Subdiffraction scattered light imaging of gold nanoparticles using structured illumination

A reflective light-scattering (RLS) microscope with structured illumination (SI) provides subdiffraction resolution and improves the image quality of gold nanoparticles in biological systems. The three-dimensional (3D)-structured pattern is rapidly and precisely controlled with a spatial light modulator and scrambled at the conjugate image plane to increase spatial incoherence. The reconstructed SI-RLS image of 100?nm gold nanoparticles reveals lateral and axial resolutions of approximately 117 and 428?nm. We present a high-resolution image of gold nanoparticles inside a HeLa cell, with improved contrast.     

Three-dimensional resolution enhancement in fluorescence microscopy by harmonic excitation

A method for increasing lateral as well as axial resolution in fluorescence microscopy is presented. A passband with a high cutoff frequency throughout reciprocal space can be achieved by illumination of the object with spatially harmonic excitation patterns generated by the interference of two collimated laser beams. Theoretical calculations show an almost isotropic point-spread function with a FWHM near 100 nm.     

Theoretical assessment of optical resolution enhancement and background fluorescence reduction by three-dimensional nonlinear structured illumination microscopy using stimulated emission depletion

Three-dimensional structured illumination microscopy (SIM) enlarges frequency cutoff laterally and axially by a factor of two, compared with conventional microscopy. However, its optical resolution is still fundamentally limited. It is necessary to introduce nonlinearity to enlarge frequency cutoff further. We propose three-dimensional nonlinear structured illumination microscopy based on stimulated emission depletion (STED) effect, which has a structured excitation pattern and a structured STED pattern, and both three-dimensional illumination patterns have the same lateral pitch and orientation. Theoretical analysis showed that nonlinearity induced by STED effect, which causes harmonics and contributes to enlarging frequency cutoff, depends on the phase difference between two structured illuminations and that the phase difference of π is the most efficient to increase nonlinearity. We also found that undesirable background fluorescence, which degenerates the contrast of structured pattern and limits the ability of SIM, can be reduced by our method. These results revealed that optical resolution improvement and background fluorescence reduction would be compatible. The feasibility study showed that our method will be realized with commercially available laser, having 3.5 times larger frequency cutoff compared with conventional microscopy.     

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.     

非平行光干涉照明显微镜三维形貌检测研究

为实现表面微观形貌快速而较简单的检测, 一种使用非平行光干涉照明的光学显微三维形貌检测方法被提出。该方法使用空间光调制器对激光光束进行衍射, 选取光强相近的2个衍射级通过显微物镜, 双光束干涉可得到周期接近图像分辨率、相位可精确调节的照明条纹, 被测样本的三维形貌可通过拍摄4帧等相位差的条纹照明图像来计算得到。该方法不需借助干涉物镜产生条纹, 不需要轴向扫描装置记录条纹变化, 相位调节精确, 成像直观。此外, 该方法所产生干涉条纹的相位随坐标线性变化, 不需对条纹周期进行修正。因为照明条纹参数调节光路独立于显微成像光路, 系统装置具有光路简洁、易于调节的优点。为验证所提出三维检测精度, 以粗糙度100 nm的粗糙度对比模块和硅片为被测样品进行了三维轮廓重建实验, 实验结果显示, 所提出方法轴向重复性测量精度为8.6 nm(2σ)。     

The Limitations of Nonlinear Fluorescence Effect in Super Resolution Saturated Structured Illumination Microscopy System

Classically, optical systems are considered to have a fundamental resolution limit due to diffraction. Many strategies for improving both axial and lateral resolutions are based on a priori information about the input signal. These strategies lead to a numerical aperture improvement. However these are still limited by the wave nature of light. By using fluorescence technique one theoretically can reach unlimited resolution. The key point is to use the nonlinear dependence of the fluorescence emission rate on the intensity of the applied illumination. In this paper we present simulation as well as experimental results which show the advantage and the problems of using the nonlinear fluorescence effect in super resolution systems as well as discussing the nonlinear phenomena concerning the fluorescence process. The results show that the nonlinear fluorescence effect is accompanied by severe quenching, bleaching and saturation phenomena. As consequence, super resolution using saturated structured illumination method in living biological samples becomes severely restricted.     

High-speed 3D imaging based on structured illumination and electrically tunable lens

In this Letter, we present a high-speed volumetric imaging system based on structured illumination and an electrically tunable lens(ETL), where the ETL performs fast axial scanning at hundreds of Hz. In the system,a digital micro-mirror device(DMD) is utilized to rapidly generate structured images at the focal plane in synchronization with the axial scanning unit. The scanning characteristics of the ETL are investigated theoretically and experimentally. Imaging experiments on pollen samples are performed to verify the optical cross-sectioning and fast axial scanning capabilities. The results show that our system can perform fast axial scanning and threedimensional(3D) imaging when paired with a high-speed camera, presenting an economic solution for advanced biological imaging applications.     

Scanning two photon fluorescence microscopy with extended depth of field

We describe the design and implementation of a light efficient binary phase-only diffraction optical element which converts a laser beam into an annular ring with the loss of less than 25% of the overall power. The use of annular illumination permits us to obtain an increase in the axial extent of the point spread function of a microscope objective without compromising the lateral resolution. The light efficiency of the system permitted two photon fluorescence images with extended depth of field to be obtained. The combination of two such extended depth of field images obtained at different parallaxes allowed the generation of high resolution two photon stereo pairs.     

Wide-field high-resolution structured illumination solid immersion fluorescence microscopy

The use of aplanatic solid immersion lenses (ASILs) made of high-refractive-index optical materials provides a route to wide-field high-resolution optical microscopy. Structured illumination microscopy (SIM) can double the spatial bandwidth of a microscope to also achieve high-resolution imaging. We investigate the combination of ASILs and SIM in fluorescence microscopy, which we call structured illumination solid immersion fluorescence microscopy (SISIM), to pursue a microscopic system with very large NA and high lateral resolution. We demonstrate that the combination can produce a wide-field high-resolution microscopic system with bandwidth corresponding to an NA of 3. Future developments of the SISIM system to make it achieve even higher resolution are proposed.     

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。     

Study of an ultrahigh-numerical-aperture fiber continuum generation source for optical coherence tomography

The axial resolution of optical coherence tomography images is primarily dependent on the bandwidth of the illumination source. Continuum generation is one way to generate the single-mode, high-bandwidth light needed for point illumination. We present an inexpensive and easy-to-implement augmentation to a Ti:sapphire laser that widens the bandwidth from 20 to over 200 nm with commerically available ultrahigh-numerical-aperture fiber. This technique can provide a readily available broad-bandwidth source for researchers and a practical enhancement to a fiber-optic optical coherence tomography system.     

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.     

Axial super-resolution by mirror-reflected stimulated emission depletion microscopy

In stimulated emission depletion (STED) microscopy, the lateral resolution is in the range of tens of nanometers depending on the sample and the instrument. The axial resolution, however, is in standard systems limited by diffraction to about 500 nm. We present an approach to three-dimensional diffraction-unlimited resolution by observing the sample at two optical angles. The system is realized by using an atomic force microscope (AFM) chip as a microreflector to deflect the STED beams near the region-of-interest (ROI), thus allowing observations at an angle ∠. Consequently, the superior lateral resolution can be utilized to resolve details in the axial direction of the main optical axis of the microscope. Here, fluorescent nanoparticles 90 nm apart and biological structures 80 nm apart along axial direction were distinguished by utilizing an off-the-shelf, commercial STED microscope, coupled with an AFM and an AFM chip micro-reflector.     

Increasing the imaging depth of coherent anti-Stokes Raman scattering microscopy with a miniature microscope objective

A miniature objective lens with a tip diameter of 1.3 mm was used for extending the penetration depth of coherent anti-Stokes Raman scattering (CARS) microscopy. Its axial and lateral focal widths were determined to be 11.4 and 0.86 microm, respectively, by two-photon excitation fluorescence imaging of 200 nm beads at a 735 nm excitation wavelength. By inserting the lens tip into a soft gel sample, CARS images of 2 microm polystyrene beads 5 mm deep from the surface were acquired. The miniature objective was applied to CARS imaging of rat spinal cord white matter with a minimal requirement for surgery.     

Enhancement of Axial Trapping Force in a Single-beam Gradient Optical Trap by a Pair of Axicons

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Enhancement of Axial Trapping Force in a Single-beam Gradient Optical Trap by a Pair of Axicons

A special kind of axicon prisms for beam transformation and enhancement of axial trapping force in a single-beam gradient optical trap is put forward. By inserting a pair of axicons into the parallel illumination path of a trapping objective, a center-weighted TEM00 Gaussian beam was transformed into an edge-weighted beam. Edge-weighted illumination for the trapping objective was thus realized, which is significant for the enhancement of axial trapping force. Based on a method of external sinusoidal excitation and lock-in detection, we precisely calibrated the axial spring constants of the same optical trap under different illumination conditions. The comparison results confirmed the feasibility of enhancing the axial trapping force by a pair of axicon. Moreover, the strength of enhancement was found to be dependent on the apodization factor for the input TEM00 Gaussian beam.     

Formation of lateral-shift speckle interferograms in diffusely scattered fields

The formation of lateral-shift speckle interferograms that characterize the axial wave aberrations of the optical system used in the coherent matte-screen illumination channel is analyzed in the Fresnel approximation based on coincidence of the objective speckle fields of two exposures in the photoplate plane. It shown theoretically and in experiment that speckle-interferometer sensitivity is doubled at a given lateral shift. Tomsk State University, Tomsk. Translated from Izvestiya Vysshikh Uchebnykh Zavedenii, Fizika, No. 9, pp. 36–42, September, 1996.     

Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator

In wide-field fluorescence microscopy, illuminating the specimen with evanescent standing waves increases lateral resolution more than twofold. We report a versatile setup for standing-wave illumination in total internal reflection fluorescence microscopy. An adjustable diffraction grating written on a phase-only spatial light modulator controls the illumination field. Selecting appropriate diffraction orders and displaying a sheared (tilted) diffraction grating allows one to tune the penetration depth in very fine steps. The setup achieves 91 nm lateral resolution for green emission.     

Talbot holographic illumination nonscanning (THIN) fluorescence microscopy

Optical sectioning techniques offer the ability to acquire three‐dimensional information from various organ tissues by discriminating between the desired in‐focus and out‐of‐focus (background) signals. Alternative techniques to confocal, such as active structured illumination, exist for fast optically sectioned images, but they require individual axial planes to be imaged consecutively. In this article, an imaging technique (THIN), by utilizing active Talbot illumination in 3D and multiplexed holographic Bragg filters for depth discrimination, is demonstrated for imaging in vivo 3D biopsy without mechanical or optical axial scanning.     

超高分辨光学相干层析成像技术与材料检测应用

本文报道了一种超高分辨率谱域光学相干层析成像(SD-OCT)系统. 该系统基于超连续谱激光光源并截取部分光谱作为宽带光源, 其中心波长为665 nm, 光谱半高全宽(FWHM) 230 nm. 系统轴向分辨率0.9 μm, 轴向扫描速率28600行/秒, 横向分辨率3.9 μm, 横向视场1 mm, 最大成像深度0.6 mm(空气中). 利用研制的超高分辨率SD-OCT系统, 对不同型号的工业砂纸精细结构进行了成像, 并与普通SD-OCT的成像结果进行对比, 充分展示了研制系统在材料无损检测中优势.