Image formation in structured illumination wide-field fluorescence microscopy

We present a theoretical analysis of the image formation in structured illumination wide-field fluorescence microscopy (SIWFFM). We show that the optically sectioned images obtained with this approach possess the optical sectioning strengths comparable to those obtained with the confocal microscope. We further show that the transfer function behaviour is directly comparable to that of the true confocal instrument. The theoretical considerations are compared with and confirmed by experimental results.     

Wide-field coherent anti-Stokes Raman scattering microscopy with non-phase-matching illumination

We have developed and tested a wide-field coherent anti-Stokes Raman scattering (CARS) microscopy technique, which provides the simultaneous imaging of an extended illuminated area without scanning. This method is based on the non-phase-matching illumination of a sample and imaging of a CARS signal with a CCD camera using conventional microscope optics. We have identified a set of conditions on the illumination and imaging optics, as well as on sample preparation. Imaging of test objects proved high spatial resolution and chemical selectivity of this technique.     

结构光照明显微中的偏振控制

结构光照明显微(structured illumination microscopy.SIM)作为一种宽场超分辨光学显微成像技术,具有成像速度快、光漂白和光毒性弱等优点,是目前主流超分辨成像方法之一.在SIM技术中,正弦强度分布的条纹结构光场的对比度决定了SIM超分辨图像的质量.低的条纹对比度将导致样品中超衍射极限的高频信息被噪声掩盖,从而无法解析出超分辨信息.结构照明入射光的偏振态调控决定了干涉条纹的对比度,是SIM的关键技术.鉴于此,本文总结对比了几种典型的SIM系统偏振控制方法,同时提出了一种使用零级涡旋半波片的偏振控制方法.实验证明,与其他方法相比,采用零级涡旋半波片法可以获得更高效的偏振控制效果,具有系统结构简单、易使用、可将光能利用率提升到接近100%的优点.     

Coherent super-resolution microscopy via laterally structured illumination

Theory describing a super-resolution microscopy experiment using temporally and spatially coherent structured illumination was developed, and used to derive a method for processing experimental data. Numerical simulations were performed to verify that the method can, in principle, produce super-resolved images that are exactly equivalent to an image processed by a system with a much larger aperture (that is, the correct weighting between different regions of the image spectrum is maintained). The process was then demonstrated experimentally, showing a factor of two improvement in resolution over a diffraction-limited, coherently illuminated, microscope.     

平场复用多焦点结构光照明超分辨显微成像

多焦点结构光照明显微技术(multifocal structured illumination microscopy, MSIM)能在50μm的成像深度内和1Hz的成像速度下实现两倍于衍射极限分辨率的提升,相比传统的宽场结构光照明显微技术,具有较大的成像深度和层析能力,更适合应用于厚样品的长时程三维超分辨成像.然而, MSIM存在成像速度慢、图像处理过程复杂等问题.本文提出了一种基于平场复用多焦点结构光照明的快速超分辨显微成像方法和系统(flat-field multiplexed MSIM, FM-MSIM),通过在照明光路中插入光束整形器件,将高斯光束转变为均为分布的平顶光束,提高激发点阵的强度均匀性和扩大视场;通过将每个衍射受限的激发点沿y方向延长,形成新的多路复用多焦点阵照明图案,提高能量利用率,减少扫描步数,进而提高成像速度和信噪比;结合基于多重测量矢量模型的稀疏贝叶斯学习图像重构算法,简化图像重构步骤,在保证空间分辨率的同时实现至少4倍于传统MSIM的成像速度.在此基础上,利用搭建的FM-MSIM系统进行了BSC细胞微管样片和小鼠肾切片标准样片的超分辨成像实验,实验结果证明...     

Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy

We describe a method of obtaining optical sectioning with a standard wide-field fluorescence microscope. The method involves acquiring two images, one with nonuniform illumination (in our case, speckle) and another with uniform illumination (in our case, randomized speckle). An evaluation of the local contrast in the speckle-illumination image provides an optically sectioned image with low resolution. This is complemented with high-resolution information obtained from the uniform-illumination image. A fusion of both images leads to a full resolution image that is optically sectioned across all spatial frequencies. This hybrid illumination method is fast, robust, and generalizable to a variety of illumination and imaging configurations.     

Wide-field imaging through scattering media by scattered light fluorescence microscopy

To obtain images through scattering media, scattered light fluorescence (SLF) microscopy that utilizes the optical memory effect has been developed. However, the small field of view (FOV) of SLF microscopy limits its application. In this paper, we have introduced a re-modulation method to achieve wide-field imaging through scattering media by SLF microscopy. In the re-modulation method, to raster scan the focus across the object plane, the incident wavefront is re-modulated via a spatial light modulator (SLM) in the updated phase compensation calculated using the optimized iterative algorithm. Compared with the conventional optical memory effect method, the re-modulation method can greatly increase the FOV of a SLF microscope. With the phase compensation theoretically calculated, the process of updating the phase compensation of a high speed SLM is fast. The re-modulation method does not increase the imaging time. The re-modulation method is, therefore, expected to make SLF microscopy have much wider applications in biology, medicine and physiology.     

Single-exposure optical sectioning by color structured illumination microscopy

Structured illumination microscopy (SIM) is a wide-field technique that rivals confocal microscopy in optical sectioning ability at a small fraction of the acquisition time. For standard detectors such as a CCD camera, SIM requires a minimum of three sequential frame captures, limiting its usefulness to static objects. By using a color grid and camera, we surpass this limit and achieve optical sectioning with just a single image acquisition. The extended method is now applicable to moving objects and improves the speed of three-dimensional imaging of static objects by at least a factor of three.     

Comment on "Wide-field coherent anti-Stokes Raman scattering microscopy with non-phase-matching illumination"

We comment on a Letter by Toytman et al. [Opt. Lett.32, 1941 (2007)] in which a novel setup for wide-field imaging in coherent anti-Stokes Raman scattering (CARS) microscopy is demonstrated. There the authors state that our phase-matching implementation of a wide-field CARS system [Appl. Phys. Lett.84, 816 (2004); New J. Phys.8, 36 (2006)] suffers from a strong background from the bulk medium. However, our results show quite the contrary, i.e., that our setup provides a very good signal contrast, due to an almost vanishing background level from the bulk solvent.     

Laser-tweezer-controlled solid immersion microscopy in microfluidic systems

We describe the creation and implementation of a near-field scanning solid immersion microscope that is specifically tailored for use in microfluidic systems. The microscope comprises a newly fabricated Weierstrass solid immersion lens (SIL), which is detached from its substrate and is free floating in the fluid, and a laser optical tweezer, which serves both as a trapping beam for alignment and positioning of the SIL and as a near-field scanning beam that images the sample through the SIL. A discussion of the SIL's fabrication method is presented along with experimental results that demonstrate the effectiveness of our microscope design.     

Resolution enhancement in digital holographic microscopy with structured illumination

We present a digital holographic microscope wherein the sample is illuminated by structured light to enable the capture of additional object spatial frequencies. Reconstructed images with increased spatial resolution are obtained by separating and synthesizing bandwidths of different frequency regions in the Fourier domain. The theoretical analysis and experimental results are presented.     

超半球形固体浸没透镜系统的亚表面显微

应用矢量衍射理论和薄膜光学方法,研究了有禁带存在时超半球形固体浸没透镜(SIL)系统的亚表面显微。模拟计算结果显示,光斑的强度,边瓣强度和光斑大小随禁带厚度发生振荡。光斑的大小和光斑的强度几乎是反相变化的。当禁带材料的折射率与固体浸没透镜(SIL)的折射率相匹配时,光斑的强度较大,分辨率较高。随着离开样品表面的距离的增加,光斑的强度不断减小,光斑的大小和边瓣强度逐渐增加。     

Full-wavelet approach for fluorescence diffuse optical tomography with structured illumination

We present a fast reconstruction method for fluorescence optical tomography with structured illumination. Our approach is based on the exploitation of the wavelet transform of the measurements acquired after wavelet-patterned illuminations. This method, validated on experimental data, enables us to significantly reduce the acquisition and computation times with respect to the classical scanning approach. Therefore, it could be particularly suited for in vivo applications.     

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.     

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.     

结合虚拟单像素成像解卷积的双边照明光片荧光显微技术

光片荧光显微术(light-sheet fluorescence microscopy,LSFM)采用薄片光束从侧面激发样品,在垂直于光片方向上进行成像,具有成像速度快、光学层析能力强以及光漂白和光毒性低等优点,适用于对较大活体生物样品进行高质量、长时间三维动态观测.然而,传统高斯光束LSFM存在分辨率低和成像视场小的...     

ESPI with structured illumination

In practical ESPI applications in industry, the object under investigation often has low reflectance. From theoretical analysis, it is shown here that the implementation of phase shifting techniques will result in a smaller fraction of acceptable measurements for a given level of tolerable phase error. The use of higher power lasers may solve this problem; but it is an expensive solution. In this work, structured lighting using diffractive optical elements is proposed as a cost-effective solution. The approach is particularly useful when the deformation phase has to be obtained from only selected areas on the object. A simple experiment conducted verifies the workability of this approach.     

散斑照明宽场荧光层析显微成像技术研究

介绍了一种新的宽场荧光层析显微方法.在传统宽场显微镜中引入散斑图案照明样品,控制散斑图案的动态变化,利用CCD相机记录对应的一系列荧光图像.由于焦平面内强度变化远比焦平面外强度变化剧烈,通过合适的算法能够获得焦平面的层析分辨的荧光显微图像.标定了系统参数,并研究了不同的图像重建算法对系统性能的影响,获得了不同生物组织样品的层析图像.实验表明,该显微方法能用于组织光学切片成像,在临床医学中具有实际应用价值. 关键词： 荧光 散斑照明 荧光显微 层析     

Three-dimensional superresolution in two-color excitation fluorescence microscopy using theta illumination method

In two-color excitation fluorescence (2CE) theta microscopy, the point spread function (PSF) of the system is the product of the two PSFs of both excitation beams and due to the ellipsoidal shape of the PSFs, the axial width of the PSF of one excitation beam is strongly reduced after multiplication with the lateral section of another PSF in nonconfocal theta microscopy. Taking the advantages of this setup, we propose using two leaky filters inserted in the two illumination arms, respectively, to compress its own lateral width of each PSF; it means, the lateral and axial superresolution (three-dimensional superresolution) of the whole system in 2CE theta microscope with two orthogonal illumination beams are realized.