Three‐dimensional bright‐field scanning transmission electron microscopy elucidate novel nanostructure in microbial biofilms

Bacterial biofilms play key roles in environmental and biomedical processes, and understanding their activities requires comprehension of their nanoarchitectural characteristics. Electron microscopy (EM) is an essential tool for nanostructural analysis, but conventional EM methods are limited in that they either provide topographical information alone, or are suitable for imaging only relatively thin (<300 nm) sample volumes. For biofilm investigations, these are significant restrictions. Understanding structural relations between cells requires imaging of a sample volume sufficiently large to encompass multiple cells and the capture of both external and internal details of cell structure. An emerging EM technique with such capabilities is bright‐field scanning transmission electron microscopy (BF‐STEM) and in the present report BF‐STEM was coupled with tomography to elucidate nanostructure in biofilms formed by the polycyclic aromatic hydrocarbon‐degrading soil bacterium, Delftia acidovorans Cs1‐4. Dual‐axis BF‐STEM enabled high‐resolution 3‐D tomographic recontructions (6–10 nm) visualization of thick (1250 and 1500 nm) sections. The 3‐D data revealed that novel extracellular structures, termed nanopods, were polymorphic and formed complex networks within cell clusters. BF‐STEM tomography enabled visualization of conduits formed by nanopods that could enable intercellular movement of outer membrane vesicles, and thereby enable direct communication between cells. This report is the first to document application of dual‐axis BF‐STEM tomography to obtain high‐resolution 3‐D images of novel nanostructures in bacterial biofilms. Future work with dual‐axis BF‐STEM tomography combined with correlative light electron microscopy may provide deeper insights into physiological functions associated with nanopods as well as other nanostructures.     

High-resolution compact X-ray microscopy

We demonstrate compact full‐field soft X‐ray transmission microscopy with sub 60‐nm resolution operating at λ= 2.48 nm. The microscope is based on a 100‐Hz regenerative liquid‐nitrogen‐jet laser‐plasma source in combination with a condenser zone plate and a micro‐zone plate objective for high‐resolution imaging onto a 2048 × 2048 pixel CCD detector. The sample holder is mounted in a helium atmosphere and allows imaging of both dry and wet specimens. The microscope design enables fast sample switching and the sample can be pre‐aligned using a visible‐light microscope. High‐quality images can be acquired with exposure times of less than 5 min. We demonstrate the performance of the microscope using both dry and wet samples.     

Propagation of surface plasmon polariton in nanometre-sized metal-clad optical waveguides

Using a local anodic‐oxidation method with a probe tip of a scanning near‐field optical microscope (SNOM) as the electrode, we have fabricated an oxide core with subwavelength dimensions on metal. The propagation of the surface plasmon polariton (SPP), which is excited at the interface between the oxide core and the metal clad, has been investigated using the same SNOM. Altering the wavelength of input light from 532 nm to 830 nm, the propagation length of the SPP extends from 2 µm to 6 µm. We carried out a simulation of the SPP propagation, and obtained a similar wavelength dependence.     

Dimensional metrology of lab‐on‐a‐chip internal structures: a comparison of optical coherence tomography with confocal fluorescence microscopy

The characterization of internal structures in a polymeric microfluidic device, especially of a final product, will require a different set of optical metrology tools than those traditionally used for microelectronic devices. We demonstrate that optical coherence tomography (OCT) imaging is a promising technique to characterize the internal structures of poly(methyl methacrylate) devices where the subsurface structures often cannot be imaged by conventional wide field optical microscopy. The structural details of channels in the devices were imaged with OCT and analyzed with an in‐house written ImageJ macro in an effort to identify the structural details of the channel. The dimensional values obtained with OCT were compared with laser‐scanning confocal microscopy images of channels filled with a fluorophore solution. Attempts were also made using confocal reflectance and interferometry microscopy to measure the channel dimensions, but artefacts present in the images precluded quantitative analysis. OCT provided the most accurate estimates for the channel height based on an analysis of optical micrographs obtained after destructively slicing the channel with a microtome. OCT may be a promising technique for the future of three‐dimensional metrology of critical internal structures in lab‐on‐a‐chip devices because scans can be performed rapidly and noninvasively prior to their use.     

Characterization and fabrication of fully metal-coated scanning near-field optical microscopy SiO2 tips

The fabrication of silicon cantilever‐based scanning near‐field optical microscope probes with fully aluminium‐coated quartz tips was optimized to increase production yield. Different cantilever designs for dynamic‐ and contact‐mode force feedback were implemented. Light transmission through the tips was investigated experimentally in terms of the metal coating and the tip cone‐angle. We found that transmittance varies with the skin depth of the metal coating and is inverse to the cone angle, meaning that slender tips showed higher transmission. Near‐field optical images of individual fluorescing molecules showed a resolution < 100 nm. Scanning electron microscopy images of tips before and after scanning near‐field optical microscope imaging, and transmission electron microscopy analysis of tips before and after illumination, together with measurements performed with a miniaturized thermocouple showed no evidence of mechanical defect or orifice formation by thermal effects.     

Phase separation in polyfluorene – polymethylmethacrylate blends studied using UV near-field microscopy

In this paper we present a near‐field microscopy study of thin films of a phase‐separated blend of the fluorescent conjugated‐polymer poly(9,9‐dioctylfluorene) [PFO] with the non‐fluorescent polymer polymethylmethacrylate [PMMA]. A scanning near‐field optical microscope (NSOM) was used to generate (blue) fluorescence from the PFO following UV excitation at 362 nm. A range of different concentrations of PFO in PMMA were studied ranging from 1 to 50% PFO in PMMA by mass. By studying both the shear force and fluorescence images we were able accurately to determine the distribution of PFO in the PMMA. We found that phase separation occurs over a number of different length‐scales between 5 µm and 250 nm. We show that at PFO concentrations of 1%, the PFO lies on top of the PMMA. At a PFO relative concentration of 50%, the PMMA phase extends through the whole thickness of the film to the underlying substrate. We use such samples to discuss the resolution of NSOM when imaging thick organic films. Furthermore, we confirm that the length‐scales of phase separation can be modified via control over spin‐casting protocols.     

A promising new wavelength region for three-photon fluorescence microscopy of live cells

We report three-photon laser scanning microscopy (3PLSM) using a bi-directional pumped optical parametric oscillator (OPO) with signal wavelength output at λ= 1500 nm. This novel laser was used to overcome the high optical loss in the infrared spectral region observed in laser scanning microscopes and objective lenses that renders them otherwise difficult to use for imaging. To test our system, we performed 3PLSM auto-fluorescence imaging of live plant cells at λ= 1500 nm, specifically Spirogyra, and compared performance with two-photon excitation (2PLSM) imaging using a femtosecond pulsed Ti:Sapphire laser at λ= 780 nm. Analysis of cell viability based on cytoplasmic organelle streaming and structural changes of cells revealed that at similar peak powers, 2PLSM caused gross cell damage after 5 min but 3PLSM showed little or no interference with cell function after 15 min. The λ= 1500 nm OPO is thus shown to be a practical laser source for live cell imaging.     

Atomic imaging using secondary electrons in a scanning transmission electron microscope: experimental observations and possible mechanisms

We report detailed investigation of high-resolution imaging using secondary electrons (SE) with a sub-nanometer probe in an aberration-corrected transmission electron microscope, Hitachi HD2700C. This instrument also allows us to acquire the corresponding annular dark-field (ADF) images both simultaneously and separately. We demonstrate that atomic SE imaging is achievable for a wide range of elements, from uranium to carbon. Using the ADF images as a reference, we studied the SE image intensity and contrast as functions of applied bias, atomic number, crystal tilt, and thickness to shed light on the origin of the unexpected ultrahigh resolution in SE imaging. We have also demonstrated that the SE signal is sensitive to the terminating species at a crystal surface. A possible mechanism for atomic-scale SE imaging is proposed. The ability to image both the surface and bulk of a sample at atomic-scale is unprecedented, and can have important applications in the field of electron microscopy and materials characterization.     

Upconversion fluorescence imaging of erbium-doped fluoride glass particles by apertureless SNOM

We have imaged fluorescent erbium‐doped fluoride glass particles by apertureless scanning near‐field optical microscopy. The optical excitation has been performed at λ = 780 nm whereas fluorescence emission has been collected around λ = 550 nm. This process, called upconversion by energy transfer, involves two erbium ions and is not linear. Besides an improvement of the lateral resolution, we have observed on some particles that the fluorescence is not homogeneously distributed, but is rather localized in some zones brighter than others. By making tip approach curves, we have also observed that the amount of fluorescence intensity scattered by the tip is increasing when the tip is approaching the sample surface.     

Precision of light intensity measurement in biological optical microscopy

Standardization and calibration of optical microscopy systems have become an important issue owing to the increasing role of biological imaging in high‐content screening technology. The proper interpretation of data from high‐content screening imaging experiments requires detailed information about the capabilities of the systems, including their available dynamic range, sensitivity and noise. Currently available techniques for calibration and standardization of digital microscopes commonly used in cell biology laboratories provide an estimation of stability and measurement precision (noise) of an imaging system at a single level of signal intensity. In addition, only the total noise level, not its characteristics (spectrum), is measured. We propose a novel technique for estimation of temporal variability of signal and noise in microscopic imaging. The method requires registration of a time series of images of any stationary biological specimen. The subsequent analysis involves a multi‐step process, which separates monotonic, periodic and random components of every pixel intensity change in time. The technique allows simultaneous determination of dark, photonic and multiplicative components of noise present in biological measurements. Consequently, a respective confidence interval (noise level) is obtained for each level of signal. The technique is validated using test sets of biological images with known signal and noise characteristics. The method is also applied to assess uncertainty of measurement obtained with two CCD cameras in a wide‐field microscope.     

A large‐field polarisation‐resolved laser scanning microscope: applications to CARS imaging

Laser‐scanning imaging techniques are frequently used to probe the molecule spatial orientation in a sample of interest by exploiting selection rules depending on the polarisation of the excitation light. For the successful implementation of these techniques the precise control of the polarisation at the sample level is of fundamental importance. Polarisation distortions induced by the optical elements are often the main limitation factor for the maximum size of the field‐of‐view in polarisation‐resolved (PR) laser‐scanning microscopy, since for large scanning angles the polarisation distortions may mask the real sample structure. Here we shall demonstrate the implementation of large‐field‐of‐view PR microscopy and show PR CARS imaging of mouse spinal cord thanks to a careful design of the laser‐beam optical path. We shall show that this design leads to strongly suppressed distortions and quantify their effects on the final images. Although the focus of this work is on CARS imaging, we stress that the approaches described here can be successfully applied to a wide range of PR laser‐scanning techniques.     

Experimental studies of surface plasmon polariton band gap effect

Surface plasmon polaritons (SPPs) propagation at a gold film surface covered by periodic arrays of ～40‐nm‐high scatterers arranged in a triangular lattice of different periods containing straight line defects is studied using collection scanning near‐field optical microscopy. The results reveal the dependence of the SPP band gap (SPPBG) effect manifested via the SPP reflection and guiding (along line defects) on the parameters of the surface structures (period, filling factor and lattice orientation). We find that the SPPBG effect is stronger along ΓK direction for all investigated periodic structures. Our results demonstrate that the SPPBG effect becomes less pronounced with a decrease of the filling factor and disappears for a filling factor close to 0.2. We also observe that the centre of the SPPBG shifts towards longer wavelengths with an increase of the lattice period.     

The application of Bayesian spectral analysis to optical sectioning using structured light imaging

We describe significant improvements to a well‐established method of obtaining optical sectioning in a conventional wide‐field microscope. This method relies on the projection of a single‐frequency grid pattern onto an object followed by mathematical manipulation of three images taken with the grid in different phases. Here, we present the use of Bayesian Spectral Analysis to determine accurate estimates of the phase of the grid pattern, permitting rapid and precise calibration of grid location. In common with previous algorithms, multiple images are combined to produce both an optical section and a wide‐field image. We describe innovations such as the use of non‐uniform phase or least‐squares solutions involving more than three images, in conjunction with direct phase estimates obtained using Bayesian Spectral Analysis, to yield images substantially free of artefacts. Auxiliary results, such as a method for determining the tilt of the grid, are also presented.     

An open‐source deconvolution software package for 3‐D quantitative fluorescence microscopy imaging

Deconvolution techniques have been widely used for restoring the 3‐D quantitative information of an unknown specimen observed using a wide‐field fluorescence microscope. Deconv , an open‐source deconvolution software package, was developed for 3‐D quantitative fluorescence microscopy imaging and was released under the GNU Public License. Deconv provides numerical routines for simulation of a 3‐D point spread function and deconvolution routines implemented three constrained iterative deconvolution algorithms: one based on a Poisson noise model and two others based on a Gaussian noise model. These algorithms are presented and evaluated using synthetic images and experimentally obtained microscope images, and the use of the library is explained. Deconv allows users to assess the utility of these deconvolution algorithms and to determine which are suited for a particular imaging application. The design of Deconv makes it easy for deconvolution capabilities to be incorporated into existing imaging applications.     

Near-field mapping of surface polariton fields

Using the general approach to image formation in collection near-field optical microscopy, I derive the symmetry relations for the amplitude coupling coefficients in the case of a weakly guiding single-mode fibre terminated with a probe tip possessing axial symmetry. It is shown that, for the symmetrical detection configuration, six elements of the coupling matrix can be expressed by using only three independent coupling coefficients. The obtained relations are further applied to describe near-field mapping of surface plasmon polariton (SPP) fields. I demonstrate that, for the symmetrical detection configuration, the near-field optical image reflects the intensity distribution of the SPP field components parallel to the surface plane, even though the strong perpendicular component is also being detected. This conclusion is supported with numerical simulations that elucidate the influence of symmetry of the fibre probe on the resulting near-field optical image. The near-field optical images simulated for scattering systems typical for SPP microoptics and localization are presented. It is found that the presence of asymmetry in the detection configuration increases the contribution of the perpendicular field component and results in the images approaching the corresponding SPP intensity distributions.     

Sub‐100‐nanometre resolution in total internal reflection fluorescence microscopy

Combining total internal reflection fluorescence microscopy with structured illumination allows optical wide‐field imaging with sub‐100‐nanometre resolution. We present a novel objective‐launch set‐up for standing wave illumination that takes advantage of a tunable transmission diffraction grating and transparent phase shifters actuated by electro‐active polymers to control the excitation pattern in three dimensions. Image acquisition is completed in less than 1 s. To reconstruct the extended image spectrum, we apply a new apodization function that results in a lateral resolution of 89 nm for green emission wavelength.     

Photonic nanopatterns of gold nanostructures indicate the excitation of surface plasmon modes of a wavelength of 50–100 nm by scanning near-field optical microscopy

Scanning near‐field optical microscopy images of metal nanostructures taken with the tetrahedral tip (T‐tip) show a distribution of dark and bright spots at distances in the order of 25–50 nm. The images are interpreted as photonic nanopatterns defined as calculated scanning near‐field optical microscopy images using a dipole serving as a light‐emitting scanning near‐field optical microscopy probe. Changing from a positive to a negative value of the dielectric function of a sample leads to the partition of one spot into several spots in the photonic nanopatterns, indicating the excitation of surface plasmons of a wavelength in the order of 50–100 nm in metal nanostructures.     

双重滤波扫频光源的研制

扫频光源是目前光学相干层析成像的关键部分,其光谱带宽和瞬时线宽分别影响着成像系统的轴向分辨率和成像深度。在单一滤波器中,这两者相互制约。针对这一情况,提出了一个利用两种滤波器组合优化的扫频光源系统。以双半导体光放大器并联作为增益介质,将声光可调滤波器(AOTF)和法布里-珀罗可调滤波器(FFP-TF)串联接入环形腔内进行双重滤波。其中AOTF的调谐范围和瞬时线宽均较宽,FFP-TF与之相反,经同步匹配设置后,两者协调工作,能够发挥各自的优势。通过搭建系统,获得了中心波长为1 316 nm的扫频激光输出,其光谱是1 235～1 380 nm,调谐范围是145 nm,瞬时线宽小于0.02 nm,扫频速度为1.35 kHz,输出光功率为0.48 mW。该扫频光源能够克服单一滤波器的固有缺陷,实现宽光谱带宽与窄瞬时线宽的有效统一,对成像综合性能的优化具有重要意义。     

含三次位相元件照相物镜的设计

设计了一个光学/数字成像系统,该成像系统利用三次位相元件辅以数字图像处理技术,在保持系统光通量及分辨率不变的前提下,提高了光学系统焦深。进行了仿真实验,实验以传统的双高斯型照相物镜为光学系统模型,选取焦点及离焦-1.6λ(λ=550 nm)近似-6.4倍焦深两个成像位置。对比分析了传统光学系统及光学/数字系统的焦点位置调制传递函数(MTF)、离焦1.6λ处调制传递函数(MTF)及在瑞利判据±100倍焦深范围内的离焦传递函数。通过光学设计软件CODEV9.7新增图像模拟功能,得到了光学系统的模拟成像结果。利用自编图像处理程序,得到最终的成像结果。结果表明,该光学/数字成像系统确实能够有效扩大光学系统焦深6倍以上。     

Simultaneous atomic-force and two-photon fluorescence imaging of biological specimens in vivo

We describe in this paper a home-built scanning-probe setup that combines the high spatial resolution of a commercial atomic-force microscope (AFM) with the high sensitivity and the discriminative power of a confocal two-photon fluorescence microscope. This scheme offers the ability of acquiring simultaneous, directly correlated topography and optical images with high sensitivity and resolution, and was successfully tested using model systems, such as dye-loaded latex beads. As a first biological application, we studied the (un)stacking of grana membranes in the envelope-free plant chloroplasts. The topographs showed two grana layers attached together in a "native unit" 15-16 nm thick and 4 nm protrusions on their surface, which we assign to Photosystem II reaction center. The optical imaging did not resolve single photosynthetic proteins, but helped in identifying the grana and indicated that the protein conformation and the chromophore binding are intact. Furthermore, our instrument allowed a direct comparison between the cell morphology and the distribution of the signaling protein H-Ras in living cells, i.e. mouse fibroblasts. With our approach the nanometer-scale resolving power of AFM is improved with the chemical identification capabilities of optical techniques, thus opening up interesting possibilities in various areas of research, including material and life sciences.