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.     

On the extinction coefficient in confocal polarization microscopy

The extinction coefficient in conventional polarized light microscopes is non-zero, even with perfect polars, solely because of the image formation process in these instruments. The imaging in confocal microscopes is different from conventional instruments and it is shown that in this case an infinite extinction coefficient results in the ideal case whereas a finite value is always to be expected with conventional instruments. Images of the same microelectronic specimen taken in both conventional and confocal polarized light microscopes are compared.     

Study of the fractal character of surfaces by scanning tunnelling microscopy: Errors and limitations

The possibility of using the intrinsic three-dimensional imaging capability of scanning tunnelling microscopes to study the fractal character of surfaces by Mandelbrot's method of ‘filling’ with water up to a given height is discussed. By plotting on a log-log plot the area against the perimeter of the ‘lakes' that appear, the fractal dimension is obtained from the slope of the straight line fitting the data points. The possible errors and limitations of the method are discussed from results obtained from both simulated and real surfaces. The effect of noise and resolution in the scanning tunnelling microscope on the calculation of the fractal dimension is also discussed.     

The use of laser scanning confocal microscopy (LSCM) in materials science

Laser scanning confocal microscopes are essential and ubiquitous tools in the biological, biochemical and biomedical sciences, and play a similar role to scanning electron microscopes in materials science. However, modern laser scanning confocal microscopes have a number of advantages for the study of materials, in addition to their obvious uses for high resolution reflected and transmitted light optical microscopy. In this paper, we provide several examples that exploit the laser scanning confocal microscope's capabilities of pseudo-infinite depth of field imaging, topographic imaging, photo-stimulated luminescence imaging and Raman spectroscopic imaging.     

Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy

Monomolecular films of polymerized dimethyl-bis[pentacosadiinoic-oxyethyl] ammonium bromide (EDIPAB) provide one- and two-photon excited fluorescence that is sufficiently high to quantify the axial resolution of 3-D fluorescence microscopes. When scanned along the optical axis, the fluorescence of these layers is bright enough to allow online observation of the axial response of these microscopes, thus facilitating alignment and fluorescence throughput control. The layers can be used for directly measuring and monitoring the axial response of 4Pi-confocal microscopes, as well as for their initial alignment and phase adjustment. The proposed technique has the potential to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Coverslips with EDIPAB-layers can be used as substrates for the cultivation of cells.     

Combining real and reciprocal space information for aberration free coherent electron diffractive imaging

Information from imaging and diffraction planes, or real and reciprocal spaces, of transmission electron microscopes (TEM) can be combined using iterative transformation algorithms to reconstruct the complex wave function, to improve image resolution and to remove residual aberrations in the case of aberration corrected TEM. Here, we describe the experimental and computation techniques needed for combining real and reciprocal space information. We demonstrate these techniques by reconstructing the complex wave function of quantum dots and carbon nanotubes beyond the microscope's resolution limit.     

Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy

Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide-field fluorescence microscope. The sample is illuminated with a series of excitation light patterns, which cause normally inaccessible high-resolution information to be encoded into the observed image. The recorded images are linearly processed to extract the new information and produce a reconstruction with twice the normal resolution. Unlike confocal microscopy, the resolution improvement is achieved with no need to discard any of the emission light. The method produces images of strikingly increased clarity compared to both conventional and confocal microscopes.     

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.     

Demonstration of high lateral resolution in laser confocal microscopy using annular and radially polarized light

The authors present the experimental result of improved lateral resolution in laser confocal microscopy (LCM) by using annular and radially polarized light as the input illumination of an existing LCM. The authors examined the lateral resolution of the LCM by imaging a single fluorescent bead and measuring the lateral width of the single bead profile appearing in the optical image. Compared to no aperture and linearly polarized light, the central peak of the single bead profile narrowed by ∼40%, being as small as 122 nm in full width at half maximum using 405 nm laser excitation in a reflection imaging. In addition, the authors showed that radial polarization helps to preserve the circular shape of the single bead profile whereas linearly polarized light tends to induce an elongation along the polarization direction. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Characterization of Local 3-D Rough Dielectric Surfaces Using Standoff GPR Measurements with Widebeam Antennas

In real life most ground surfaces are not flat but rough. The observation of surface roughness depends on the wavelength and angle of the incident wave. In order to be able to detect shallow subsurface objects, on one hand we need to use higher frequencies to achieve better range resolution. One the other hand we have to deal with rough surfaces relative to shorter wavelengths. In this paper a wideband ground-penetrating radar (GPR) phase measurement and processing technique for characterizing three-dimensional (3-D) rough dielectric surfaces is presented. The method is based on the measurement of phase data by a standoff GPR with wide-beam antennas at short range over 3-D rough ground surfaces. The principle of this method was verified experimentally in the measurement of a composite surface. The height of the composite surface varies from 0 to 8 cm. The antennas are open-ended waveguide antennas whose frequency range is 2.3 GHz to 4.3 GHz. They are broadband, have low gain and wide beamwidth. The experimental tests demonstrate that the 3-D rough surfaces can be characterized locally by using a monochromatic and multifrequency broadband phase processing and imaging method. The results show good agreement between the imagery of the surface height distribution obtained by this method and the actual geometry of the 3-D rough surfaces.     

适合于微细加工的外差探测技术及应用

本文详细地讨论了外差干涉仪的两个主要问题,即干涉仪的横向定位问题和非线性误差分析及其误差补偿问题.首先,提出了一种新颖的解析方法实现干涉仪亚微米级的高精度定位.该方法首先建立了测量光束扫过台阶边缘时测量相位渐变数学模型,并讨论了它与激光束分布的关系.文章利用以上数学模型对测量相位数据进行了详细地分析,实现了在一般激光束径时,干涉仪的定位精度为亚微米量级.另一方面,文章详细地分析了共光路干涉仪三个主要误差源.分析结果表明:由Wollaston棱镜引起的误差主要是二阶误差,而由激光束的椭圆偏振化引起的误差为一阶误差.同时我们发现:金属反射镜的方位误差可以使线偏振光经反射后变为椭圆偏振光,该椭圆偏振光具有不正交性和不相等偏心度,文章首次详细地分析了这种不正交性和不相等偏心度与反射镜方位误差的关系及其由此产生的非线性误差.最后,文章分析了干涉仪的误差补偿措施以提高整个干涉仪的测量精度.     

Comparison of optical and stylus methods for measurement of surface texture

Optical methods are increasingly used for measurement of surface texture, particularly for areal measurements where the optical methods are generally faster. A new Working Group under Technical Committee (TC) 213 in the International Organization for Standardization is addressing standardization issues for areal surface texture measurement and characterization and has formed a project team to address issues posed by the optical methods. In this paper, we review the different methods of measuring surface texture and describe a classification scheme for them. We highlight optical methods and describe some of their characteristics as well as compare surface-profiling results obtained from three optical methods with those obtained from stylus profiler instruments. For moderately rough surfaces (Ra?≈?500 nm), roughness measurements obtained with white light interferometric (WLI) microscopy, confocal microscopy, and the stylus method seem to provide close agreement on the same roughness samples. For surface roughness measurements in the 50 to 300 nm range of Ra, discrepancies between WLI and the stylus method are observed. In some cases the discrepancy is as large as about 75% of the value obtained with the stylus method. By contrast, the results for phase shifting interferometry over its expected range of application are in moderately good agreement with those of the stylus method.     

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.     

双测头复合型微纳米测量仪的研制

针对当前微纳米测量中存在的大范围高精度测量及复杂微结构几何参数表征难题,基于多测头传感和精密定位平台复用技术,开发了一台具有多种测量尺度和测量模式的复合型微纳米测量仪。为使其具备大范围快速扫描测量和小范围精细测量功能,仪器集成了白光干涉和原子力显微镜两种测头,通过设计适用于两种测头集成的桥架结构及宏/微两级驱动定位平台,实现整机的开发。为保证仪器测量结果的准确性和溯源性,利用标准样板对开发完成的仪器进行了校准。仪器搭载的白光干涉测头可以达到横向500 nm,纵向1 nm的分辨力;原子力显微镜测头横向和纵向分辨力均可达到1 nm。最后,利用目标仪器对微球样品进行了测量,通过大范围成像和小范围精细扫描,获得了微球的表面特征,验证了仪器对复杂微结构的测量能力。     

Surface plasmon fluorescence microscopy: an analysis

This paper examines the imaging performance of surface plasmon microscopes operating in the far field. Until recently the accepted view has been that the achievable lateral resolution with a surface plasmon imaging system is limited by propagation decay length of the surface plasmons rather than by diffraction. We show by simulation that the lateral resolution of a surface plasmon imaging system can be comparable to that of the best optical microscopes. We also show that new imaging modes such as two‐photon and also second harmonic surface plasmon microscopy are extremely promising imaging modes that may be expected to be powerful techniques for the analysis of surface structures.     

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.     

Influence of the elliptical illumination on acquisition and correction of coherent aberrations in high-resolution electron holography

In high-resolution off-axis electron holography, the interpretable lateral resolution is extended up to the information limit of the electron microscope by means of a correcting phase plate in Fourier space. A plane illuminating electron wave is generally assumed. However, in order to improve spatial coherence, which is essential for holography, the object under investigation is illuminated with an elliptically shaped electron source. This special illumination imposes a variation of beam directions over the field of view. Therefore, due to the interaction of beam tilt and coherent wave aberration, the effective aberrations vary over the field of view yielding a loss of isoplanicity. Consequently, in the past the aberrations were only corrected successfully for a small part of the field of view. However, a thorough analysis of the holographic imaging process shows that the imaging artifacts introduced by the elliptical illumination can be corrected under reconstruction by means of a phase curvature, which models the illuminating wave front. Applied in real space, this phase curvature is seamlessly incorporated into the correction process for coherent wave aberration resulting in an improvement of interpretable lateral resolution up to the information limit for the whole field of view.     

The effect of deformation on the lateral resolution of atomic force microscopy

A computer model based on the elastic properties of rubber is introduced for the evaluation of the lateral resolution in atomic force microscopy of deformable specimens. The computational results show that, if the full width at half-height can be defined as the lateral resolution, it is continuously improved at greater probe forces, at the expense of a reduced molecular height. In fact, even for a probe that is bigger than the molecule, the real size of the molecule can be 'recovered' at about 25% compression. This result demonstrates that for a better lateral resolution, a greater probe force can be beneficial, provided that the molecule is not moved or damaged and the response remains elastic. Measurements on isolated low-density lipoproteins (LDL) show that with 26% vertical compression, the lateral size measured in atomic force microscopy is only about 72% of the value predicted by a simple convolution, and is only slightly larger (≈ 13%) than the known size of LDL. Therefore, the results on LDL provide a direct support for the conclusions of the computational model.     

The theory of scanning microscopes with Gaussian pupil functions

The theory of imaging in scanning microscopes with lenses, source and detector all having Gaussian pupil function is developed. This assumption is useful as the expressions may be evaluated analytically. It is shown that Type 2 microscopes exhibit superior performance to those of Type 1. Effects of defocus are considered. It is found that defocus can be used in a Type 2 microscope to observe phase information without the limitation in resolution associated with stopping down the collector of a conventional microscope. It is also found that a Type 2 microscope discriminates against light scattered by parts of the object outside of the focal plane, allowing observation of detail within a thick object.     

PHOEBE,a prototype scanning laser-feedback microscope for imaging biological cells in aqueous media

Based on the principle of laser-feedback interferometry (LFI), a laser-feedback microscope (LFM) has been constructed capable of providing an axial (z) resolution of a target surface topography of ～ 1 nm and a lateral (x, y) resolution of ～ 200 nm when used with a high-numerical-aperture oil-immersion microscope objective. LFI is a form of interferometry in which a laser's intensity is modulated by light re-entering the illuminating laser. Interfering with the light circulating in the laser resonant cavity, this back-reflected light gives information about an object's position and reflectivity. Using a 1-mW He–Ne (λ = 632·8 nm) laser, this microscope (PHOEBE) is capable of obtaining 256 × 256-pixel images over fields from (10 μm × 10 μm) to (120 μm × 120 μm) in ～ 30 s. An electromechanical feedback circuit holds the optical pathlength between the laser output mirror and a point on the scanned object constant; this allows two types of images (surface topography and surface reflectivity) to be obtained simultaneously. For biological cells, imaging can be accomplished using back-reflected light originating from small refractive-index changes (> 0·02) at cell membrane/water interfaces; alternatively, the optical pathlength through the cell interior can be measured point-by-point by growing or placing a cell suspension on a higher-reflecting substrate (glass or a silicon wafer). Advantages of the laser-feedback microscope in comparison to other confocal optical microscopes include: the simplicity of the single-axis interferometric design; the confocal property of the laser-feedback microscope (a virtual pinhole), which is achieved by the requirement that only light that re-enters the laser meeting the stringent frequency, spatial (TEM₀₀), and coherence requirements of the laser cavity resonator mode modulate the laser intensity; and the improved axial resolution, which is based on interferometric measurement of optical amplitude and phase rather than by use of a pinhole as in other types of confocal microscopes.