Spectrally encoded slit confocal microscopy

A simple and cost-effective method for real-time imaging in confocal microscopy is proposed. Spectrally encoded slit confocal microscopy (SESCoM) uses a spectral encoding technique together with a confocal slit aperture to achieve two-dimensional images. Simulation and experimental results of the SESCoM's axial and lateral performances are presented. The measured FWHM of the axial response is 1.15 mum when an objective with a NA of 0.95 is used. FWHMs of the lateral line spread functions are measured to be 236 and 244 nm along the x and y directions, respectively. Both the axial and the lateral experimental results agree well with the simulation results.     

具有高空间分辨力的整形环形光式差动共焦测量法

提出一种新的具有高空间分辨力的整形环形光式差动共焦测量方法。该方法通过整形环形光式共焦测量法和锐化爱里斑主瓣，改善系统横向分辨力；通过差动共焦测量法改善系统的轴向分辨力，最终达到提高系统空间分辨能力的目的。理论分析和实验表明：整形环形光内孔归一化半径ε越大，横向分辨力改善越明显，量程扩展范围越宽；当入射光波长λ=632.8nm，物镜数值孔径取NA=0.85，ε=0.5时，该系统的横向分辨力优于0.2μm，轴向分辨力优于2nm。该方法为光触针测量系统空间分辨力的提高提供了1种新的方法，可广泛应用于超精密三维微细结构工件的超精密测量。     

微分干涉差共焦显微膜层微结构缺陷探测系统

多层膜极紫外光刻掩模"白板"缺陷是制约下一代光刻技术发展的瓶颈之一,为提高对掩模"白板"上的膜层微结构缺陷的分辨能力,提出了一种微分干涉差共焦显微探测系统方案。基于标量衍射理论,计算了系统横向和轴向分辨率。利用MATLAB建模仿真,在数值孔径为0.65、工作波长为405 nm时,分析比较了微分干涉差共焦显微系统、传统显微系统和共焦显微系统的分辨率。结果表明微分干涉差共焦显微系统具有230 nm的横向分辨率和25 nm轴向台阶高度差的分辨能力(对应划痕等缺陷形式)。此外,仿真和分析了实际应用中探测器尺寸、样品轴向偏移等的影响,模拟分析了膜层微结构缺陷的探测,结果表明本系统可以探测200 nm宽、10 nm高的微结构缺陷,较另外两种系统有更好的探测能力。     

A lateral super-resolution differential confocal technology with phase-only pupil filter

In order to achieve a higher lateral resolution required for ultraprecision measurement of microstructural workpieces, phase-only pupil filtering differential confocal microscopy (PFDCM), a new approach is proposed based on the differential confocal microscopy (DCM), which uses a three-zone phase-only pupil filter with lateral super-resolution capability obtained through optimized design to change the distribution of DCM three-dimensional point spread function, so that the DCM lateral resolution is therefore significantly improved while its axial resolution is slightly improved. Preliminary experimental comparison and analyses indicate that, the lateral and axial resolutions of PFDCM are better than 0.2 μm and 2 nm, respectively, when wavelength of incidence laser beam , numerical aperture of measuring lens NA=0.85, and lateral spot size with a three-zone phase-only pupil filter G_T=0.65. It is therefore concluded that PFDCM is a new approach to further improvement of lateral resolution in laser probe measurement systems.     

A shaped annular beam tri-heterodyne confocal microscope with good anti-environmental interference capability

A shaped annular beam tri-heterodyne confocal microscope has been proposed to improve the anti-environmental interference capability and the resolution of a confocal microscope. It simultaneously detects far-, on-, and near-focus signals with given phase differences by dividing the measured light path of the confocal microscope into three sub-paths (signals). Pair-wise real-time heterodyne subtraction of the three signals is used to improve the anti-environmental interference capability, axial resolution, and linearity; and a shaped annular beam super-resolution technique is used to improve lateral resolution. Theoretical analyses and preliminary experiments indicate that an axial resolution of about 1 nm can be achieved with a shaped annular beam tri-heterodyne confocal microscope and its lateral resolution can be better than 0.2 $\mu $m for $\lambda =632.8$~nm, the numerical aperture of the lens of the microscope is NA $=0.85$, and the normalized radius $\varepsilon =0.5$.     

Scanning optical reconstruction of coded aperture images

A scanning optical technique is described and used to reconstruct coded aperture images obtained with four different kinds of arrays. The feasibility and advantages of the technique are demonstrated experimentally. The results are used to compare the imaging properties of these arrays, i.e.: lateral resolution, background noise and axial (tomographic) resolution.     

Confocal pattern period in multiple-aperture confocal imaging systems with coherent illumination

We have built a programmable multiple-aperture confocal imaging system that uses a spatial light modulator (a Texas Instruments digital micromirror device). Excellent axial resolution and confocal imaging quality have been experimentally demonstrated with this system, even when the distance between adjacent ON pixels is four times the size of the pixel aperture. By contrast, typical pinhole-based systems (e.g., a Nipkow disk) employ an adjacent aperture distance-to-size ratio of 10x . We have achieved improvement over such systems by 6.25x in both light-utilization efficiency and confocal image rate without adding more components and extra processing time.     

Aberration-free optical refocusing in high numerical aperture microscopy

We describe a method of optical refocusing for high numerical aperture (NA) systems that is particularly relevant for confocal and multiphoton microscopy. This method avoids the spherical aberration that is common to other optical refocusing systems. We show that aberration-free images can be obtained over an axial scan range of 70 mum for a 1.4 NA objective lens. As refocusing is implemented remotely from the specimen, this method will enable high axial scan speeds without mechanical interference between the objective lens and the specimen.     

Wavelength division multiplexed confocal microscopy using supercontinuum

We investigate both theoretically and experimentally wavelength division multiplexed confocal imaging by using white light supercontinuum. We show that with the optimized pinhole diameter an axial resolution of 0.75 μm and detection efficiency of 80% can be achieved. In addition, we applied the axial WDM confocal system to 3D surface measurement and the result agreed well with that measured by commercially available surface profilometer. The measured sensitivity of the system is 3.25 nm. Finally, we demonstrated lateral confocal imaging by using supercontinuum. An effective lateral scanning range of 130 μm was obtained.     

反射式光纤共焦扫描成像的研究

建立了反射式光纤共焦扫描成像系统,分析了光纤－集光透镜参数Ａ及物透镜有效数值孔径等对系统成像分辨率的影响。并在此基础上选择了合适参数的透镜,获得了优化的反射式光纤共焦扫描成像系统,测试结果表明,该系统具有亚微米级横向成像能力,微米级向层析能力,成像稳定性那,它将应用于材料及生物组织三维成像检测中。     

Resolution enhancement in a light-sheet-based microscope (SPIM)

Light-sheet-based microscopy [single-plane illumination microscope (SPIM)] performs very well at low numerical apertures. It complements conventional (FM), confocal (CFM), and two-photon fluorescence microscopy (2hnu-FM) currently used in modern life sciences. Lateral and axial SPIM point spread function (PSF) extents are measured by using fluorescent beads to determine the 3D resolution. The results are compared with values derived from an analytical theory and numerical simulations. The discrepancies are found to be less than 5%. The axial extent of a SPIM-PSF (10x/0.3 W) is approximately 5.7 microm. This value is almost a factor of 2 smaller than in CFM, more than 2.5 times smaller than in FM, and more than three times smaller than in 2hnu-FM. SPIM outperforms 2hnu-FM and FM, while CFM has a better axial resolution at NAs above 0.8.     

A study of the optical transfer function for annular binary filters

The annular binary pupil filters consist of two transparent annuli obstructed by an annular mask. The optical transfer functions for them are analytically derived under paraxial approximation, and three- and two- as well as one-dimensional numerical plots are presented. It is shown that by changing the parameters of transparent regions it is possible to improve the axial resolution whilst the transverse resolution remains unchanged, compared with an annular lens. Results indicate, as is well known, that this kind of filters can be applied to three-dimensional confocal microscopy.     

Measurement of lateral and axial resolution of confocal Raman microscope using dispersed carbon nanotubes and suspended graphene

A confocal Raman microscope (CRM) facilitates visualization of the spatial distribution of molecular bonds or phonon modes at the submicron level and has been extensively used in the characterization of nanomaterials and devices. The lateral and axial resolution is a key specification that defines the performance of CRM, however, the interpretation of spatial resolution in the literature is often ambiguous, making it often difficult to directly compare Raman images obtained under different conditions. In this report, a convenient and reliable measurement protocol using dispersed carbon nanotubes and suspended graphene as test specimens is proposed to facilitate the determination of the lateral and axial resolutions of a CRM. Spatial resolution values comparable to the results based on Rayleigh criterion calculations were obtained using Raman mapping images of test specimens. This was achieved without the need for complex deconvolution processes or the consideration of an asymmetric dielectric environment.     

Imaging biological specimens with the confocal scanning laser microscope/macroscope

A new confocal scanning laser microscope/macroscope (cslm/M) has recently been developed. It combines in one instrument the high resolution capability of a confocal scanning beam microscope for imaging small specimens, with good resolution confocal imaging of macroscopic specimens. Some of its main features include: (a) 0.25 μm lateral resolution in the microscope mode and 5 μm lateral resolution in the macroscope mode; (b) a field of view that can vary from 25 μm × 25 μm to 75,000 μm × 75,000 μm; (c) capability for acquiring large data sets from 512 × 512 pixels to 2048 × 2048 pixels; (d) 0.5 μm depth resolution in the microscope mode and 200 μm depth resolution in the macroscope mode.

In this work the cslm/M was used to image whole biological specimens (> 5 m diameter), including insects which are ideal specimens for the macroscope. Specimens require no preparation, unlike scanning electron microscope (SEM) specimens which require a conductive coating. The specimens described in this paper are too large to be imaged in their entirety by a scanning beam laser microscope, however they can be imaged by slower scanning stage microscopes. In the macroscope mode the cslm/M was used to acquire a large number (e.g. 20–40) of confocal image slices which were then used to reconstruct a three-dimensional image of the specimen. High resolution images were collected by the cslm/M by switching to the microscope mode where high numerical aperture (NA) objectives were used to image a small area of interest. Reflected-light and fluorescence images of plant and insect specimens are presented which demonstrate the morphological details obtained in various imaging modes. A process for three-dimensional visualization of the data is described and images are shown.     

利用变迹术和相干门相结合实现光学相干层析成像术轴向超分辨

光学相十层析成像（光学相干层析成像术）的轴向分辨力由光源带宽和探测光束的聚焦条件共同决定。提高光学相干层析成像术轴向分辨力的方法主要基于带宽光源技术。提出了一种将变迹术与光学相十层析成像术相干门有机结合的方法来提高其轴向分辨力。通过适当形式的光瞳滤波器．使光学相干层析成像术系统轴向响应的主瓣宽度缩小到相干门之内,而其旁瓣则处于相干门之外．不对相干成像产生有效贡献。这样．就能在光源带宽不变的条件下,有效提高光学相十层析成像术的轴向分辨力,避免了采用宽带光源所带来的费用昂贵和系统复杂等缺陷。     

用于高数值孔径物镜的可调光瞳滤波器

根据矢量衍射理论，提出了一种用于提高高数值孔径显微物镜纵向分辨率的可调二元环形光瞳滤波器，通过改变滤波器的环半径获得不同的纵向分辨率，并利用共焦显微术点扩散函数的乘积性大大地降低了旁瓣相对主瓣的比值。模拟结果表明，这种新型滤波器不仅可以提高高数值孔径物镜的纵向分辨率，同时基本上不影响横向分辨率。     

激光分光瞳共焦显微镜系统研制

为了满足生物类等样品对大工作距和高分辨率共焦显微镜的需求,将分光瞳技术与激光共焦显微技术结合应用到成像系统上。阐述了激光分光瞳共焦显微成像原理,首次成功搭建了相应的显微镜成像测量系统。理论分析和实验表明:分光瞳共焦显微技术独特的非共轴结构使系统的轴向分辨力是相同数值孔径物镜单轴系统的3倍以上,对理论高度为100nm的台阶样品进行成像测试,得到的样品三维形貌,成像质量良好。     

Combined confocal/en face T-scan-based ultrahigh-resolution optical coherence tomography in vivo retinal imaging

Combined confocal scanning ophthalmoscopy/en face T-scan-based ultrahigh-resolution optical coherence tomography (OCT) of the human retina in vivo is reported for the first time to our knowledge. The system uses a superluminescent diode-based broadband source, which gives an axial resolution of 3.2 microm in the retina. We demonstrate acquisition of T-scan-based OCT B-scan and simultaneous confocal/C-scan images of the human retina of large lateral size (covering a field of up to 20 degrees ) at a frame rate of 2Hz.     

An approach to achieve lateral superresolution for small probe confocal measurement system and its element

A shaped annular beam superresolution approach is proposed to improve a lateral resolution of a small probe laser confocal measurement system (LCMS). The approach proposed enables lateral superresolution measurement of LCMS to be achieved by using a binary optical diffractive element to shape a He–Ne Gaussian laser beam into an annular beam with an inner diameter of 0.87 mm and an outer diameter of 1.8 mm required for superresolution measurement, and shift the beam spatial frequency from low to high. And a binary optical element (BOE) with 16 phase levels is designed and fabricated to shape a Gaussian laser beam into an annular beam. Preliminary experimental results indicate that an intensity distribution of a shaped annular beam is in agreement with simulation results, the diffractive efficiency is 87.2%; LCMS lateral and axial resolutions of 0.2 μm and 3 nm are achieved, respectively, and its measurement range is expanded nearly to double, when BOE is used in LCMS and , NA=0.85.     

Reflection confocal microscope readout system for three-dimensional photochromic optical data storage

We propose to use a reflection confocal microscope (RCM) as a readout system for digital data stored in a three-dimensional (3D) photochromic optical memory. We describe the merits and the difficulties for 3D optical memory that are associated with the use of such a RCM.It is shown by means of 3D Fourier space analysis that successful reading of 3D data can be obtained by selection of the appropriate parameter for the RCM.The system parameters include the numerical aperture of the objective lens and the wavelength of the light used for analysis (reading). Experimental results of multilayer recording and reflection confocal reading in photochromic-molecule-doped poly(methyl methacrylate) are presented. Good-contrast images are obtained.