相干反斯托克斯拉曼散射显微成像技术及其在生物医学领域的应用

相干反斯托克斯拉曼散射（ CARS）成像技术是一种具有高信号强度、高化学选择性、无需外源标记的显微成像新技术。本文在简介CARS成像基本原理的基础上,综述了CARS显微成像技术的发展、与其它技术相结合等研究新方向,以及在生物医学领域的应用进展。     

基于深度学习的多分辨显微关联成像系统设计

显微成像技术作为研究细胞和生物组织的主要工具,对生物医学的发展起到了极大的推动作用。生物样本的复杂化和生物医学领域对时间和空间分辨率的多样化需求决定了单一功能生物成像系统应用的局限性。为满足生物医学领域的多样化需求,解决成像质量与成像时间之间的矛盾,设计了一种基于深度学习的多分辨显微关联成像系统。该系统通过对显微镜进行硬件设计改造和软件处理,将深度学习与关联成像技术有效结合,当采样率仅为60%时,成像系统能够较好地恢复图像细节,大幅降低欠采样带来的噪声,同时显著提升系统成像的时间分辨率。另外,为了满足所设计的小型多分辨显微关联成像系统的实际需求,采用基于重参数化思想的超高效轻量超分网络,在资源受限的设备下实现实时高质量成像。所提出的成像系统可以在保证成像质量的同时显著缩短成像时间和减少内存占用。不同类型生物样本和分辨率板的测试结果进一步表明了系统的鲁棒性和抗噪性能,研究结果对生物医学领域具有重要意义。     

超分辨显微技术在活细胞中的应用与发展

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。     

光谱及光谱成像技术在生物医学领域应用简介

光谱技术和光谱成像技术因其可以获取观测目标的化学组分信息,在生物医学领域具有极广阔的应用前景。介绍了光谱及光谱成像技术在生物医学领域七种应用的最新进展。其中,荧光多标记共定位、染色体核型分析及眼底病变检测技术已成熟,并已进入临床应用;光谱成像癌变检测、生物芯片检测、无损血糖检测及拉曼癌变检测四类应用虽然临床意义重大,但因检测精度较低或普适性较差等原因,处于发展初期阶段,仍需开展进一步研究。     

相干拉曼散射显微技术及其在生物医学领域的应用

相干拉曼散射显微技术作为一种新型的成像技术,具有无标记、高特异性、非侵入等优点,已被广泛用于化学结构及物质成分分析。近年来,光子学、生物医学和显微成像技术等领域的相互交叉和融合发展,极大地推动了相干拉曼散射显微成像技术在生物医学领域的应用。简要介绍了相干拉曼散射显微成像的基本原理、分类、系统构成,同时概述了相干拉曼散射显微成像技术近年来在生物医学领域的应用,包括检测、脂类分析和蛋白质构象变化等,最后对其未来发展进行了展望。     

激光微束显微切割植物染色体的研究

激光微束是六十年代激光技术问世后在生物学和医学领域中出现的一项新技术。该技术利用激光方向性好、光色单一和亮度高等独一无二的特点,把激光束通过光路系统引入显微镜并聚焦成很小的光点对细胞或细胞器进行精细的细胞或亚细胞水平的显微外科术。染色体是细胞核里一种极重要的结构。每一种真核生物都有一定数目的染色体;每条染色体上都有排列次序一定的基因。任何染色体数目的变化和染色体上基因排列次序的变化,都会引起生物遗传性的变异。激光微束遗传操作真核生物染色体是激光生物学中重要和活跃的研究课题。Berns等以两栖类和哺乳动物细胞为材料进行了激光显微照射染色体及其子细胞遗传分析的大量研究。梁宏等用氩离子激光显微切割长鼻(鼠菐)培养细胞有丝分裂染色体或使受照染色体区域DNA失活都取得了成功。Monajembashi和Cremer建立了用激光微束显微切割人淋巴细胞染色体的技术。本文报道用激光微束切割高等植物染色体的实验结果,其目的是建立起对农作物染色体进行激光遗传操作的有效技术方法。     

蛋白酶荧光探针及新型显微成像技术的生物医学应用

研究上正常生理条件蛋白酶的活化情况仍然存在很多困难,将蛋白酶荧光探针技术和基于各种光学平台的显微成像技术有机地结合起来,可以最大限度地记录活细胞生理条件下蛋白酶活性变化的时空信息.综述了蛋白酶荧光探针技术及其与该类探针应用相关的新型显微成像技术在生物医学光子学领域的应用进展.     

光学投影断层成像技术及其研究进展

光学投影断层成像技术是一种新颖的针对毫米至厘米量级的介观尺寸的三维荧光成像技术,具有经济、快速、分辨率高和成像范围广等优异性能,是当今生物医学光子学领域的研究热点之一。综述了光学投影断层成像的技术原理、发展研究现状以及其广泛的应用领域,阐述了不同光学投影断层成像技术的特点。目前光学投影断层成像技术的发展主要集中在系统自身(包括焦平面扫描、角度复用、角度滤波等方法)的改进、后期图像重组算法的改进以及该技术结合多维度荧光成像技术三个方面。通过技术细节的改进和发展,光学投影断层成像在生物医学、组织形态学和组织病理学、活体成像和荧光标记追踪等研究领域获得广泛应用。     

Perkin Elmer公司推出激光诱导活细胞快速示踪装置

Perkin Elmer公司最近推出一种激光诱导活细胞动态过程快速示踪装置（Photo Kinesis）。该装置可附加在著名的Ultra View ERS或RS系列活细胞高速成像系统上。采用该装置，可完成活细胞样本上多达100个目标区域（ROI，region of interest）的光漂白后荧光恢复（FRAP，fluorescence recovery after photobleaching）、     

扫描离子电导显微镜的研制与实现

在微纳米尺度上对活细胞高分辨率成像对生命科学研究具有重要的意义,其将有助于再现正在发生的生命过程、检测细胞对外界刺激做出的响应,甚至观测某些蛋白簇在细胞膜表面的运动。然而直到今天,仍然没有很好的实现上述目标。扫描离子电导显微镜（SICM）由于其真正的非接触、高分辨、无损独特成像方式,规避了扫描过程中探针与样品表面发生力的接触,得到越来越多的关注和广泛的应用。从系统的角度阐述自制SICM 系统的设计、硬件集成及跳跃模式扫描算法的实现,并通过对聚二甲基硅氧烷（PDMS）栅格成像以及与原子力显微镜（AFM）成像结果的对比,验证了系统功能的正确性和有效性;最后开展了生理环境下活体细胞的原位扫描成像实验,初步获取了活体神经细胞轴突结构的三维形貌图像。SICM 的成功搭建,将为人们深入了解生理条件下活体生物样品表面微观结构与功能机理等提供有效的研究方法与手段。     

A Smart DNA Nanoassembly Containing Multivalent Aptamers Enables Controlled Delivery of CRISPR/Cas9 for Cancer Immunotherapy

CRISPR/Cas9 system is promising for the reversal of tumor immunosuppression in immunotherapy, but the controlled delivery of CRISPR/Cas9 remains challenging. Herein, the study reported a smart DNA nanoassembly containing multivalent aptamers, realizing the controlled delivery of Cas9/sgRNA ribonucleoprotein (RNP) for enhanced cancer immunotherapy. A single-stranded DNA complementary to sgRNA in the Cas9/sgRNA RNP can initiate a cascade-clamped hybridization chain reaction (C-HCR) to wrap the Cas9/sgRNA RNP up in the DNA nanoassembly. After selective internalization of DNA nanoassembly by cancer cells, Cas9/sgRNA RNP is released to cytoplasm in response to endogenous RNase H and enters the nuclei to knock out β-catenin. The expression of the programmed death-ligand one gene is effectively suppressed, and the immunosuppressive tumor microenvironment is reprogrammed. Meanwhile, the migration of cancer cells is inhibited, and the apoptosis of cancer cells is promoted. In a breast cancer mouse model, the administration of DNA nanoassembly effectively increased the infiltration of CD8⁺ T cells, eventually achieving high therapeutic efficacy.     

Targeted Delivery of CRISPR/Cas9‐Mediated Cancer Gene Therapy via Liposome‐Templated Hydrogel Nanoparticles

Due to its simplicity, versatility, and high efficiency, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has emerged as one of the most promising approaches for treatment of a variety of genetic diseases, including human cancers. However, further translation of CRISPR/Cas9 for cancer gene therapy requires development of safe approaches for efficient, highly specific delivery of both Cas9 and single guide RNA to tumors. Here, novel core–shell nanostructure, liposome‐templated hydrogel nanoparticles (LHNPs) that are optimized for efficient codelivery of Cas9 protein and nucleic acids is reported. It is demonstrated that, when coupled with the minicircle DNA technology, LHNPs deliver CRISPR/Cas9 with efficiency greater than commercial agent Lipofectamine 2000 in cell culture and can be engineered for targeted inhibition of genes in tumors, including tumors the brain. When CRISPR/Cas9 targeting a model therapeutic gene, polo‐like kinase 1 (PLK1), is delivered, LHNPs effectively inhibit tumor growth and improve tumor‐bearing mouse survival. The results suggest LHNPs as versatile CRISPR/Cas9‐delivery tool that can be adapted for experimentally studying the biology of cancer as well as for clinically translating cancer gene therapy.     

Magnetic-Activated Nanosystem with Liver-Specific CRISPR Nonviral Vector to Achieve Spatiotemporal Liver Genome Editing as Hepatitis B Therapeutics

Chronic hepatitis B infection remains incurable due to the stable presence of various forms of hepatitis B virus (HBV) genome, especially the HBV covalently closed circular DNA (cccDNA). The emergence of clustered regularly interspaced short palindromic repeat (CRISPR) technology provides a new opportunity to potentially cure the HBV infection. However, the efficiency and specificity remain unsatisfactory, especially for nonviral CRISPR/Cas9 delivery. To tackle these, a liver-specific CRISPR/Cas9 magnetic nanosystem FMNP_pAG333/sgXPP is constructed based on fluorinated polyethylenimine-coated magnetic nanoparticles and liver-specific ApoE.HCR.hAAT promoter-driven Cas9-T2A-EGFP plasmid with dual sgRNAs. The elaborate system enables magnetic field-induced spatially specific distribution and hepatocyte-specific promoter-driven liver-specific gene editing. Moreover, this CRISPR/Cas9 magnetic nanosystem is designed to disrupt the two conserved sites in X opening reading frame and Pol opening reading frame of the HBV genome, thereby significantly inactivating the HBV genome without showing off-target effects. Treatment with FMNP_pAG333/sgXPP for 7 days reduces serum HBsAg levels by 76% with a total editing efficiency of ≈20% in the two conserved sites. Collectively, this study demonstrates spatiotemporal liver genome editing as well as the feasibility of applying a nonviral CRISPR/Cas9 vector for HBV treatment, which may open up a new application for CRISPR therapeutics.     

Precise RNA Editing: Cascade Self-Uncloaking Dual-Prodrug Nanoassemblies Based on CRISPR/Cas13a for Pleiotropic Immunotherapy of PD-L1-Resistant Colorectal Cancer

CRISPR/Cas13a is a powerful genome editing system for RNA knockdown that holds enormous potential for cancer treatment by targeting currently undruggable oncogenes or immune checkpoints. However, the precise intratumoral activation of CRISPR/Cas13a to maximize the therapeutic efficiency while guaranteeing biosafety remains a daunting challenge. Here, a cascade self-uncloaking nanoassembly (SRC) based on a dual-prodrug comprising SN38 and Cas13a/RNP is developed, and the external encapsulation is performed by coating with a ROS-responsive probe, which is stimulated by the tumor microenvironment to achieve the efficient NIR-II imaging by CH10055 due to disaggregation into single molecules, while the second release of prodrug in the hypoxic environment enables targeted controlled release. SN38 not only induces immunogenic cell death (ICD), but significantly combats the immunosuppressive microenvironment of colorectal cancer in combination with the RNA editing targeting the novel immune checkpoint TIM3 to regulate the cGAS-STING pathways, resulting in synergistic activation of both innate and adaptive immunity. The treatment of SRC exhibits a tenfold increase in tumor regression of α-PD-L1 in PD-L1-resistant orthotopic and xenograft models by inducing effective tumor immune infiltration. These results demonstrate the feasibility of using CRISPR/Cas13a in cancer treatment, and SRC holds immense promise as a neoadjuvant strategy for enhancing CRC immunotherapy.     

Cascade-Responsive “Oxidative Stress Amplifiers” Simultaneously Destroy Lysosomes and Co-Deliver CRISPR/Cas9 to Enhance Oxidative Damage in Tumor

Amplifying intracellular oxidative stress by organelle-targeted reactive oxygen species (ROS) production combined with tumor cell-specific gene disruption is a promising strategy for tumor treatment. However, due to the vulnerability of CRISPR/Cas9 ribonucleoproteins (RNPs) to ROS, co-delivery of CRISPR/Cas9 RNPs and ROS generators to enhance the sensitivity of tumor cells to oxidative stress remains challenging. Herein, a cascade-responsive “oxidative stress amplifier” (named DR-TAF-pHT/FA) is proposed, which can successively respond to cathepsin B, localized laser irradiation and ATP to generate ROS on the lysosomal membrane of tumor cells and release Cas9/sgNrf2 RNPs for efficient gene disruption. It is demonstrated that, under near infrared (NIR) irradiation, DR-TAF-pHT/FA achieves targeted rupture of lysosomal membranes, inducing significant intracellular oxidative stress. Meanwhile, due to the protective function of TAF coating (TA-Fe³⁺ coordination self-assembled networks), Cas9/sgNrf2 RNPs can safely escape into the cytoplasm and be released in response to ATP, further amplifying oxidative stress and promoting tumor cell apoptosis through efficient Nrf2 gene disruption. Treatment with DR-TAF-pHT/FA + NIR significantly improves tumor ablation efficiency and extends median survival time (over 70 days) in Hela xenograft models. This “oxidative stress amplifier” provides a new paradigm for multimodal and synergistic tumor therapy through precise lysosomal membrane bursting together with efficient Nrf2 gene disruption.     

In Vitro Prostate Cancer Treatment via CRISPR-Cas9 Gene Editing Facilitated by Polyethyleneimine-Derived Graphene Quantum Dots

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 (CRISPR associated protein 9) is a programmable gene editing tool with a promising potential for cancer gene therapy. This therapeutic function is enabled in the present study via the non-covalent delivery of CRISPR ribonucleic protein (RNP) by cationic glucosamine/PEI-derived graphene quantum dots (PEI-GQDs) that aid in overcoming physiological barriers and tracking genes of interest. PEI-GQD/RNP complex targeting the tumor protein 53 (TP53) gene mutation overexpressed in ∽50% of cancers successfully produces its double-stranded breaks in solution and in prostate cancer (PC-3) cells. Restoring this cancer “suicide” gene can promote cellular repair pathways and lead to cancer cell apoptosis. Its repair to the healthy form performed by simultaneous PEI-GQD delivery of CRISPR RNP and a gene repair template leads to a successful therapeutic outcome: 40% apoptotic cancer cell death, while having no effect on non-cancerous (HeK293) cells. The translocation of PEI-GQD/RNP complex into PC-3 cell cytoplasm is tracked via GQD intrinsic fluorescence, while enhanced green fluorescent protein (EGFP)-tagged RNP is detected in the cell nucleus, showing the successful detachment of the gene editing tool upon internalization. Using GQDs as non-viral delivery and imaging agents for CRISPR-Cas9 RNP sets the stage for image-guided cancer-specific gene therapy.     

基于低秩矩阵近似的低噪宽幅热红外成像技术

为解决强背景弱信号场景下热红外成像系统噪声制约图像信噪比的问题,提出了一种基于低秩矩阵近似理论的低噪宽幅热红外成像技术.利用面阵摆扫方式实现宽幅扫描成像并构建严格的观测矩阵,通过加权核范数最小化方法求解去噪的低秩矩阵形式.试验证明该技术具有较高的峰值信噪比与降噪鲁棒性,在宽幅成像的同时也提高了探测灵敏度.研究成果在红外弱目标识别、广域侦查等领域具有一定应用价值.     

星源照射双/多基地SAR成像

星源照射双/多基地合成孔径雷达(SAR),采用卫星发射,卫星、临近空间、飞机、地面等平台接收,实现对地海面场景和目标的高分辨成像。该技术具有可成像范围广、隐蔽性好、抗干扰能力强等优点,且可以通过波束调控实现扫描、聚束、滑动聚束等多种组合成像模式,从而获取更加丰富的成像信息,具有十分广阔的民用和军事应用前景。目前,国内外针对星源照射双/多基地SAR成像技术开展了多年的研究,积累了诸多研究成果。该文分别从系统组成、构型方法、回波模型、成像方法、收发同步与试验验证等方面对该技术进行阐述与分析,同时对相关的研究工作进行较系统的回顾,并展望了星源照射双/多基地SAR成像技术未来的发展方向。     

A 128-kb FeRAM macro for contact/contactless smart-card microcontrollers

This paper describes a 128-kb FeRAM macro for smart-card microcontrollers. This macro, which was designed and fabricated using a 0.35-/spl mu/m three-metal CMOS and a Capacitor-on-Metal/Via-stacked-Plug (CMVP) process technology, is ideally suited for recent system LSIs such as smart-card microcontrollers. It has a flexible memory size ranging from 32 to 128 kb, a low consumption current of 0.3 mA, and endurance of more than 10/sup 8/ write/read cycles under a wide range of supply voltages, from 2.7 to 5.5 V. These characteristics, which are required of not only contact-type smart-card microcontrollers but also contactless-type ones, were achieved by using four newly developed circuit technologies: 1) a three-metal CMVP memory cell; 2) a voltage-regulating architecture; 3) a main/sub bitline and wordline structure; and 4) a dynamic-type offset sense amplifier.