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Optical fiber integrated unlabeled differential super-resolution microscopic imaging system
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摘要:
得益于荧光标记技术，远场超分辨显微成像技术的不断发展为人们打开了一道微观世界的大门。远场超分辨光学显微镜也成为现代医学、生命科学等领域研究发展的重要工具和设备。但是相比之下，远场无标记显微成像技术发展却相对缓慢。为此，本文提出了一种采用光纤器件的集成化差分显微成像方法。该方法通过特制的光纤模式选择耦合器(MSC)实现了差分成像系统中空心光斑的生成，并解决了空心光斑和实心光斑严格对准困难的问题。搭建了一套结构简单紧凑，高度集成化的无标记显微成像系统。实验中，采用直径为150 nm的金颗粒和最小间距约50 nm的无标记聚合物线对结构对系统进行成像测试，分辨率相较传统扫描共聚焦显微镜大幅提升。
Abstract:Far-field super-resolution microscopic imaging technology based on fluorescent labels opened a gate to the microscopic world, which has become an important tool in the research of modern medicine and life science. However, the development of far-field unlabeled super-resolution microscopy is relatively slow. Here, an integrated differential microscopic imaging method using optical fiber devices is proposed in this article. The generation of hollow spots in the differential imaging system is realized by a special fiber mode selection coupler (MSC). The problem of strict alignment between hollow and solid spots is naturally solved in this method. A highly integrated label-free microscopic imaging system was established. In experiments, gold particles with a diameter of 150 nm and unlabeled polymer lines with a minimum spacing of about 50 nm were imaged to test the imaging system. The resolution of the imaging system shows great improvement compared to conventional scanning confocal microscopy.
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Overview: Fluorescence emission difference (FED) microscopy was first applied to fluorescent samples. However, given its basic principle, this microscopy also can perform super-resolution imaging of non-fluorescent samples. In this method, solid and hollow light spots of the same wavelength are used to image the samples respectively. An image with super-resolution details can be achieved through data processing of the two images. In traditional methods, the generation and modulation of hollow and solid light spots are based on a spatial light modulator or vortex phase plate and multiple groups of spatial optical devices, resulting in a complex system that is susceptible to external interference.
In this article, we proposed a modified FED microscopy based on a special Optical Fiber Mode Selection Coupler (MSC). The coupler has two input ports: Gaussian light beam input through one of the input ports will be converted into a vortex beam which has circular optical field distribution; Gaussian light beam input through the other input port will be converted into the fundamental mode of the fiber which remains Gaussian optical field distribution. Since the two output beams are emitted through the same fiber, the two beams naturally propagate along the same optical axis in the subsequent optical path, which solved the problem of strict alignment between hollow spot and solid spot in a traditional FED microscopy system.
In the MSC-FED system, a continuous laser beam at a wavelength of 532 nm was emitted by a semiconductor laser. The beam was firstly divided into two channels through a single-mode fiber 1×2 coupler. Two Acousto-Optic Modulators (AOM) were integrated into two fiber optical paths to achieve high-speed switching control with rising/falling edge times of less than 10 ns. After passing through AOM, light in the two channels incident into two input ports of the MSC respectively. By the switch control of AOM, light beams in two channels alternately output solid spot and hollow spot from the special MSC. The solid spot or hollow spot was used to scan the sample through the subsequent optical elements in the system.
In experiments, gold particles with a diameter of 150 nm and unlabeled polymer lines with a minimum spacing of about 50 nm were imaged to test the imaging system. The resolution of the imaging system shows great improvement compared to conventional scanning confocal microscopy.
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图 1 光纤模式选择耦合器原理图。当基模形式激光分别从(a)单模光纤，以及(b)少模光纤输入端输入MSC时，单模光纤输出端与少模光纤输出端分别输出的光场分布形式
Figure 1. Schematic diagram of the fiber mode selection coupler. Output of the single-mode fiber and the few-mode fiber, when the fundamental mode laser is separately launched into (a) the single-mode fiber, and (b) the few-mode fiber
图 2 对于532 nm工作波长，单模光纤中LP01模式和少模光纤中LP11模式的等效折射率与纤芯直径的对应关系
Figure 2. Mode effective index curves for the LP01 mode in SMF and the LP11 mode in FMF as functions of core radius, at the wavelength of 532 nm
图 3 在MSC的耦合区域中，单模光纤中的LP01模式和少模光纤中的LP11模式周期性转化规律的仿真图
Figure 3. Simulation of periodic transformation between the LP01 mode in the single-mode fiber and the LP11 mode in the few-mode fiber, in the coupling region of MSC
图 4 在满足匹配条件的情况下，宽谱激光从MSC的单模光纤输入端注入时，从单模光纤输出端(黑色曲线)和少模光纤输出端(红色曲线)输出的光谱图。在工作波长532 nm下，绝大部分功率从少模光纤端输出
Figure 4. Spectrum diagram of the output of the single-mode fiber (black curve) and the output of the low-mode fiber (red curve) when the wide-spectrum laser is injected from the single-mode fiber input of the MSC under the matching conditions. At the working wavelength of 532 nm, most of the power output is from the few-mode fiber end
图 5 基于光纤MSC的FED成像系统原理图
Figure 5. Schematic diagram of the FED imaging system based on the fiber MSC
图 6 光纤MSC中少模光纤输出端支持的前两阶本征标量模式的光场分布图。(a) LP01模式；(b) LP11模式
Figure 6. Optical field distribution of the first two order eigenmodes supported by the LFM output end of MSC. (a) LP01 mode; (b) LP11 mode
图 7 基于光纤MSC的FED成像系统中不同加权系数下，在MSC输出口获得的实心光斑(紫色虚线)、空心斑光场(蓝色虚线)、等效合成光斑光场(红色实线)分布图。(a) r=0.5；(b) r=0.7；(c) r=1
Figure 7. Optical field distribution of solid spot (purple dashed line), hollow spot (blue dashed line), and equivalent spot (red solid line) in the FED imaging system based on the fiber MSC with different weighting coefficients, obtained at the output end of MSC. (a) r =0.5；(b) r =0.7；(c) r =1
图 8 应用基于光纤MSC的FED系统对金颗粒的扫描成像结果。(a)通过实心光斑扫描；(b)通过环形光斑扫描；(c)将图(a)和图(b)进行加权相减后获得的金颗粒样品像。图(a1-c1)、(a2-c2)分别为图(a-c)中两个局部的放大图
Figure 8. Imaging result of gold particles using the FED system based on the fiber MSC. (a) Solid spot scanning; (b) Donut-shaped spot scanning; (c) Image of gold particle samples obtained by weighted subtraction of Figs. (a,b). Figures (a1-c1) and (a2-c2) are enlarged views of two different areas in Figs. (a-c), respectively
图 9 应用基于光纤MSC的FED系统对纳米级线对的扫描成像结果。(a)通过实心光斑扫描；(b)通过环形光斑扫描；(c)通过将图(a)和图(b)进行加权相减后获得的纳米线对样品像。图(a1-c1)，分别为图(a-c)中局部的放大图
Figure 9. Imaging result of nano-scale wire pairs using the FED system based on fiber MSC. (a) Solid spot scanning; (b) Donut-shaped spot scanning; (c) Image of nano-scale wire pairs obtained by weighted subtraction of Figs. (a, b)
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