基于涡旋光照明的暗场数字全息显微方法研究

提出了一种基于涡旋光照明的暗场数字全息显微方法. 从理论上阐述了涡旋光环形照明原理和暗场数字全息显微原理, 分析了涡旋光的准无衍射特性对成像的影响; 搭建了相应的数字全息显微成像系统, 采用690 nm的聚苯乙烯小球作为实验样品; 最后通过对小球明暗场下数字全息显微再现像的分析对比, 证明该方法可以有效地提高数字全息系统的分辨率, 同时增强了再现像的对比度. 关键词： 全息 暗场数字全息显微 涡旋光 分辨率     

Lensless digital holographic microscope with light-emitting diode illumination

We demonstrate the operation of a digital in-line microscope with LED illumination. We show with a practical example that, for typical setups, the limited temporal coherence and the spatial incoherence of the source do not affect the resolving power of the system. On the contrary, important advantages are obtained in terms of signal-to-noise ratio and alignment simplification.     

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.     

Full off-axis red-green-blue digital holographic microscope with LED illumination

We developed a new full off-axis red-green-blue (RGB) digital holographic microscope with an LED illumination. A decisive advantage of the use of LED illumination is a large image quality improvement due to its partially coherent nature. The off-axis configuration enables the fast recording of the holographic data in each spectral channel. The digital holographic refocusing and the optical phase map computation are successfully demonstrated. The multiwavelength operation provides a significant improvement of the collected information for colored samples.     

Immersion digital in-line holographic microscopy

Digital in-line holographic microscopy is a promising new tool for high resolution imaging. We demonstrate, by using latex beads, that a considerable increase in numerical aperture, and, therefore, resolution can be achieved if the space between a source and a CCD camera chip is filled with a high refractive index medium. The high refractive index medium implies a shorter effective wavelength so that submicrometer resolution can be obtained with laser light in the visible range.     

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.     

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.     

预放大数字全息显微系统的特点

为了提高预放大数字全息显微系统的成像质量,采用理论分析与实验验证相结合的方法,分别对采用平面及球面参考光记录的预放大数字全息显微系统进行了研究和比较。结果表明：在通常的实验条件下,系统的横向分辨力主要取决于显微物镜的成像分辨力;记录距离较小时,两种系统的横向分辨力均随着记录距离的增大略有降低;但当记录距离较大时,球面参考光预放大数字全息系统的横向分辨力降低得更为明显,即平面参考光预放大数字全息显微系统较为优越;在记录距离为0的情况下,即像面数字全息成像情况下,两种系统的再现像均具有最高的分辨力,在利用普通工业用传感器条件下,横向分辨力远超过了2.19 m,且像质较好。因此,尽可能减小全息图的记录距离,或者采用像面数字全息系统,可以有效提高数字全息系统的成像分辨力。     

High-numerical-aperture microlens shape measurement with digital holographic microscopy

In this Letter, we introduce an algorithm that overcomes limitations in shape measurement by holographic microscopic methods in cases of micro-optical elements with high NA, such as microlenses. The presented algorithm provides a simple method for shape reconstruction from interferometrically measured phase. The algorithm is based on the analysis of local ray transition through the measured object. We develop algorithms for holographic configurations working in transmission and reflection. The accuracy of the developed algorithms is proved by experiments and extensive simulations. We present an experiment in a holographic Mach-Zehnder configuration where we have measured and successfully reconstructed the height distribution of spherical and cylindrical microlenses with NA up to 0.3.     

Color lensless digital holographic microscopy with micrometer resolution

Color digital lensless holographic microscopy with micrometer resolution is presented. Multiwavelength illumination of a biological sample and a posteriori color composition of the amplitude images individually reconstructed are used to obtain full-color representation of the microscopic specimen. To match the sizes of the reconstructed holograms for each wavelength, a reconstruction algorithm that allows for choosing the pixel size at the reconstruction plane independently of the wavelength and the reconstruction distance is used. The method is illustrated with experimental results.     

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...     

预放大数字全息显微系统的特点

为了提高预放大数字全息显微系统的成像质量,采用理论分析与实验验证相结合的方法,分别对采用平面及球面参考光记录的预放大数字全息显微系统进行了研究和比较。结果表明:在通常的实验条件下,系统的横向分辨力主要取决于显微物镜的成像分辨力;记录距离较小时,两种系统的横向分辨力均随着记录距离的增大略有降低;但当记录距离较大时,球面参考光预放大数字全息系统的横向分辨力降低得更为明显,即平面参考光预放大数字全息显微系统较为优越;在记录距离为0的情况下,即像面数字全息成像情况下,两种系统的再现像均具有最高的分辨力,在利用普通工业用传感器条件下,横向分辨力远超过了2.19 m,且像质较好。因此,尽可能减小全息图的记录距离,或者采用像面数字全息系统,可以有效提高数字全息系统的成像分辨力。     

Resolution enhancement of digital laser scanning fluorescence microscopy with a dual-lens optical pickup head

The resolution of the cell fluorescence image captured by a digital laser scanning microscopy with a modified dual-lens BD-ROM optical pickup head is enhanced by image registration and double sample frequency. A dual objective lens of red (655 nm) and blue (405 or 488 nm) laser sources with numerical apertures of 0.6 and 0.85 is used for sample focusing and position tracking and cell fluorescence image capturing, respectively. The image registration and capturing frequency are based on the address-coded patterns of a sample slide. The address-coded patterns are designed as a string of binary code, which comprises a plurality of base-straight lands and grooves and data-straight grooves. The widths of the base-straight lands, base-straight grooves, and data-straight grooves are 0.38, 0.38, and 0.76 μm, respectively. The numbers of sample signals in the x-direction are measured at every intersection point by intersecting the base intensity of the push–pull signal of the address-coded patterns, which has a minimum spacing of 0.38 μm. After taking a double sample frequency, the resolution of the measured cell fluorescence image is enhanced from 0.38 μm to the diffraction limit of the objective lens.     

Polarization phase shifting in digital holographic microscopy

In the present work we have made use of polarization phase shifting in digital holographic microscopy (DHM) for three dimensional phase profiling of transmissive and reflecting microscopic samples. The Mach–Zehnder arrangement with proper polarizing elements (polarizer-masked cube beam splitter, quarter wave plate and a linear polarizer) is used for recording the phase-shifted digital holograms. The suggested procedure is simple and accurate and obviates the need of piezo devices for phase shifting. The phase profile of the specimen is reconstructed from the holograms by using standard phase shifting algorithms.     

Multiframe full-field heterodyne digital holographic microscopy

Digital holographic microscopy using multiframe full-field heterodyne technology is discussed in which two acousto-optic modulators are applied to generate low-frequency heterodyne interference and a high-speed camera is applied to acquire multiframe full-field holograms. We use a temporal frequency spectrum analysis algorithm to extract the object's information. The twin-image problem can be solved and the random noise can be significantly suppressed. The relationship between the frame number and the reconstruction accuracy is discussed. The typical objects of microlenses and biology cells are reconstructed well with 100-frame holograms for illustration.     

Dual plane in-line digital holographic microscopy

We report a dual plane in-line digital holographic microscopy technique that exploits the method of subtraction of average intensity of the entire hologram to suppress the zero-order diffracted wave. Two interferograms are recorded at different planes to eliminate the conjugate image. The experimental results demonstrate successful reconstruction of phase objects as well as of amplitude objects. The two interferograms can be recorded simultaneously, using two CCD or CMOS sensors, in order to increase the acquisition rate. This enhanced acquisition rate, together with the improved reconstruction capability of the proposed method, may find applications in biomedical research for visualization of rapid dynamic processes at the cellular level.     

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.     

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.     

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.     

Phase reconstruction in lensless digital in-line holographic microscopy

The phase reconstruction in a digital in-line holographic microscopy is compared using two numerical reconstruction methods. The first method uses one Fourier transform and second one uses three Fourier transforms. It is shown that the latter method gives improved object phase reconstruction as compared to the former.