Prospects for 3D, nanometer-resolution imaging by confocal STEM

Confocal STEM is a new electron microscopy imaging mode. In a microscope with spherical aberration-corrected electron optics, it can produce three-dimensional (3D) images by optical sectioning. We have adapted the linear imaging theory of light confocal microscopy to confocal STEM and use it to suggest optimum imaging conditions for a confocal STEM limited by fifth-order spherical aberration. We predict that current or near-future microscopes will be able to produce 3D images with 1 nm vertical resolution and sub-Angstrom lateral resolution. Multislice simulations show that we will need to be cautious in interpreting these images, however, as they can be complicated by dynamical electron scattering.     

SPP tomography: A simple wide‐field nanoscope

We explore the wide‐field optical nanoimaging capabilities of the surface plasmon polariton (SPP) tomography technique. We show that nanofeatures with lateral dimensions smaller than λ/20 can be observed in the surface emission (SE) images of plasmonic crystals with a period of 300 nm. Moreover, as a proof‐of‐concept, we demonstrate that SPP tomography permits to resolve two single objects with a center‐to‐center separation of 200 nm and edge‐to‐edge separation as small as λ/7. We present a comprehensive discussion about the nanoimaging capabilities of the SPP tomography technique. In contrast to other optical subwavelength resolution techniques, in our approach for imaging nanosize features, enhanced evanescent waves are coupled to the far‐field via leakage radiation associated with SPPs excited by near‐field fluorescence; therefore wide‐field images, which are not out‐of‐plane diffraction‐limited, are formed directly in the microscope's camera. We also discuss additional imaging processing capabilities associated with the fact that SPP tomography SE images are formed by the microscope lenses through an analog tomography process. SCANNING 35: 246‐252, 2013. © 2012 Wiley Periodicals, Inc.     

Analytic derivation of optimal imaging conditions for incoherent imaging in aberration-corrected electron microscopes

The optimal lens parameters for incoherent imaging using third and fifth-order aberration-corrected electron microscopes are derived analytically. We propose simple models for the point spread function (PSF) and transfer function that give analytic formulae for the lateral resolution and depth resolution. We also derive an analytic formula for the contrast transfer function (CTF) in three dimensions and show that depth sectioning has an information limit equivalent to tomography with a missing cone of 90 degrees minus the aperture angle.     

Surface plasmon and guided optical wave microscopies

This article summarizes some recent developments in the field of surface plasmon and guided optical wave microscopies. It is shown that these imaging techniques based on evanescent light allow for a quantitative optical characterization of ultrathin films with a thickness sensitivity of a few Ångstroms and a lateral resolution of μm.     

Surface plasmon and guided optical wave microscopies

This article summarizes some recent developments in the field of surface plasmon and guided optical wave microscopies. It is shown that these imaging techniques based on evanescent light allow for a quantitative optical characterization of ultrathin films with a thickness sensitivity of a few Ångstroms and a lateral resolution of μm.     

Image sharpness and contrast transfer in coherent confocal microscopy

Confocal microscopes provide clear, thin optical sections with little disturbance from regions of the specimen that are not in focus. In addition, they appear to provide somewhat greater lateral and axial image resolution than with non-confocal microscope optics. To address the question of resolution and contrast transfer of light microscopes, a new test slide that enables the direct measurement of the contrast transfer characteristics (CTC) of microscope optics at the highest numerical aperature has been developed. With this new test slide, the performance of a confocal scanning laser microscope operating in the confocal reflection mode and the non-confocal transmission mode was examined. The CTC curves show that the confocal instrument maintains exceptionally high contrast (up to twice that with non-confocal optics) as the dimension of the object approaches the diffraction limit of resolution; at these dimensions, image detail is lost with non-confocal microscopes owing to a progressive loss of image contrast. Furthermore, we have calculated theoretical CTC curves by modelling the confocal and non-confocal imaging modes using discrete Fourier analysis. The close agreement between the theoretical and experimental CTC curves supports the earlier prediction that the coherent confocal and the incoherent non-confocal imaging mode have the same limit of resolution (defined here as the inverse of the spatial frequency at which the contrast transfer converges to zero). The apparently greater image resolution of the coherent confocal optics is a consequence of the improved contrast transfer at spacings which are close to the resolution limit.     

High-resolution wide-field surface plasmon microscopy

This paper describes the application of a Köhler illuminated high‐resolution wide‐field microscope using surface plasmons to provide the image contrast. The response of the microscope to a grating structure in both the Fourier and the image planes is presented to demonstrate image formation by surface waves. The effect of spatial filtering in the back focal (Fourier) plane to enhance image constrast is described. We also discuss how the surface wave contrast mechanism affects the imaging performance of the microscope and discuss factors that can be expected to lead to even greater improvements in lateral resolution and sensitivity.     

3-D Optical Interference Microscopy at the Lateral Resolution

For applications in micro- and nanotechnologies the lateral resolution of optical 3-D microscopes becomes an issue of increasing relevance. However, lateral resolution of 3-D microscopes is hard to define in a satisfying way. Therefore, we first study the measurement capabilities of a highly resolving white-light interference (WLI) microscope close to the limit of lateral resolution. Results of measurements and simulations demonstrate that better lateral resolution seems to be achievable based on the envelope evaluation of a WLI signal. Unfortunately, close to the lateral resolution limit errors in the measured amplitude of micro-structures appear. On the other hand, results of interferometric phase evaluation seem to be strongly low-pass filtered in this case.

Furthermore, the instrument transfer characteristics and the lateral resolution capabilities of WLI instruments are also affected by polarization. TM polarized light is less sensitive to edge diffraction and thus systematic errors can be avoided. However, apart from ghost steps due to fringe order errors, the results of phase evaluation seem to be closer to the real surface topography if TE polarized light is used. The lateral resolution can be further improved by combining WLI and structured illumination microscopy. Since the measured height of rectangular profiles close to the lateral resolution limit is generally too small compared to the real height, we introduce a method based on phase evaluation which characterizes the heights of barely laterally resolved rectangular gratings correctly.     

Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes

A theory for multiphoton fluorescence imaging in high aperture scanning optical microscopes employing finite sized detectors is presented. The effect of polarisation of the fluorescent emission on the imaging properties of such microscopes is investigated. The lateral and axial resolutions are calculated for one-, two- and three-photon excitation of p-quaterphenyl for high and low aperture optical systems. Significant improvement in lateral resolution is found to be achieved by employing a confocal pinhole. This improvement increases with the order of the multiphoton process. Simultaneously, it is found that, when the size of the pinhole is reduced to achieve the best possible resolution, the signal-to-noise ratio is not degraded by more than 30%. The degree of optical sectioning achieved is found to improve dramatically with the use of confocal detection. For two- and three-photon excitation axial full width half-maximum improvement of 30% is predicted.     

Depth discrimination in scanned heterodyne microscope systems

The optical transfer function of several scanning microscope systems is derived, using a physically intuitive approach. The technique allows a wide range of systems to be modelled with only minor modifications to the basic formulation. The results are then used to determine the response of various scanning microscopes for objects both in and out of the focal plane. The possibility of performing extended-focus phase imaging in heterodyne microscopes by scanning the sample along the optical axis is also examined. This mode of operation should allow measurements of minute topographical and phase variations on tilted or warped samples with the same lateral resolution as would be obtained when the sample is in focus throughout the entire scan.     

Scanning probe microscopy of biological samples and other surfaces

Scanning probe microscopes derived from the scanning tunnelling microscope (STM) offer new ways to examine surfaces of biological samples and technologically important materials. The surfaces of conductive and semiconductive samples can readily be imaged with the STM. Unfortunately, most surfaces are not conductive. Three alternative approaches were used in our laboratory to image such surfaces. 1. Crystals of an amino acid were imaged with the atomic force microscope (AFM) to molecular resolution with a force of order 10^?8 N. However, it appears that for most biological systems to be imaged, the atomic force microscope should be able to operate at forces at least one and perhaps several orders of magnitude smaller. The substitution of optical detection of the cantilever bending for the measurement by electron tunnelling improved the reliability of the instrument considerably. 2. Conductive replicas of non-conductive surfaces enabled the imaging of biological surfaces with an STM with a lateral resolution comparable to that of the transmission electron microscope. Unlike the transmission electron microscope, the STM also measures the heights of the features. 3. The scanning ion conductance microscope scans a micropipette with an opening diameter of 0·04-0·1 μm at constant ionic conductance over a surface covered with a conducting solution (e.g., the surface of plant leaves in saline solution).     

Two-colour two-photon confocal microscopy with isotropic three-dimensional resolution and parallel excitation

We demonstrate a novel design of two-colour two-photon fluorescence microscope in which isotropic three-dimensional imaging resolution and high scanning speed can be achieved simultaneously. In our scheme, a three-dimensional optical lattice constructed by multi-beam interference is used for two-colour two-photon fluorescence excitation. Our simulation results show that a resolution of 113.5 nm can be achieved in both transverse and axial directions with two pump pulses at the wavelengths of 400 and 800 nm, respectively; meanwhile, imaging speed can be greatly improved compared with that of traditional two-photon scanning fluorescence microscopes.     

SPR生物传感技术的研究进展与应用

由于具有实时、免标记、高灵敏度等优点,表面等离体子共振(surface plasmon resonance,SPR)技术被广泛应用于生物分子相互作用的研究分析。在SPR能够检测的分子量范围内,基本所有的具有特异性反应的生物分子都可以被SPR生物传感器检测。因此,SPR技术越来越展示出其重要的应用价值,成为近几年来研究的热点。本文重点从原理、技术改进以及应用等三个角度对SPR技术进行阐述,并从技术改进方面重点介绍了SPR技术的两个发展:表面等离子体子共振成像(SPRi)技术与局域表面等离子体子技术(LSPR)。     

Kelvin probe force microscopy in application to biomolecular films: Frequency modulation,amplitude modulation,and lift mode

Kelvin probe force microscopy (KPFM) is a powerful technique to visualize the differences of work function in metals and lateral surface potential distribution in thin organic films. Earlier we have shown that frequency modulated-Kelvin probe force microscopy has significant advantages in both sensitivity and resolution when applied to metal and inorganic interfaces in vacuum. High resolution, high sensitivity, and performance in ambient conditions are required in order to study biologically relevant samples. In this work we compared the resolution of frequency modulation (FM-KPFM), amplitude modulation (AM-KPFM), and lift modes KPFM for imaging the local electrical surface potential of complex biomolecular films and demonstrated that FM-KPFM mode has superior resolution for biological applications. The power of the method was illustrated on pulmonary surfactant films, revealing nm spatial resolution and mV potential sensitivity in ambient air. At this level of resolution this method can provide critical insight into the molecular arrangement and function of complex biosystems in a way that other KPFM modes cannot do. Based on the observed changes in the local surface potential we discovered that excess cholesterol produces nm size electrostatic domains and change the electric fields.     

Three-dimensional imaging in confocal imaging systems with finite sized detectors

We consider the effect of the finite size of the detector on both the lateral and axial resolution of the confocal system. The use of a finite sized detector means that the imaging is no longer truly coherent. We find that the lateral resolution is considerably more sensitive to the detector size than is the axial response. The question of the rejection of flare light is also considered. Experimental results are shown and we find that acceptable extended-focus, auto-focus and height images may be obtained from non truly-confocal systems. We also find that lens apodization has a far greater effect on the axial resolution than the lateral resolution.     

Image correlation microscopy for uniform illumination

Image cross-correlation microscopy is a technique that quantifies the motion of fluorescent features in an image by measuring the temporal autocorrelation function decay in a time-lapse image sequence. Image cross-correlation microscopy has traditionally employed laser-scanning microscopes because the technique emerged as an extension of laser-based fluorescence correlation spectroscopy. In this work, we show that image correlation can also be used to measure fluorescence dynamics in uniform illumination or wide-field imaging systems and we call our new approach uniform illumination image correlation microscopy. Wide-field microscopy is not only a simpler, less expensive imaging modality, but it offers the capability of greater temporal resolution over laser-scanning systems. In traditional laser-scanning image cross-correlation microscopy, lateral mobility is calculated from the temporal de-correlation of an image, where the characteristic length is the illuminating laser beam width. In wide-field microscopy, the diffusion length is defined by the feature size using the spatial autocorrelation function. Correlation function decay in time occurs as an object diffuses from its original position. We show that theoretical and simulated comparisons between Gaussian and uniform features indicate the temporal autocorrelation function depends strongly on particle size and not particle shape. In this report, we establish the relationships between the spatial autocorrelation function feature size, temporal autocorrelation function characteristic time and the diffusion coefficient for uniform illumination image correlation microscopy using analytical, Monte Carlo and experimental validation with particle tracking algorithms. Additionally, we demonstrate uniform illumination image correlation microscopy analysis of adhesion molecule domain aggregation and diffusion on the surface of human neutrophils.     

Near-field imaging of surface plasmon on gold nano-dots fabricated by scanning probe lithography

We obtained scanning near‐field optical microscopy images to study the excitation of surface plasmons on metallic dots fabricated using scanning probe lithography. Gold nano‐dots were fabricated by applying electric voltages to conducting probes installed in an atomic force microscope using the mechanism of field‐induced diffusion and nano‐oxidation plus Au‐coating. High spatial resolution of scanning near‐field optical microscopy revealed a ‘bifold’ pattern of surface plasmon mode on fabricated Au dots in the polarization direction of incident light. We found that scanning near‐field optical microscopy imaging combined with scanning probe lithography is able to provide a systematic study of surface plasmon excitation on nano‐metallic structures.     

表面等离子体激元透镜设计及其数值计算

提出了一种新的表面等离子体激元透镜的设计方案,该方案通过在两个亚波长小孔的外表面放置电介质光栅实现对入射光束的有效会聚。利用遗传算法研究了波导中表面等离子体激元的色散关系,结果表明,通过调节亚波长小孔的宽度和介电常数可以有效地调控有效折射率,从而实现对亚波长金属平板波导结构中表面等离子体激元传播特性的调控。利用时域有限差分方法(FDTD)结合完美匹配层(PML)边界条件数值模拟了此结构中的光场分布,讨论了光栅周期数对成像特性的影响,从而深入理解了纳米聚焦效应的物理机制。结果显示,随着表面光栅数的增多,焦距和焦斑大小都在增加。光栅数从5增加至11时,焦距由1.715μm增大至2.325μm,焦斑大小由0.615μm增大至1.715μm,这一结构有可能被用作未来集成光路中的纳米聚焦器件。     

Quantitative detection of gold nanoparticles on individual, unstained cancer cells by scanning electron microscopy

Gold nanoparticles are rapidly emerging for use in biomedical applications. Characterization of the interaction and delivery of nanoparticles to cells through microscopy is important. Scanning electron microscopes have the intrinsic resolution to visualize gold nanoparticles on cells. A novel sample preparation protocol was developed to enable imaging of cells and gold nanoparticles with a conventional below lens scanning electron microscopes. The negative influence of 'charging' on the quality of scanning electron microscopes' images could be limited by deposition of biological cells on a conductive (gold) surface. The novel protocol enabled high-resolution scanning electron microscopes' imaging of small clusters and individual gold nanoparticles on uncoated cell surfaces. Gold nanoparticles could be counted on cancer cells with automated routines.