fibres in structural batteries are multifunctional by acting both as structural reinforcement and as lithium (Li)-ion battery electrode. The relationship between the microstructure and mechanical capabilities of carbon fibres are well established, but much remains unexplored regarding their electrochemical properties. Specifically needed is a nanoscale understanding of how Li atoms distribute and interact in the carbon fibres. Atom probe tomography (APT) is uniquely positioned to provide subnanometre resolution in three dimensions. However, it has previously been hampered by undesirable Li migration during analysis. Here, we show that APT is successfully used to analyse electrochemically cycled polyacrylonitrile-based carbon fibres, through electrostatic shielding by means of conductive coating. We measure ∼1.5 at% Li in the carbon fibres after full delithiation, and thus identify trapped Li to constitute a substantial part of the initial capacity fade. After lithiation, Li accounts for ∼9 at% and according to frequency distribution analysis tend to agglomerate on the atomic scale. With nearest neighbour analysis, Li agglomeration is shown independent of heteroatom dopants such as nitrogen. Thus, the agglomeration is more likely induced by differing accessibility for Li in the crystalline and amorphous domains in the carbon fibre. The method used in this study can inform APT experiments on other type of Li-containing carbon electrodes. The findings of the study can be used to guide design of novel carbon fibres for structural batteries with enhanced electrochemical properties.

Imaging Valley Excitons in a 2D Semiconductor with Scanning Tunneling Microscope-Induced Luminescence.

Valley excitons dominate the optoelectronic response of transition-metal dichalcogenides and are drastically affected by structural and environmental inhomogeneities localized in these materials. Critical to understanding and controlling these nanoscale excitonic changes is the ability to correlate the imaging of excitonic states with crystalline structures on the atomic scale. Here, we apply scanning tunneling microscope-induced luminescence microscopy to image valley excitons in a semiconducting transition-metal dichalcogenide monolayer decoupled by a 10 nanometer-thick hexagonal-boron-nitride flake incorporated in a lateral homojunction on an Au electrode surface. This design enables the observation of chiral excitonic emission arising from neutral and charged valley excitons of the monolayer semiconductor at ambipolar voltages with a quantum efficiency up to ∼10-5 photon/electron. The measured light helicity demonstrates considerable circular polarization dependent on the sample voltage, reaching as much as 40%. The real-space luminescence imaging maps─at subnanometer resolution─of the valley excitons reveal striking spatial variations associated with localized inhomogeneities, including surface impurities and possibly nanoscale dielectric and/or potential disorders in the monolayer. Our study introduces a promising format for 2D materials to explore and tailor their optoelectronic processes at the atomic scale.

Full-length single-molecule protein fingerprinting.

Proteins are the primary functional actors of the cell. While proteoform diversity is known to be highly biologically relevant, current protein analysis methods are of limited use for distinguishing proteoforms. Mass spectrometric methods, in particular, often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate fluorescence resonance energy transfer (FRET)-based single-molecule protein fingerprinting to map the location of individual amino acids and post-translational modifications within single full-length protein molecules. Our data show that both intrinsically disordered proteins and folded globular proteins can be fingerprinted with a subnanometer resolution, achieved by probing the amino acids one by one using single-molecule FRET via DNA exchange. This capability was demonstrated through the analysis of alpha-synuclein, an intrinsically disordered protein, by accurately quantifying isoforms in mixtures using a machine learning classifier, and by determining the locations of two O-GlcNAc moieties. Furthermore, we demonstrate fingerprinting of the globular proteins Bcl-2-like protein 1, procalcitonin and S100A9. We anticipate that our ability to perform proteoform identification with the ultimate sensitivity may unlock exciting new venues in proteomics research and biomarker-based diagnosis.