受激拉曼散射显微技术在生物科学中的应用

受激拉曼散射(stimulated Raman scattering,SRS)显微技术,可以通过探测分子的特定振动模式,实现对活细胞的无标记和新型非荧光标记成像。由于SRS显微术具有较高的化学特异性,在生命科学成像领域具有极大的应用潜力。本文简要介绍了SRS显微技术的原理,同时总结了其在脂类、核酸和蛋白质等生物分子,以及在组织结构、小分子药物和食品分析中的应用。     

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。     

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。     

基于拉曼晶体的多波长激光技术研究进展

利用受激拉曼散射效应,以拉曼晶体作为介质,可产生同轴输出的多波长激光信号,该种激光器具有结构紧凑、脉冲能量高和波长可调谐等特点,在全色激光成像与显示、光电对抗等领域有着重要的应用前景。本文介绍了受激拉曼散射基本原理和常用拉曼激光器结构,研究了国内外基于拉曼晶体的多波长激光技术的研究进展,总结了利用受激拉曼散射产生多波长激光存在的不足。针对目前受激拉曼散射高阶散射光较难生成,生成的多波长激光信号覆盖谱段较窄,输出功率较低,调谐方式单一等问题,提出了今后多波长激光技术发展方向。     

三维拉曼显微成像技术研究进展（特邀）

拉曼显微成像技术无需样本制备,具有无损、无创、对水溶液不敏感的优点,可在微米或纳米尺度下表征样本的生化组分及分布,成为生命科学领域重要的研究工具。随着对复杂生物样本研究的不断深入,拉曼显微成像也被期待能够实现对生物样本中的分子组成与分布的动态立体观测。首先,系统性地梳理近年来三维拉曼显微成像技术的研究进展,包括基于自发拉曼散射、相干拉曼散射、表面增强拉曼散射以及拉曼标签的不同三维成像方法的技术手段、改进策略与实验结果。然后,总结了不同成像技术在细胞生物学、发育生物学等方面的应用进展。最后展望了不同三维拉曼显微成像技术在生物医学光学显微成像技术应用中所面临的挑战和发展前景。     

新的生物医学成像技术——光学相干显微术及其应用

光学相干显微术是一种新的成像技术，它能对高散射介质，如生物组织进行非介入的快速成像，其分辨率远大于Ｂ超，因而在活体生物组织的微结构分析和疾病诊断方面有重要的应用价值。     

自适应光学技术在深层动态荧光显微成像中的应用和发展

荧光显微成像技术是开展微观生命科学研究的重要手段和工具,使用该技术可以观察生物体内的精细结构、动态追踪生物体内组织、细胞、细胞核、蛋白、小分子等不同尺度的生命活动过程。其中,研究深层组织高时空分辨率荧光显微成像技术,是当前成像领域一个前沿问题。应用自适应光学技术实时补偿经由不透明散射、非均匀生物组织传播而引入的复杂波前畸变已被证实是实现上述技术的一种有效途径。文中首先归纳了深层动态荧光显微成像的需求和特点,随后分别介绍了自适应光学技术近几年在共聚焦显微成像、随机光学重构显微成像、光激活定位显微成像、受激辐射光淬灭显微成像、双光子/多光子激发显微成像中的相关应用,并对今后的研究问题和发展方向提出展望。     

“2013中国光学重要成果”入选成果介绍

正(排序不分先后)基于等离激元增强拉曼散射的单分子化学成像中国科学技术大学侯建国院士研究团队(doi:10.1038/nature12151)在高分辨化学识别与成像领域取得重大突破,在国际上首次实现了亚纳米分辨的单个卟啉分子的拉曼成像。他们将具有低温超高真空环境的高分辨扫描隧道显微技术与具有单光子检测灵敏度的拉曼光谱技术结合在一起,利用隧道结中纳腔等离激     

包层抽运掺镱光纤激光器中受激拉曼散射和受激布里渊散射效应

高功率光纤激光器大多选用掺镱双包层光纤作为增益介质,由于光纤尺寸较小,极易在光纤谐振腔中产生受激布里渊散射、受激拉曼散射效应。包层掺镱双包层光纤激光器中一旦发生受激拉曼散射和受激布里渊散射效应,其产生高强度信号成为高功率光纤激光器的主要噪声来源,影响激光输出的特性和稳定性。对包层抽运掺镱光纤激光器中的受激布里渊散射和受激拉曼散射进行了实验研究,在单模双包层光纤中观察到受激布里渊散射和受激拉曼散射。实验结果表明,在光纤谐振腔中,抽运方式、谐振腔输出镜损耗、受激瑞利散射对受激布里渊散射的影响较大,尤其是受激瑞利散射为谐振腔提供了附加反馈,不仅压窄激光信号的线宽,而且使得受激布里渊散射的阈值迅速降低。     

双向泵浦受激拉曼散射增益谱平坦光纤放大器

研究现有的拉曼光纤放大器(Raman Fiber Amplifier,RFA)增益谱平坦化技术的不足,提出一种新型的拉曼光纤放大器的设计,即双向泵浦受激拉曼散射增益谱平坦光纤放大器。利用受激拉曼散射增益谱几乎在450cm-1波数处形成的对称结构,利用前向泵浦与信号光产生的频移对信号光进行放大作用,同时利用后向泵浦与信号光产生的频移对信号光进行补偿性的放大作用,从而实现双向泵浦受激拉曼散射增益谱平坦光纤放大器的设计。通过MATLAB的仿真验证,所设计的放大器增益平坦度高,信号光功率增益大,可实现双向泵浦受激拉曼散射光纤放大器的平坦化。     

Framework for parsing, visualizing and scoring tissue microarray images.

Increasingly automated techniques for arraying, immunostaining, and imaging tissue sections led us to design software for convenient management, display, and scoring. Demand for molecular marker data derived in situ from tissue has driven histology informatics automation to the point where one can envision the computer, rather than the microscope, as the primary viewing platform for histopathological scoring and diagnoses. Tissue microarrays (TMAs), with hundreds or even thousands of patients' tissue sections on each slide, were the first step in this wave of automation. Via TMAs, increasingly rapid identification of the molecular patterns of cancer that define distinct clinical outcome groups among patients has become possible. TMAs have moved the bottleneck of acquiring molecular pattern information away from sampling and processing the tissues to the tasks of scoring and results analyses. The need to read large numbers of new slides, primarily for research purposes, is driving continuing advances in commercially available automated microscopy instruments that already do or soon will automatically image hundreds of slides per day. We reviewed strategies for acquiring, collating, and storing histological images with the goal of streamlining subsequent data analyses. As a result of this work, we report an implementation of software for automated preprocessing, organization, storage, and display of high resolution composite TMA images.     

Measuring the progression of foreign-body reaction to siliconeimplants using in vivo MR microscopy

We used in vivo magnetic resonance (MR) microscopy to follow the growth of fibrous capsule as a foreign body reaction to silicone implants in rats. Anesthetized rats were imaged 1, 7, 14, and 28 days after silicone-coated MR imaging coils were sutured to their neck muscles. On the twenty eighth day, rats were sacrificed and coils and adjacent tissues were removed en bloc and fixed in formalin, reimaged with MR, and sectioned for conventional histology. Three-dimensional (3-D) spin-echo [3DFT] acquisition gave in-plane resolution of 32×32 μm in vivo and 16×16 μm ex vivo. All MR images showed a diffuse band of elevated signal intensity between the silicone of the coil and adjacent tissue. The border of the hyperintense band was thin and not well defined at seven days post-implantation. From 7-28 days, the band showed relatively homogeneous signal intensity and its thickness increased 44% on the rectus muscle side and 78% on the subcutaneous side. The capsule thickness determined either by MR in vivo and ex vivo microscopy or conventional histology was not significantly different, and there was a significant correlation between thickness measurements among those methods. MR in vivo microscopy provides sufficient resolution and spatial information to serially evaluate the growth of the foreign body fibrous capsule over time, thus achieving greater accuracy and consistency in measurements     

Quantitative Segmentation of Principal Carotid Atherosclerotic Lesion Components by Feature Space Analysis Based on Multicontrast MRI at 1.5 T

The purpose of this paper is to evaluate the capability of feature space analysis (FSA) for quantifying the relative volumes of principal components (thrombus, calcification, fibrous, normal intima, and lipid) of atherosclerotic plaque tissue in multicontrast magnetic resonance images (mc-MRI) acquired in a setup resembling clinical conditions ex vivo. Utilizing endogenous contrast, proton density, T1-weighted, and T2-weighted images were acquired for 13 carotid endarterectomy (CEA) tissues under near-clinical conditions (human 1.5 T GE Excite scanner with sequence parameters comparable to an in vivo acquisition). An FSA algorithm was utilized to segment and quantify the principal components of atherosclerotic plaques. Pilot in vivo mc-MRI images were analyzed in the same way as the ex vivo images for exploring the possible adaptation of this technique to in vivo imaging. Relative abundance of principal plaque components in CEA tissues as determined by mc-MRI/FSA were compared to those measured by histology. Mean differences plusmn standard deviations were 5.8 plusmn 4.1% for thrombus, 1.5 plusmn1.4 % for calcification, 4.0 plusmn2.8% for fibrous, 8.2 plusmn 10% for normal intima, and 2.4 plusmn 2.2% for lipid. Reasonable quantitative agreement between the classification results obtained with FSA and histological data was obtained for near-clinical imaging conditions. Combination of mc-MRI and FSA may have an application for determining atherosclerotic lesion composition and monitoring treatment in vivo.     

Quantitative laser scanning confocal autofluorescence microscopy ofnormal, premalignant, and malignant colonic tissues

Laser scanning confocal autofluorescence microscopy (LSCAM) using 351- to 364-nm excitation light was used to quantitatively compare fluorescent spectral emission of unstained, frozen histological sections of normal, premalignant, and malignant colonic tissues. To identify the spatial origins of fluorescent signals accurately, the same frozen section slides used for microscopy were fixed and histochemically stained immediately following LSCAM imaging. Tissue fluorescence emission was quantified in terms of the intrinsic fluorescence coefficient beta (lambda), defined as the fluorescence power per unit tissue volume per unit wavelength (centered at lambda) divided by the incident light irradiance. Over all emission wavelengths, colonic tissues emitted autofluorescence ranging from beta (lambda) approximately 10(-1.5) to 10(-3.0) cm-1. In the 530- to 610-nm spectral region, markedly increased autofluorescence (beta up to 10(-2.5)) was observed in the dysplastic cells of adenomatous polyps, as compared to normal epithelial cells. Compared to adenomatous polyps, decreased dysplastic cell autofluorescence was observed in adenocarcinoma. The brightest fluorescence in the lamina propria, which was attributed to eosinophils (beta approximately 10(-2.5)) in previous studies, was also observed in other granular structures (beta up to 10(-1.4)). LSCAM reveals quantitative significant differences in fluorescence emission between normal and diseased colonic tissues.     

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。     

Vibro-acoustic tissue mammography

A novel method for detection and imaging of microcalcifications in breast tissue is presented. The method, called vibro-acoustography, uses the radiation force of ultrasound to vibrate tissue at low (kHz) frequency and utilizes the resulting response to produce images that are related to the hardness of the tissue. The method is tested on human breast tissues. The resulting vibro-acoustographic images are in agreement with corresponding X-ray mammography images of the specimens. The existence of microcalcifications in locations indicated by vibro-acoustography is confirmed by histology. Microcalcifications as small as 110 microm in diameter are detected by this method. Resulting vibro-acoustographic images show microcalcifications with high contrast with respect to the background soft tissue. Structures such as dense sclerotic tissue do not seem to interfere with detection of microcalcifications.     

Chromophore-Free Sealing and Repair of Soft Tissues Using Mid-Infrared Light-Activated Biosealants

Poor strength, infection, leakage, long procedure times, and inflammation limit the efficacy of common tissue sealing devices in surgeries and trauma. Light-activated sealing is attractive for tissue sealing and repair, and can be facilitated by the generation of local heat following absorption of nonionizing laser energy by chromophores. Here, the inherent ability of biomaterials is exploited to absorb nonionizing, mid-infrared (midIR) light in order to engender rapid photothermal sealing and repair of soft tissue wounds. In this approach, the biomaterial simultaneously acts as a photothermal convertor as well as a biosealant, which dispenses the need for exogeneous light-absorbing nanoparticles or dyes. Biomechanical recovery, mathematical modeling, histopathology analyses, tissue strain mapping using digital imaging correlation, and visualization of the biosealant-tissue interface using hyperspectral imaging indicate superior performance of midIR sealing in live mice compared to conventional sutures and glue. The midIR-biosealant approach demonstrates rapid sealing of soft tissues, improves cosmesis, lowers potential for scarring, obviates safety concerns because of the nonionizing light used, and allows adoption of a wide diversity of biomaterials. Taken together, the studies demonstrate a novel advance both in biomaterials for surgical sealing along with the use of nonionizing midIR light, with high potential for clinical translation.     

Application of real-time confocal laser scanning microscopy to observe living cells in tissue specimens

Fluorescence microscopy imaging has developed into an important tool for the study of cell structure and function in cell biology. This non-invasive technique permits the characterization, localization and qualitative quantification of free ions, messengers, pH, voltage and other molecules in living cells. The regulation of cytosolic Ca2+ homeostasis is essential for cells. However, most investigations have used cultured or isolated cells as an experimental model and, consequently, provide only limited insight into the mechanisms that operate in tissue in situ. More useful information could be obtained by studying intact tissue specimens. The calcium dynamics of some tissue specimens, such as arteriole smooth muscle cells, supra cervical ganglia and peripheral nerve bundles, were analysed in this study. Real-time confocal microscopy revealed that individual cells exhibited different [Ca2+]i dynamics and the responses to transmitters/modulators were heterogeneous. It is important that the confocal microscopes have good detection performances, due to the reduction of stray light. We conclude that real-time confocal microscopy is a useful tool for investigating structural and functional changes of cells in living tissues, although suitable tissue-preparation is important for these measurements.     

A Bright Two-Photon Lipid Droplets Probe with Viscosity-Enhanced Solvatochromic Emission for Visualizing Lipid Metabolic Disorders in Deep Tissues

The polarity of lipid droplets (LDs) plays an important role in pathological processes associated with abnormal lipid metabolism. Monitoring the variation of LDs polarity in cells and tissues is of great importance in biomedical research and clinical diagnosis. However, developing fluorescent LDs-specific probes with high polarity sensitivity, brightness, and permeability for deep tissue imaging is still challenging. Herein, a push–pull fluorescent luminogen (DPBT) with aggregation-induced emission, strong solvatochromism, large Stokes shift, high solid-state fluorescence efficiency and superior two-photon absorption is facilely developed. The lipophilic DPBT can specifically stain LDs with high biocompatibility and good photostability. The viscosity-enhanced solvatochromic emission property enables DPBT to visualize LDs polarity with high brightness and imaging contrast, and deep penetration depth under two-photon microscopy. DPBT can specifically stain lipids in various mouse tissues (atherosclerotic plaque, liver, and mesenteric adipose tissues) and map their polarity distribution to reflect lipid metabolic states within those tissues. It is found that the lipids deposition as well as their polarity distribution in tissues of hyperlipoidemia mouse are clearly different from the tissues of the normal mouse. Its excellent properties make DPBT a promising candidate for investigating LDs-associated physiological and pathological processes in live biological samples.     

沿传输方向浓度渐变掺铒光纤放大器的模型

提出了两种掺铒浓度渐变的分布式光纤放大器（ｄ－ＥＤＦＡ）模型：沿传输方向，掺杂浓度单调线性下降与掺杂浓度线性由大到小，由小到大两次变化的ｄ－ＥＤＦＡ，用传输方程研究了不同渐变系数下，ｄ－ＥＤＦＡ的各种特性。