Image formation in scanned heterodyne microscope systems

Several new scanning microscopic techniques have recently been developed which rely on modulation of the optical beam to enhance the imaging performance. These systems have the common feature that the image is formed by detection or demodulation of an a.c. signal. Techniques have been developed which are sensitive to both intensity and phase information in the sample. We refer to such microscopes as heterodyne imaging systems (and by analogy single-frequency microscopes, operating at d.c. only, are referred to as homodyne) although as we will point out this term is only strictly applicable to the interferometric-based methods. Although the theory for the conventional homodyne scanning optical microscope is well developed there has been no systematic study of heterodyne microscopic techniques, which is the purpose of this paper. Several techniques are discussed and compared and the different ways of extracting amplitude and phase information are considered in detail with reference to experimental systems which have been demonstrated to have good imaging performance.     

Theoretical comparison of illumination and collection mode images of magneto-optical dots

We compare theoretical images of the same sample obtained with two different scanning near-field optical microscopes. The sample is a two-dimensional periodic array of magnetic sub-micrometric dots. The magnetization is perpendicular to the sample plane (polar magnetization). The first configuration is a scanning tunnelling optical near-field microscope (STOM) where the tip is used in the detection mode and the sample is illuminated by total internal reflection. The second configuration is an inverted STOM: the tip is used in the emission mode and the diffracted field is far-field detected in one direction. We present the models used to describe the two configurations and then explain the main lines of the formalism used to calculate the diffracted fields by a magneto-optical sample.     

一种基于剪切力的距离控制方法在扫描近场光学显微镜中的应用

介绍了扫描近场光学显微镜中基于剪切力的样品、探针间距离控制的方法。当受振动激励的光纤探针由远处逐渐接近样品表面时,样品与针尖间的剪切力使针尖的振动振幅减小,通过检测探针振幅的变化从而控制针尖与样品间的距离。此种方法可以方便地将光纤探针导入工作区域内并在扫描过程中保持适当的高度。我们测量了探针系统的幅频特性和力曲线,并用该方法获得4μm×4μm的范围内光盘表面的形貌信息。     

SNOM/STM using a tetrahedral tip and a sensitive current-to-voltage converter

Scanning near-field optical microscopes (SNOM) using the tetrahedral-tip (T-tip) with scanning tunnelling microscopy (STM) distance control have been realized in transmission and reflection mode. Both set-ups used ordinary STM current-to-voltage converters allowing measurement of metallic samples. In the transmission mode, a resolution of 10 nm to 1 nm with regard to material contrast can be achieved on binary metal samples. Because of the great near-field optical potential of the T-tip with respect to the optical resolution, it is a challenging task to find out whether these results can be transferred to non-metallic sample systems as well. This paper reports on a newly designed SNOM/STM transmission mode set-up using the tetrahedral-tip. It implements a sensitive current-to-voltage converter to widen the field of measurable sample systems. Beyond this, mechanical and optical measuring conditions are substantially improved compared to previous set-ups. The new set-up provides a basis for the routine investigation of metal nanostructures and adsorbed organic monolayers at resolutions in the 10 nm range.     

Inexpensive, high-quality optical relay for use in confocal scanning beam imaging

An inexpensive, high optical-quality relay lens made up of two eyepieces arranged in an afocal assembly for use in confocal scanning laser imaging is described. In the past we have used relays, within our confocal microscopes, made up of achromats with long focal lengths (> or = 10 cm), which take up large optical tracks and suffer from significant amounts of astigmatism and curvature of field. We quantify aberrations associated with achromat and eyepiece relays using CODE V optical design and analysis software. The eyepiece relay is found to be more compact, better corrected, and not significantly more expensive than its achromat counterpart. In addition to being used to interconnect two scanning mirrors optically as well as scanning mirrors with microscope objectives, it can form part of the optics in a confocal scanning laser MACROscope-Microscope system (Biomedical Photometrics, Inc., Waterloo, Ontario, Canada). Due to design constraints, the MACROscope-Microscope system cannot incorporate a conventional wide-field microscope into its structure such as is done in most commercial confocal microscopes. The eyepiece relay is used as a stand-alone, compact optical link between the scanning mirrors and the microscope objective. This consequently makes the MACROscope-Microscope system more compact and easier to commercialize.     

Optical characterization of probes for photon scanning tunnelling microscopy

The photon scanning tunnelling microscope is a well-established member of the family of scanning near-field optical microscopes used for optical imaging at the sub-wavelength scale. The quality of the probes, typically pointed uncoated optical fibres, used is however, difficult to evaluate in a direct manner and has most often been inferred from the apparent quality of recorded optical images. Complicated near-field optical imaging characteristics, together with the possibility of topographically induced artefacts, however, has increased demands for a more reliable probe characterization technique. Here we present experimental results obtained for optical characterization of two different probes by imaging of a well-specified near-field intensity distribution at various spatial frequencies. In particular, we observe that a sharply pointed dielectric probe can be highly suitable for imaging when using p -polarized light for the illumination. We conclude that the proposed scheme can be used directly for probe characterization and, subsequently, for determination of an optical transfer function, which would allow one to deduce from an experimentally obtained image of a weakly scattering sample the field distribution existing near the sample surface in the absence of the probe.     

A self‐adaptive and nonmechanical motion autofocusing system for optical microscopes

For the design of a passive autofocusing (AF) system for optical microscopes, many time‐consuming and tedious experiments have been performed to determine and design a better focus criterion function, owing to the sample‐dependence of this function. To accelerate the development of the AF systems in optical microscopes and to increase AF speed as well as maintain the AF accuracy, this study proposes a self‐adaptive and nonmechanical motion AF system. The presented AF system does not require the selection and design of a focus criterion function when it is developed. Instead, the system can automatically determine a better focus criterion function for an observed sample by analyzing the texture features of the sample and subsequently perform an AF procedure to bring the sample into focus in the objective of an optical microscope. In addition, to increase the AF speed, the Z axis scanning of the mechanical motion of the sample or the objective is replaced by focusing scanning performed by a liquid lens, which is driven by an electrical current and does not involve mechanical motion. Experiments show that the reproducibility of the results obtained with the proposed self‐adaptive and nonmechanical motion AF system is better than that provided by that of traditional AF systems, and that the AF speed is 10 times faster than that of traditional AF systems. Also, the self‐adaptive function increased the speed of AF process by an average of 10.5% than Laplacian and Tenegrad functions.     

Imaging an optical disc by the combined use of scanning tunnelling microscopy and scanning electron microscopy

We present the data obtained by scanning tunnelling microscopy combined with scanning electron microscopy of the digitally encoded structure on a stamper used to fabricate optical discs. The combination allows us to focus the STM tip on a preselected spot with a precision of ?0·3 μm. The data show the superiority of STM for a more detailed characterization of shape, width, length, height and fine structure appearing on the sample. We also show the influence of tip shape on STM resolution. Simultaneous use of both microscopes is possible but high electron doses produce an insulating layer of contaminants thick enough to make STM operation impossible.     

Optical sectioning microscope with a binary hologram based beam scanning

We describe the development of a beam scanning microscope that can perform optical sectioning based on the principle of confocal microscopy. The scanning is performed by a laser beam diffracted from a dynamic binary hologram implemented using a liquid crystal spatial light modulator. Using the proposed scanning mechanism, unlike the conventional confocal microscopes, scanning over a two-dimensional area of the sample can be obtained without the use of a pair of galvo mirror scanners. The proposed microscope has a number of advantages, such as superior frame to frame repeatability, simpler optical arrangement, increased pixel dwell time relative to the time between two pixels, illumination of only the sample points without pulsing the laser, and absolute control over the amplitude and phase of the illumination beam on a pixel to pixel basis. The proposed microscope can be particularly useful for applications requiring very long exposure time or very large working distance objective lenses. In this paper we present experimental implementation of the setup using a nematic liquid crystal spatial light modulator and proof-of-concept experimental results.     

Fabrication of a versatile substrate for finding samples on the nanometer scale

With increasing interest in nanometer scale studies, a common research issue is the need to use different analytical systems with a universal substrate to relocate objects on the nanometer scale. Our paper addresses this need. Using the delicate milling capability of a focused ion beam (FIB) system, a region of interest (ROI) on a sample is labelled via a milled reference grid. FIB technology allows for milling and deposition of material at the sub 20-nm level, in a similar user environment as a standard scanning electron microscope (SEM). Presently commercially available transmission electron microscope (TEM) grids have spacings on the order 100 μm on average; this technique can extend this dimension down to the submicrometre level. With a grid on the order of a few micrometres optical, FIBs, TEMs, scanning electron microscopes (SEMs), and atomic force microscopes (AFM) are able to image the ROI, without special chemical processes or conductive coatings required. To demonstrate, Au nanoparticles of ∼ 25 nm in size were placed on a commercial Formvar^®- and carbon-coated TEM grid and later milled with a grid pattern. Demonstration of this technique is also extended to bulk glass substrates for the purpose of sample location. This process is explained and demonstrated using all of the aforementioned analytical techniques.     

Theoretical comparison of illumination and collection mode images of magneto-optical dots

We compare theoretical images of the same sample obtained with two different scanning near-field optical microscopes. The sample is a two-dimensional periodic array of magnetic sub-micrometric dots. The magnetization is perpendicular to the sample plane (polar magnetization). The first configuration is a scanning tunnelling optical near-field microscope (STOM) where the tip is used in the detection mode and the sample is illuminated by total internal reflection. The second configuration is an inverted STOM: the tip is used in the emission mode and the diffracted field is far-field detected in one direction. We present the models used to describe the two configurations and then explain the main lines of the formalism used to calculate the diffracted fields by a magneto-optical sample.     

基于时域结构的光学相干层析系统的调试

基于时域结构的光学相干层析系统由于光线多次通过,按照光路可逆原理调整会很困难.在理论分析和多次实验基础上,现总结出一套快速调试方法,大大缩短光路调试时间.详细分步骤描述了纵向傅里叶快速扫描光学延迟线和横向扫描部分的调试方法,给出快速寻找干涉系统光程匹配点的调试方法.利用构建好的光学相干层析系统进行层析成像实验调试,纵向...     

A method for evaluating microscope objectives to optimize performance of confocal systems

A method for evaluating the performance of microscope objectives on two types of confocal scanning optical microscope is presented. Of these two confocal microscope types, off-axis beam-scanning systems are found to require microscope objectives which have been corrected for flatness of field as well as for spherical aberration and astigmatism in order to obtain maximum axial and laterial resolution. In the case of on-axis specimen-scanning microscopes, less highly corrected objective lenses (not corrected for flatness of field) may in practice prove to have superior resolving properties.     

Fibre-optic nonlinear optical microscopy and endoscopy

Nonlinear optical microscopy has been an indispensable laboratory tool of high‐resolution imaging in thick tissue and live animals. Rapid developments of fibre‐optic components in terms of growing functionality and decreasing size provide enormous opportunities for innovations in nonlinear optical microscopy. Fibre‐based nonlinear optical endoscopy is the sole instrumentation to permit the cellular imaging within hollow tissue tracts or solid organs that are inaccessible to a conventional optical microscope. This article reviews the current development of fibre‐optic nonlinear optical microscopy and endoscopy, which includes crucial technologies for miniaturized nonlinear optical microscopy and their embodiments of endoscopic systems. A particular attention is given to several classes of photonic crystal fibres that have been applied to nonlinear optical microscopy due to their unique properties for ultrashort pulse delivery and signal collection. Furthermore, fibre‐optic nonlinear optical imaging systems can be classified into portable microscopes suitable for imaging behaving animals, rigid endoscopes that allow for deep tissue imaging with minimally invasive manners, and flexible endoscopes enabling imaging of internal organs. Fibre‐optic nonlinear optical endoscopy is coming of age and a paradigm shift leading to optical microscope tools for early cancer detection and minimally invasive surgery.     

Calibration and standardization of the emission light path of confocal microscopes

Recently, there has been a large expansion in the usage of optical microscopes for obtaining quantitative information from biological samples in order to determine fundamental biological information such as molecular kinetics and interaction, and heterogeneity within cell populations. Consequently, we built a highly stable, uniform, isotropically emitting and convenient‐to‐use light source, and designed image analysis procedures for calibrating the emission light path of optical microscopes. We used the source and procedures to analyse the quantitative imaging properties of a widely used model of laser scanning confocal microscope. Results showed that the overall performance was as high as could be expected given the inherent limitations of the optical components and photomultiplier tubes. We observed that the photon detection efficiency did not vary with photomultiplier tube gain and that the highest dynamic range was achieved with relatively low gain and 12‐bit digitization. Practical applications of the light source for checking the transmission of optical components in the emission light path are presented.     

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.     

Direct interpretation of near-field optical images

The interpretation of the detection process in near-field optical microscopy is reviewed on the basis of a discussion about the possibility of establishing direct comparisons between experimental images and the solutions of Maxwell equations or the electromagnetic local density of states. On the basis of simple physical arguments, it is expected that the solutions of Maxwell equations should agree with images obtained by collecting mode near-field microscopes, while the electromagnetic local density of states should be considered to provide a practical interpretation of illumination mode near-field microscopes.
We review collecting mode near-field microscope images where the conditions to obtain good agreement with the solutions of Maxwell equations have indeed been identified. In this context of collecting mode near-field microscopes, a fundamentally different functionality between dielectric and gold-coated tips has been clearly identified experimentally by checking against the solutions of Maxwell equations. It turns out that dielectric tips detect a signal proportional to the optical electric field intensity, whereas gold-coated tips detect a signal proportional to the optical magnetic field intensity. The possible implications of this surprising phenomenon are discussed.     

Optical characterization of probes for photon scanning tunnelling microscopy

The photon scanning tunnelling microscope is a well-established member of the family of scanning near-field optical microscopes used for optical imaging at the subwavelength scale. The quality of the probes, typically pointed uncoated optical fibres, used is however, difficult to evaluate in a direct manner and has most often been inferred from the apparent quality of recorded optical images. Complicated near-field optical imaging characteristics, together with the possibility of topographically induced artefacts, however, has increased demands for a more reliable probe characterization technique. Here we present experimental results obtained for optical characterization of two different probes by imaging of a well-specified near-field intensity distribution at various spatial frequencies. In particular, we observe that a sharply pointed dielectric probe can be highly suitable for imaging when using p-polarized light for the illumination. We conclude that the proposed scheme can be used directly for probe characterization and, subsequently, for determination of an optical transfer function. which would allow one to deduce from an experimentally obtained image of a weakly scattering sample the field distribution existing near the sample surface in the absence of the probe.     

Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy

Layer‐by‐layer technique is used to adsorb a uniform ultrathin layer of fluorescently labelled polyelectrolytes on a glass cover slip. Due to their thickness, uniformity and fluorescence properties, these ultrathin layers may serve as a simple and applicable standard to directly measure the z‐response of different scanning optical microscopes. In this work we use ultrathin layers to measure the z‐response of confocal, two‐photon excitation and 4Pi laser scanning microscopes. Moreover, due to their uniformity over a wide region, i.e. cover slip surface, it is possible to quantify the z‐response of the system over a full field of view area. This property, coupled with a bright fluorescence signal, enables the use of polyelectrolyte layers for representation on sectioned imaging property charts: a very powerful method to characterize image formation properties and capabilities (z‐response, off‐axis aberration, spherical aberration, etc.) of a three‐dimensional scanning system. The sectioned imaging property charts method needs a through‐focus dataset taken from such ultrathin layers. Using a comparatively low illumination no significant bleaching occurs during the excitation process, so it is possible to achieve long‐term monitoring of the z‐response of the system. All the above mentioned properties make such ultrathin layers a suitable candidate for calibration and a powerful tool for real‐time evaluation of the optical sectioning capabilities of different three‐dimensional scanning systems especially when coupled to sectioned imaging property charts.     

Near- and far-field second-harmonic imaging of quasi-phase-matching crystals

Second-harmonic scanning near- and far-field optical microscopy of an electric-field poled KTiOPO₄ quasi-phase-matching crystal has been accomplished. This has been done in order to reveal the walls that form the intersections between inverted and non-inverted crystal domains. The domain walls are seen clearly only in images recorded by means of second-harmonic generation because of a large nonlinear contrast, and they appear as bright stripes when studied in a reflection geometry but they are dark when studied in transmission. The images show that the duty cycle of the quasi-phase-matching crystal differs from the ideal and that the walls are not completely smooth. These effects, in combination with the observed scattering from the domain walls, are expected to lower the output of the crystal when used for frequency doubling. We conclude that the wall thickness is no more than approximately 100 nm, which makes it a suitable test object for the resolution capabilities of scanning near-field optical microscopes that are used for nonlinear imaging.