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.     

Optical sectioning in fluorescence microscopy

We review the origins of optical sectioning in fluorescence microscopy in terms of the structure of the illumination used to generate the fluorescence within the specimen. We note that the conventional microscope using essentially uniform illumination does not exhibit optical sectioning whereas the confocal microscope using point (many spatial frequencies) illumination does. We show that the optical sectioning strength of a confocal microscope is not optimal and discuss the advantages of using a single spatial frequency for the structure of the illumination and the detection. In this case the optical sectioning strength is shown to be up to 25% narrower than in the ideal confocal case.     

Three-dimensional reconstruction of the guinea pig inner ear, comparison of OPFOS and light microscopy, applications of 3D reconstruction

Three-dimensional (3D) reconstruction of anatomical structures can give additional insight into the morphology and function of these structures. We compare 3D reconstructions of the guinea pig inner ear, using light microscopy and orthogonal plane fluorescence optical sectioning microscopy. Applications of 3D reconstruction of the inner ear are further explored. For each method two bullas were prepared for 3D reconstruction. Both methods are explained. In general, the 3D reconstructions using orthogonal plane fluorescence optical sectioning microscopy are superior to light microscopy. The exact spiral shape of the cochlea could be reconstructed using orthogonal plane fluorescence optical sectioning microscopy and the length of the basilar membrane measured. When a resolution of 20 μm is sufficient, orthogonal plane fluorescence optical sectioning microscopy is a superior technique for 3D reconstruction of inner ear structures in animals.     

Optical sectioning in confocal fluorescent microscopes

We discuss the three-dimensional imaging properties of confocal fluorescence scanning optical microscopes with particular reference to their optical sectioning property. We consider the practically important cases of the use of finite-sized circular and slit detectors and show how they effect the strength of the sectioning. We find, inter alia, that the performance is always better if the excitation and fluorescent wavelengths are close to each other, indeed as the fluorescent wavelength becomes very large the sectioning disappears altogether. We also compare the strength of the sectioning with that obtained in non-fluorescent confocal microscopy as this has implications in immuno-gold labelling studies.     

Direct-view microscopy: optical sectioning strength for finite-sized, multiple-pinhole arrays

Direct-view microscopes use multiple-aperture arrays in the source and detector planes. We develop a theory for brightfleld and fluorescence direct-view microscopy which allows us to determine the optical sectioning strength for finite-sized, multiple-pinhole arrays with an arbitrary distribution of apertures. We specialize to the cases of square, hexagonal and interleaving Archimedean spiral arrays and consider the implications of the array configuration on both the optical sectioning strength and the light budget.     

Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system

The bilateral imaging approach known from confocal applications operating in the line mode was used to realize real-time two-photon imaging. It is shown that the sectioning inherent to two-photon imaging could be improved by the introduction of a confocal line aperture in the imaging path. Using a high-power, low-repetition-rate amplified Ti:sapphire system, various biological objects were visualized including live boar sperm.     

A confocal laser microscope scanner for digital recording of optical serial sections

A confocal laser microscope scanner developed at our institute is described. Since an ordinary microscope is used, it is easy to view the specimen prior to scanning. Confocal imaging is obtained by laser spot illumination, and by focusing the reflected or fluorescent light from the specimen onto a pinhole aperture in front of the detector (a photomultiplier tube). Two rotating mirrors are used to scan the laser beam in a raster pattern. The scanner is controlled by a microprocessor which coordinates scanning, data display, and data transfer to a host computer equipped with an array processor. Digital images with up to 1024 × 1024 pixels and 256 grey levels can be recorded. The optical sectioning property of confocal scanning is used to record thin (～ 1 μm) sections of a specimen without the need for mechanical sectioning. By using computer-control to adjust the focus of the microscope, a stack of consecutive sections can be automatically recorded. A computer is then used to display the 3-D structure of the specimen. It is also possible to obtain quantitative information, both geometric and photometric. In addition to confocal laser scanning, it is easy to perform non-confocal laser scanning, or to use conventional microscopic illumination techniques for (non-confocal) scanning. The design has proved reliable and stable, requiring very few adjustments and realignments. Results obtained with this scanner are reported, and some limitations of the technique are discussed.     

光学综合孔径干涉成像技术

闭合相位技术、U-V覆盖技术和像重构技术是光学综合孔径干涉成像的三个关键技术.文中详细介绍了闭合相位技术的原理、U-V覆盖技术(包括即时覆盖和通过孔径旋转的非即时覆盖两种方法)和用于图像重构的常用方法以及用于光学综合孔径像重构的混合迭代方法,最后讨论了光学综合孔径干涉成像技术的应用.     

Optical microcantilever consisting of channel waveguide for scanning near-field optical microscopy controlled by atomic force

We develop a novel optical microcantilever for scanning near-field optical microscopy controlled by atomic force mode (SNOM/AFM). The optical microcantilever has the bent channel waveguide, the corner of which acts as aperture with a large tip angle. The resonance frequency of the optical microcantilever is 9 kHz, and the spring constant is estimated to be 0.59 N/m. The optical microcantilever can be operated in contact mode of SNOM/AFM and we obtain the optical resolution of about 200 nm, which is as same size as the diameter of aperture. We confirm that the throughput of optical microcantilever with an aperture of 170 nm diameter would be improved to be more than 10(-5).     

Optical microcantilever consisting of channel waveguide for scanning near-field optical microscopy controlled by atomic force

We develop a novel optical microcantilever for scanning near-field optical microscopy controlled by atomic force mode (SNOM/AFM). The optical microcantilever has the bent channel waveguide, the corner of which acts as aperture with a large tip angle. The resonance frequency of the optical microcantilever is 9 kHz, and the spring constant is estimated to be 0.59 N/m. The optical microcantilever can be operated in contact mode of SNOM/AFM and we obtain the optical resolution of about 200 nm, which is as same size as the diameter of aperture. We confirm that the throughput of optical microcantilever with an aperture of 170 nm diameter would be improved to be more than 10⁻⁵.     

Modified fabrication process for aperture probe cantilevers

We report the development of cantilever- and fibre-based probes for scanning near-field optical microscopy. Both probe concepts rely on the integration of a microfabricated aperture tip with reproducible optical and mechanical properties. Numerical calculations were carried out using a finite integration code to investigate the polarization-sensitive transmission behaviour of aperture tips. In order to establish technological guidelines for the optimization of the properties of the optical tip the distinct influence of the tip geometry on the intensity distribution in the vicinity of the aperture is studied in detail.     

Observation of three-dimensional internal structure of steel materials by means of serial sectioning with ultrasonic elliptical vibration cutting

A three-dimensional (3D) internal structure observation system based on serial sectioning was developed from an ultrasonic elliptical vibration cutting device and an optical microscope combined with a high-precision positioning device. For bearing steel samples, the cutting device created mirrored surfaces suitable for optical metallography, even for long-cutting distances during serial sectioning of these ferrous materials. Serial sectioning progressed automatically by means of numerical control. The system was used to observe inclusions in steel materials on a scale of several tens of micrometers. Three specimens containing inclusions were prepared from bearing steels. These inclusions could be detected as two-dimensional (2D) sectional images with resolution better than 1 μm. A three-dimensional (3D) model of each inclusion was reconstructed from the 2D serial images. The microscopic 3D models had sharp edges and complicated surfaces.     

Microfabricated silicon dioxide cantilever with subwavelength aperture

We have developed a microfabricated SiO₂ cantilever with subwavelength aperture for scanning near-field optical microscopy (SNOM), to overcome the disadvantages of conventional optical fibre probes such as low reproducibility and low optical throughput. The microcantilever, which has a SiO₂ cantilever and an aperture tip near the end of the cantilever, is fabricated in a reproducible batch process. The circular aperture with a diameter of 100–150 nm is formed by a focused ion-beam technique. Incident light is directly focused on the aperture from the rear side of the cantilever using a focusing objective, and high optical throughput (10⁻² to 10⁻³) is obtained. The microcantilever can be operated as a SNOM probe in contact mode or in dynamic mode.     

大型光学设备检测中大口径平行光管波像差的消除

使用大口径平行光管检测大型光学设备或元件时,平行光管的自身误差会影响检测结果,故本文提出了一种消除光学检测结果中平行光管引入误差的新方法。该方法使用干涉仪获取平行光管和光学检测系统的出射波前信息并以37项Standard Zernike Phase多项式进行拟合;通过两组系数相减分离平行光管引入误差,再配合ZEMAX中建立的等效被检光学系统的仿真模型模拟真实系统的出射光锥,最终获得被检光学系统真实的出射波前信息。利用ZEMAX中的光学系统模型验证了该方法在大口径光学检测工作中的可行性;使用焦距为1 597mm,口径为150mm的小型平行光管、焦距为50mm的光学镜头进行了实验。实验结果表明:使用该方法获得的被检光学系统出射波前与真实波前的PV值误差小于0.005λ,RMS值误差小于0.001λ,可以满足实验室中对被检光学系统成像质量参数检测的精度要求。     

Modified fabrication process for aperture probe cantilevers

We report the development of cantilever- and fibre-based probes for scanning near-field optical microscopy. Both probe concepts rely on the integration of a microfabricated aperture tip with reproducible optical and mechanical properties. Numerical calculations were carried out using a finite integration code to investigate the polarization-sensitive transmission behaviour of aperture tips. In order to establish technological guidelines for the optimization of the properties of the optical tip the distinct influence of the tip geometry on the intensity distribution in the vicinity of the aperture is studied in detail.     

多次函数拟合法计算光纤模场直径

光纤的模场直径是评价光纤性能的一个重要参数。测量单模光纤模场直径的常用方法是远场可变孔径法。通过测量给定单模光纤透过不同尺寸孔径光阑的多组远场辐射光功率数据并对实测数据进行处理,可得到被测光纤的模场直径。在实际测量过程中,为了消除被测光纤模场中心与孔径光阑中心偏离引起的测量误差,应用多次函数拟合法对远场可变孔径法测得的数据进行处理,以此自动识别并排除测量数据中的个别不合理的误差数据,从而有效提高模场直径的测量精度。     

Single molecule mapping of the optical field distribution of probes for near-field microscopy

The most difficult task in near-field scanning optical microscopy (NSOM) is to make a high quality subwavelength aperture probe. Recently, we have developed high definition NSOM probes by focused ion beam (FIB) milling. These probes have a higher brightness, better polarization characteristics, better aperture definition and a flatter end face than conventional NSOM probes. We have determined the quality of these probes in four independent ways: by FIB imaging and by shear-force microscopy (both providing geometrical information), by far-field optical measurements (yielding throughput and polarization characteristics), and ultimately by single molecule imaging in the near-field. In this paper, we report on a new method using shear-force microscopy to study the size of the aperture and the end face of the probe (with a roughness smaller than 1.5 nm). More importantly, we demonstrate the use of single molecules to measure the full three-dimensional optical near-field distribution of the probe with molecular spatial resolution. The single molecule images exhibit various intensity patterns, varying from circular and elliptical to double arc and ring structures, which depend on the orientation of the molecules with respect to the probe. The optical resolution in the measurements is not determined by the size of the aperture, but by the high optical field gradients at the rims of the aperture. With a 70 nm aperture probe, we obtain fluorescence field patterns with 45 nm FWHM. Clearly, this unprecedented near-field optical resolution constitutes an order of magnitude improvement over far-field methods like confocal microscopy.     

A technique for revealing tissue embedded within resin blocks

A technique is described for revealing in fine detail large pieces of tissue still embedded within rough, scratched or partly-trimmed blocks of epoxy and other resin before sectioning. The pieces of tissue immersed in baths of oil or glycerol and illuminated by an appropriate optical system, may be photographed to provide a guide to subsequent sectioning, aiding the precise location of elements in the sections back to their position in the parent tissue, and for tracing the connections of the element between serial sections. The method has been used by the authors in sectioning for light microscopy, but could equally be used with sections for electron microscopy.     

大口径数字波面检测技术的研究

现代光学系统多采用大口径的光学元件来提高系统的测试精度。数字波面干涉仪是一种高精度光学元件检测设备,但目前传统的技术无法满足检测大口径光学元件的要求。在普通数字波面干涉仪的基础上,采用目标函数多孔径拼接技术,能很好地解决大口径光学元件检测问题,最终得到较完整的波前信息。     

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.