Hematologic malignancies are one of the most common malignant tumors caused by the clonal proliferation and differentiation of hematopoietic and lymphoid stem cells. The examination of bone marrow cells combined with immunodeflciency typing is of great signiflcance to the diagnostic type, treatment and prognosis of hematologic malignancies. Super-resolution fluorescence microscopy (SRM) is a special kind of optical microscopy technology, which breaks the resolution limit and was awarded the Nobel Prize in Chemistry in 2014. With the development of SRM, many related technologies have been applied to the diagnosis and treatment of clinical diseases. It was reported that a major type of SRM technique, single molecule localization microscopy (SMLM), is more sensitive than flow cytometry (FC) in detecting cell membrane antigens' expression, thus enabling better chances in detecting antigens on hematopoietic cells than traditional analytic tools. Furthermore, SRM may be applied to clinical pathology and may guide precision medicine and personalized medicine for clone hematopoietic cell diseases. In this paper, we mainly discuss the application of SRM in clone hematological malignancies.

为了进一步认知复杂环境中的细胞生物学过程, 研究人员发展了各种各样的生物成像技术。在这些技术中, 生物荧光成像因简单的成像条件以及对生物样品的相容性而得到了广泛的发展。然而, 传统的荧光成像技术受到了光学衍射极限的限制, 无法分辨低于200 nm的空间结构, 阻碍了对亚细胞结构的生物学过程研究。超分辨荧光显微镜技术突破了传统光学衍射对成像分辨率的限制, 能够获取纳米尺度的细胞动态过程。除了对传统的宽场荧光显微镜框架的改进及升级改造之外, 目前典型的超分辨成像显微镜技术通常依赖于荧光探针材料的光物理性质。常用的荧光探针材料包括荧光蛋白、有机荧光分子和纳米荧光材料等。本文介绍了几种主流的超分辨荧光显微成像技术并总结了已经成功应用到超分辨生物荧光成像中的荧光探针材料的应用进展。

Optical microscopy promises researchers to see most tiny substances directly. However, the resolution of conventional microscopy is restricted by the diffraction limit. This makes it a challenge to observe subcellular processes happened in nanoscale. The development of superresolution microscopy provides a solution to this challenge. Here, we briefly review several commonly used super-resolution techniques, explicating their basic principles and applications in biological science, especially in neuroscience. In addition, characteristics and limitations of each technique are compared to provide a guidance for biologists to choose the most suitable tool.

细胞是生命体的基本单位和功能单位, 对活细胞内部结构及其功能的研究是了解掌握生命本质的基础之一, 因此活细胞的实时观测对生命科学的发展具有重要意义。传统的光学显微技术受衍射极限的限制, 无法观测200 nm以下的生物结构细节。近20年来, 随着超衍射极限光学理论、技术、器件和荧光探针等方面的快速发展, 超分辨显微成像技术已成为应用于生命科学研究的重要手段。然而, 大多数超分辨显微方法或测量耗时长, 或易引起荧光蛋白漂白/细胞损伤, 在活细胞研究中受到极大限制, 已成为超分辨显微领域重点攻关的方向之一。为此, 文中结合作者在快速超分辨显微技术研究的基础上, 介绍了基于单分子成像的光激活定位显微技术和随机光学重构显微技术、基于荧光非线性可饱和光转换的受激发射显微技术以及基于结构光照明的超分辨显微技术, 并探讨了在活细胞成像中的发展应用。最后, 文中展望了超分辨显微成像技术在活细胞成像中的未来发展趋势。

受激发射损耗显微技术(STED)作为一种远场超分辨显微成像技术, 具有几十纳米甚至几纳米的空间分辨率, 是细胞生物学等研究领域的重要成像工具。圆环形空心损耗光在物镜焦点附近的光场强度分布对STED空间分辨率起决定性作用。在高数值孔径物镜聚焦下, 光场的偏振态会对聚焦光场的强度分布产生显著的影响, 此外, 显微系统的轴外像差会严重破坏空心损耗光焦斑的中心对称性。基于矢量衍射理论, 理论模拟了在高数值孔径物镜聚焦条件下, 入射涡旋光的偏振态和光学系统中的彗差和像散对空心损耗光焦场强度分布的影响。实验上使用纯相位型空间光调制器来校准光学系统相差, 优化变形的损耗光, 利用纳米探针扫描焦点区域, 测量了其焦场强度分布。测量结果与由矢量稍微理论观测的结果一致。

Far-field fluorescence microscopy has made great progress in the spatial resolution, limited by light diffraction, since the super-resolution imaging technology appeared. And stimulated emission depletion (STED) microscopy and structured illumination microscopy (SIM) can be grouped into one class of the super-resolution imaging technology, which use pattern illumination strategy to circumvent the diffraction limit. We simulated the images of the beads of SIM imaging, the intensity distribution of STED excitation light and depletion light in order to observe effects of the polarized light on imaging quality. Compared to fixed linear polarization, circularly polarized light is more suitable for SIM on reconstructed image. And right-handed circular polarization (CP) light is more appropriate for both the excitation and depletion light in STED system. Therefore the right-handed CP light would be the best candidate when the SIM and STED are combined into one microscope. Good understanding of the polarization will provide a reference for the patterned illumination experiment to achieve better resolution and better image quality.