自适应光学在双光子显微成像技术中的应用

光学显微镜是生物医学研究必不可少的工具，其中双光子显微成像技术具有大深度三维显微成像功能，被认为是深层生物组织研究的首选工具。但是，在双光子成像系统使用过程中，光学系统的装配偏差、光学元件不理想以及生物样品的不均匀性都会在成像过程中引入像差，从而降低成像质量。通过在双光子显微成像系统中引入自适应光学技术，可实现对像差的有效校正，从而提高成像的分辨率、深度和视场。介绍了双光子显微成像中的像差来源和特点，概述了自适应光学技术中不同的探测和校正方法，综述了近年来自适应光学技术在双光子显微成像中不同的应用成果，最后对自适应光学在双光子显微成像中的发展进行了展望。     

自适应光学高分辨率共聚焦显微成像技术

生物样品折射率的空间变化导致了光学畸变的产生,这种畸变对于共聚焦显微镜观察厚的生物样品和活体体内组织成像是一种严重的限制。自适应光学(AO)技术是通过快速反应的变形镜使镜面发生形变来补偿像差,在共聚焦显微镜中应用自适应光学技术可以校正光学畸变,观察深层组织活动,进行活体成像和实时检测。详细分析了共聚焦显微镜中像差的来源及光学畸变的特点,讨论了目前在共聚焦显微镜中自适应光学校正的方案及研究现状,讨论了共聚焦显微镜中自适应光学的波前传感器、畸变测量和波前校正器,并探讨了目前超高分辨率显微成像技术的发展方向。     

自适应宽场高分辨率显微成像技术的研究进展北大核心CSCD

光学显微成像技术可以用来观察微小物体的结构细节,但在生物样品的显微成像领域中,像差的存在使得任何显微成像技术的成像质量都无法达到理论预期。为了解决这一问题,自适应光学技术被应用于不同类型的显微成像系统中进行像差的探测和校正。着重总结了自适应宽场高分辨率显微成像技术的研究动态,阐明了数字全息自适应光学技术和非相干数字全息自适应光学技术的特点、优势以及存在的问题。     

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。     

生物医学光声显微成像:技术和应用进展

光声成像兼具光学成像对比度高和超声成像在深层生物组织中分辨率高等优点,是近年来迅速发展起来的一种生物医学成像模态。光声显微成像(PAM)是光声成像的一种重要实现方式,利用其可以无创提供活体生物组织结构和功能信息的优点,研究人员已开展了临床前和临床应用研究。为了使不同领域的研究人员了解这一快速发展的成像技术,本文综述了光声显微成像的发展现状、最新技术和研究进展。文章首先介绍了PAM的基本原理和典型的系统实现,然后概述了包括空间分辨率、成像深度、扫描方式、信号探测手段和多模态成像等方面的重要研究进展,接着阐述了PAM在生物医学领域的应用现状,最后总结了其未来发展面临的挑战。     

深组织光片荧光显微成像研究进展（特邀）

随着生物医学研究对复杂组织结构和功能的深入探索,高分辨率、高信噪比的深组织成像技术变得愈加重要。传统的显微镜技术往往局限于二维、透明的生物薄样本的观测,这在很大程度上无法满足当前生物医学领域对三维深组织体成像的研究需求。光片荧光显微镜凭借其低光损伤、高采集速率、大视场、体成像等优点被生物学家广泛使用。然而,生物组织固有的高散射特性仍然为深层成像带来了巨大的挑战。本文重点介绍了光片荧光显微成像技术在深组织成像领域的最新进展,特别是应对高散射样本挑战的解决策略,旨在为相关领域的研究人员提供有价值的参考,助力其对该前沿技术的最新进展和应用前景的理解。首先,阐述了光片荧光显微镜的基本原理和高散射吸收特性的形成原因及影响;然后,进一步阐明了增加组织穿透深度、应对光散射和吸收等问题的最新进展;最后,探讨了具有大穿透深度和强抗散射能力的光片荧光显微成像技术的发展前景以及潜在应用。     

计算光学成像在散射中的应用

自然界中普遍存在光散射现象。如何通过散射介质实现高分辨率成像是光学成像领域亟待解决的重要问题。在早期研究中,多重光散射被认为是雾霾、云层、生物组织等复杂介质成像中的障碍。然而,最近研究表明,散射并不是成像的基本限制:光子在经过多次散射后仍然包含了大量信息。为了深入了解新兴的计算光学成像是如何解决多重光散射问题的,文中主要介绍了波前整形、散斑相关及深度学习等方法在散射成像领域中的研究进展。最新的研究成果表明:波前整形可以实现动态散射介质内部的高分辨率快速聚焦;散斑相关能够利用单帧散斑实现非侵入式成像;基于深度学习的成像技术能恢复出隐藏在光学厚度为13.4的白色聚苯乙烯平板背后的物体。     

利用波前调制技术提升光透明样品的双光子成像分辨率

组织光透明技术结合双光子显微成像(TPM)技术能够有效提升生物样品的显微成像深度,然而现有的光透明剂与常用显微物镜的浸润介质折射率并不匹配,会引入球差从而降低深层组织成像的荧光强度和分辨率。针对该问题分析了球差对TPM荧光强度和分辨率的影响,建立了由物镜特性(数值孔径和浸润介质)、聚焦深度和物体折射率等参数构成的球差补偿模型,进而指导空间光调制器进行球差补偿。对荧光小球仿体样品和光透明脑组织样品的双光子成像结果显示,该球差补偿方法能显著提升样品信号量和系统纵向分辨率。另外,该方法在校正过程中无需多次成像,操作简单且耗时短,对光透明剂和显微物镜无特殊要求,具有较强的通用性。     

可抑制生物组织散射效应的光学聚焦技术研究进展

生物组织对光的散射使得光束通过透镜后无法在组织的深层(大于1 mm处)聚焦,制约了需要光能聚焦的成像技术(如共聚焦显微、双光子显微)在生物医学领域的应用。为了抑制生物组织的散射效应,将光聚焦到深层组织,需要对入射光的波前进行调制。基于此要求,以下三种光学聚焦技术得以提出并发展:用待聚焦区的光强作为反馈信号的波前整形技术;将声光调制和时间反演(或光学相位共轭)技术相结合进而在散射介质内部实现光学聚焦的技术;对散射介质传输矩阵进行测量的光学聚焦技术。本文对上述光学聚焦技术的研究进展进行了综述,比较并展望了其在生物医学领域中的应用前景。     

自适应光学相干层析在视网膜高分辨成像中的应用

视网膜光学相干层析（OCT）技术利用外部低相干光源照射人眼眼底,并将人眼眼底散射信号进行干涉成像,获得人眼视网膜的断层图像信息,以实现人眼视网膜无创、实时、在体的光学活检。传统光学相干层析在视网膜成像时的轴向分辨率可达3 μm以上,但由于人眼个体差异和不可避免的像差限制了视网膜OCT的横向分辨率,只能达到约15~20 μm。而自适应光学作为一项波前校正的先进技术,可以校正OCT色差以及人眼有限视场和眼球运动导致的像差,将OCT横向分辨率提高到低于2 μm,以实现视网膜细胞及微细血管近衍射极限成像,及时发现患者眼底存在的早期病变。在介绍自适应光学和视网膜光学相干层析的技术特点基础上,对自适应光学在视网膜光学相干层析成像应用的国内外发展现状进行了论述,总结了自适应光学OCT视网膜高分辨成像在宽带光源色差校正、眼球运动伪影减少、自适应光学视场扩大和波前传感与校正系统简化的关键技术和未来发展趋势,以实现大视场、高效率、高灵敏度、高分辨率的高速人眼视网膜成像,为未来自适应光学OCT视网膜成像技术的研究和应用提供参考和借鉴。     

A Non‐Mulberry Silk Fibroin Protein Based 3D In Vitro Tumor Model for Evaluation of Anticancer Drug Activity

The limitations of clinical chemotherapy are credited primarily to drug resistance. Effective development and screening of new drugs require appropriate in vitro tumor models that resemble the in vivo situation to evaluate drug efficiency and to decrease the use of experimental animals. 3D in vitro model systems that are able to mimic in vivo microenvironments are now highly sought after in cancer research. Here, the characteristics of breast cancer cell line MDA‐MB‐231 cells on 3D, and 2D Antheraea mylitta silk matrices and tissue culture plates are compared. After long term culture of breast cancer cells in the silk scaffold, the engineered tumor construct shows different zones of cell proliferation, such as an avascular tumor. Silk fibroin matrix 3D tumor models are studied for the evaluation of various anticancer drugs. The cytotoxic effects of three different drugs (Paclitaxel, Celecoxib, and ZD6474) at different concentrations are evaluated for MDA‐MB‐231 grown on 2D films as well as on a 3D fibroin scaffold. Higher drug concentrations are required to achieve a comparable reduction in cell viability and invasive potential in 3D culture. Combinatorial treatment of drugs at IC₅₀ concentrations result in up to 84% death of cancer cells. The results indicate that 3D in vitro tumor models may be better systems to evaluate cancer treatment strategies.     

Microfabricated Porous Silk Scaffolds for Vascularizing Engineered Tissues

There is critical clinical demand for tissue‐engineered (TE), 3D constructs for tissue repair and organ replacements. Current efforts toward this goal are prone to necrosis at the core of larger constructs because of limited oxygen and nutrient diffusion. Therefore, critically sized 3D TE constructs demand an immediate vascular system for sustained tissue function upon implantation. To address this challenge the goal of this project was to develop a strategy to incorporate microchannels into a porous silk TE scaffold that could be fabricated reproducibly using microfabrication and soft lithography. Silk is a suitable biopolymer material for this application because it is mechanically robust, biocompatible, slowly degrades in vivo, and has been used in a variety of TE constructs. Here, the fabrication of a silk‐based TE scaffold that contains an embedded network of porous microchannels is reported. Enclosed porous microchannels support endothelial lumen formation, a critical step toward development of the vascular niche, while the porous scaffold surrounding the microchannels supports tissue formation, demonstrated using human mesenchymal stem cells. This approach for fabricating vascularized TE constructs is advantageous compared to previous systems, which lack porosity and biodegradability or degrade too rapidly to sustain tissue structure and function. The broader impact of this research will enable the systemic study and development of complex, critically‐sized engineered tissues, from regenerative medicine to in vitro tissue models of disease states.     

4D Printing of Multi-Responsive Membrane for Accelerated In Vivo Bone Healing Via Remote Regulation of Stem Cell Fate

Dynamic regulation of substrate micro-structures is an effective strategy to control stem cell fate in tissue engineering. Translating this into in vivo tissue repair in a clinical setting remains challenging, which requires precise temporal control of multi-scale structural features. Using 4D printing technique, a multi-responsive bilayer morphing membrane consisting of a shape memory polymer (SMP) layer and a hydrogel layer, is fabricated. The SMP layer is featured with responsive surface micro-structures, which can switch the phase between proliferation and differentiation precisely, thus promoting the bone formation. The hydrogel layer endows the membrane with the ability to digitally regulate its 3D geometry, matching the specific macroscopic bone shape in clinical scenario. The authors’ in vivo experiments show that the 4D shape-shifting membrane exhibits over 30% improvement in new bone formation in comparison to a reference membrane with static micro-structure. More importantly, the 4D membrane can conformally wrap a bone defect model in a non-invasive way and this strategy can be extended to repairs involving complex tissue defects.     

数字全息显微中自动调焦技术及其应用 （封面文章） （特邀）

数字全息显微技术(Digital Holographic Microscopy, DHM)将光学干涉和光学显微技术相结合,为微观物体的三维形貌、透明物体的厚度/折射率分布提供了一种快速、无损测量手段。数字全息显微可以通过计算机模拟物光波的衍射传播以实现对被测样品的数字调焦。然而,这一过程需要事先知道全息图到物体像面的距离(文中称为离焦距离)。如何自动获得离焦距离一直是数字全息显微技术中的研究热点。为此,文中着重介绍了基于锐度度量、能量集中度、振幅模量分析、稀疏度测量以及不同照明调制的离焦量获取方法。利用该离焦量可以实现对运动样品或动态过程的自动调焦,为运动物体或动态过程的跟踪观测和实时干预提供了有力手段。此外,文中还介绍了数字全息显微自动调焦技术在细胞、三维粒子场上成像、识别和追踪以及生物组织三维成像等方面的应用。     

In Situ Porous Structures: A Unique Polymer Erosion Mechanism in Biodegradable Dipeptide-based Polyphosphazene and Polyester Blends Producing Matrices for Regenerative Engineering

Synthetic biodegradable polymers serve as temporary substrates that accommodate cell infiltration and tissue in-growth in regenerative medicine. To allow tissue in-growth and nutrient transport, traditional three-dimensional (3D) scaffolds must be prefabricated with an interconnected porous structure. Here we demonstrated for the first time a unique polymer erosion process through which polymer matrices evolve from a solid coherent film to an assemblage of microspheres with an interconnected 3D porous structure. This polymer system was developed on the highly versatile platform of polyphosphazene-polyester blends. Co-substituting a polyphosphazene backbone with both hydrophilic glycylglycine dipeptide and hydrophobic 4-phenylphenoxy group generated a polymer with strong hydrogen bonding capacity. Rapid hydrolysis of the polyester component permitted the formation of 3D void space filled with self-assembled polyphosphazene spheres. Characterization of such self-assembled porous structures revealed macropores (10-100 μm) between spheres as well as micro- and nanopores on the sphere surface. A similar degradation pattern was confirmed in vivo using a rat subcutaneous implantation model. 12 weeks of implantation resulted in an interconnected porous structure with 82-87% porosity. Cell infiltration and collagen tissue in-growth between microspheres observed by histology confirmed the formation of an in situ 3D interconnected porous structure. It was determined that the in situ porous structure resulted from unique hydrogen bonding in the blend promoting a three-stage degradation mechanism. The robust tissue in-growth of this dynamic pore forming scaffold attests to the utility of this system as a new strategy in regenerative medicine for developing solid matrices that balance degradation with tissue formation.     

Discovery and Evaluation of a Functional Ternary Polymer Blend for Bone Repair: Translation from a Microarray to a Clinical Model

Skeletal tissue regeneration is often required following trauma, where substantial bone or cartilage loss may be encountered and is a significant driver for the development of biomaterials with a defined 3D structural network. Solvent blending is a process that avoids complications associated with conventional thermal or mechanical polymer blending or synthesis, opening up large areas of chemical and physical space, while potentially simplifying regulatory pathways towards in vivo application. Here ternary mixtures of natural and synthetic polymers were solvent blended and evaluated as potential bone tissue engineering matrices for osteoregeneration by the assessment of growth and differentiation of STRO‐1+ skeletal stem cells. Several of the blend materials were found to be excellent supports for human bone marrow‐derived STRO‐1+ skeletal cells and fetal skeletal cells, with the optimized blend exhibiting in vivo osteogenic potential, suggesting that these polymer blends could act as suitable matrices for bioengineering of hard tissues.     

Improved Schedules by Using Data Collected Under Preventive Maintenance

Most preventive maintenance (PM) models require, as inputs, information on the behaviour of the equipment under failure-only maintenance (FM). Once a schedule of PM has been applied, data arising from failures are affected by the PM. Over life the behaviour that would occur under FM changes. But PM also tends to delay aging processes. This paper examines: 1) how data collected under four kinds of PM policy can be modified to re-assess the FM characteristics, and 2) how to tell whether an apparent change in them is important. The approach takes account of engineering as well as statistical factors. It concludes that: 1. Estimation of distributions and costs as they would be under FM from data collected under PM cannot be accurate but a suboptimal policy can be worse. 2. A different type of PM policy might be required following changes to costs and/or distribution. The optimum under one model might not be the overall optimum. 3. The solutions suggested in the paper remain unproved due to the ``data problem'. Mathematical research alone can never produce workable procedures. A viable methodology will need years of experimentation with real systems. 4. The potential savings are huge.     

Improved performance of coded digital FM

The performance of convolutionally encoded narrow-band digital FM with Viterbi decoding was considered in some detail by Simon (1983) for a noncoherent limiter/discriminator (L/D) with integrate and dump (I&D) bit detection. Employing a new threshold receiver which averages the output of the I&D detector with the output of a sample and hold (S&H) detector, a 3-dB improvement over Simon's results for the bit error probability with FM clicks is shown to be achievable. At low error rates, the performance of this new receiver is, moreover, comparable to that obtained when the clicks are exactly removed by Simon's hypothetical “genie”     

Bioinspired Development of an In Vitro Engineered Fracture Callus for the Treatment of Critical Long Bone Defects

Cell-based regenerative constructs provide hope for the restoration of tissue function in compromised biological conditions such as complex bone defects. A strategy mimicking the cascade of events of postnatal fracture healing suggests an implant design where progenitor cells provide the driving force for the construct's tissue forming capacity, while framing biomaterials provide cells with 3D cues to direct cellular processes. Large bone defects mainly heal through the formation of an intermediate endochondral fracture callus. The authors aimed to develop an in vitro engineered fracture callus manufactured by bioprinting to provide a spatially organized tissue construct based on: i) in vitro 3D primed human periosteum derived cells and ii) biocompatible thiol-ene alginate hydrogels, mimicking the cells and extracellular matrix present in the different zones of the callus. Cell viability and maintained osteochondrogenic differentiation upon bioprinting is confirmed in vitro. In vivo assessment displays that the developed biomaterials provided essential 3D cues that further guided the cells in their tissue forming process in the absence of additional stimulatory molecules. The reported findings confirm the appeal of a biomimetic approach to steer tissue development of in vitro engineered constructs and illustrate the suitability of bioprinting methodologies for the fabrication of living regenerative implants.     

Blade Coating of Alloy as Top Electrodes for Efficient All-Printed Organic Photovoltaics

All printing of organic photovoltaics (OPVs) including the top electrode is highly desirable for achieving cost-effective, high-throughput, and large-area photovoltaic manufacturing. Here, the printing of a low-melting-point alloy as top electrodes in OPVs via blade coating is investigated. The Field's metal (FM) with the melting point of 62 °C is adopted for the top electrodes, because FM can be printed under moderate temperatures without harming the active layers while remaining solid state under solar irradiation. The correlations between the processing parameters and properties of the blade-coated electrodes are elucidated. OPVs based on the D18:Y6 active layer and blade-coated FM electrodes achieve a highest power conversion efficiency of 17.28%. The OPVs with FM-electrode demonstrate much higher thermal stability than that of the Ag-electrode devices. All-printed OPVs, in which the FM electrode is blade coated and the other layers are prepared by flexible micro-comb printing, exhibit an efficiency of 16.07%. The results represent the records of evaporation-free and all-printed OPVs, demonstrating that printing FM as OPV electrodes is a cost-effective and time-saving strategy to substitute the vacuum-evaporated metals, as well as a feasible route toward high-performance all-printed OPVs.