v多模态非线性光学显微系统报告人王若曦吕卓明余建曾佑君 v 王若曦共焦显微技术 Confocal

v多模态非线性光学显微系统报告人：王若曦吕卓明余建曾佑君

v 王若曦：共焦显微技术（Confocal Microscopy） v 吕卓明：二次谐波，双光子，多光子三种显微成像系统 v 余建：相干拉曼散射显微术(Coherent Raman Scattering Microscopy) v 曾佑君：Multimodal nonlinear optical microscopy

Confocal Microscopy

Confocal Microscopy v Principles of Confocal Microscopy v Scan Modes v Advantages

Principles of Confocal Microscopy v In confocal system: 1. A point light source for illumination 2. A point light focus within the specimen 3. A pinhole at the image detecting plane These three points are optically conjugated together and aligned accurately to each other in the light path of image formation. 黄永宁. 激光共焦扫描显微镜简介. 现代仪器使用与维修. 1998年 03期

Principles of Confocal Microscopy Laser source Detector Dichroic beamsplitter Scanners Objective Specimen Fluorescence photons 黄永宁. 激光共焦扫描显微镜简介. 现代仪器使用与维修. 1998年 03期

Scan modes ØSingle-beam scanning ( Single-point ) Ømultiple-beam scanning

Scan modes - Single-beam Scanning I. Single-beam scanning

Scan modes - Multiple-beam Scanning I. II. multiple-beam scanning 1 Schematic diagram of parallel confocal microscopy 3 D image reconstruction for parallel confocal microscopy 涂龙等. 并行共焦显微镜检测技术及其研究进展. 中国激光. 2012

Scan modes - Multiple-beam Scanning I. II. multiple-beam scanning Schematic diagram of Nipkow spinning-disk confocal microscopic system 涂龙等. 并行共焦显微镜检测技术及其研究进展. 中国激光. 2012

Scan modes - Multiple-beam Scanning I. II. multiple-beam scanning Arrangement for confocal three-dimensional analysis with a microlens array H. J. Tiziani et al. Theoretical analysis of confocal microscopy with microlenses[J]. Appl. Opt. , 1996, 35(1)

Scan modes - Multiple-beam Scanning I. II. multiple-beam scanning Schematic diagram of confocal microscopic system based on Dammann grating Rong Yang et. al. Parallel confocal systems for biomedical application[C]. SPIE, 2001

Advantages : 1. shallow depth of field (100 nm) 2. the ability to collect serial optical sections from thick specimens 3. void of interference from lateral stray light: higher contrast. 4. images derived from optically sectioned slices. (depth discrimination, 3 D-view) 1.

Advantages

参考文献： [1] 黄永宁. 激光共焦扫描显微镜简介. 现代仪器使用与维修. 1998年 03 期 [2] T. M. Wilson. Confocal Microscopy [M]. New York: Academic Press, 1990 [3] 田维坚，陈波，郭履荣等. 并行更加三维检测方法的理论分析 [J]. 光学学报， 1999, 19 （10）： 1381~1385 [4] Thomas C. Trusk. 3 D Reconstruction of Confocal Image Data[M]. Charleston: Springer, 2011. 243~272

二次谐波，双光子，多光子三种显微成像系统

v 目录 § 非线性光学效应的产生机制 § 二次谐波显微成像的原理及装置 § 双光子 § 多光子

v 非线性光学效应的产生机制 n 在线性光学的范畴内，介质中的粒子在外光电场作用下产生感应的电极化强度，这种电极化强度正比于入射的光电场强度，即： n 在非线性光学的范畴内，电极化强度不再正比于入射的光电场强度，即：

其中为光电场。分别为一阶又叫线性 )、二阶、三阶. . . 极化强度。化率(又叫线性极化率 ), 它是二阶张量；极化率, 它是三阶张量; ( 为一阶极为二阶为三阶极化率, 它是四阶张量. . . 。上式右边出现第二项第三项以及更高次项，即非线性光学效应的根源。

v 二次谐波显微成像的原理及装置 u 原理：二次谐波产生(又称倍频)过程是一个非线性光学过程。二次谐波产生过程可以看作是不同频率光子的交换过程。两个频率为 ω 的光子在谐波产生过程中湮灭，同时形成一个新的频率为 2ω 的光子。它是一个纯粹的量子力学过程。图 1 二次谐波产生示意图

二次谐波产生属于二阶非线性行为, 可以写作: 即为二次谐波的极化强度。二次谐波的产生需要两个条件, 一是材料有非中心对称结构, 二是必须满足相位匹配关系。[1]

从上式还可以看出, 介质与激光电场相互作用产生的二次谐波不仅与光电场强度有关, 而且与介质的极化率 x( n )相联系, 在物理学上, x( n)这些线性和非线性的极化率表征了介质的微观特性, 反映了介质的电子态、分子的对称性、旋向及排列等, 因此可以通过对介质非线性光学现象的探测来了解介质的微观结构信息。

u装置: 图 2 二次谐波成像装置示意图整个系统由光源、显微镜、探测器、计算机处理系统等主要部分构成。[2]

v 二次谐波显微成像的特点 u SHG(second harmonic generation)是生物组织的原发性信号 u SHG具有高度的方向性 u SHG信号背景优于荧光成像, 成像较荧光清晰, 图像信噪比很高, 能进行三维成像 u SHG还具有较好的膜特异性, 可以作为膜探针;

v 双光子显微成像的原理及装置 u 原理：图 3 单光子激发能级图图 4 双光子激发能级图

以上两张能级图可以用薛定谔方程描述，即：方程解的第一项对应单光子激发，多光子跃迁由高阶解描述的。

u装置: 图 5 双光子荧光显微成像装置示意图

图 6 双光子荧光显微镜结构示意图

v 多光子显微成像技术是基于双光子荧光和二次谐波产生的。图 7 多光子荧光激发示意图

l [1]庄正飞, 郭周义, 刘汉平, 喻碧英, 吴淑莲, 卓双木, 邓小元二次谐波显微成像技术 [ J]. 激光生物学报, 2008, 17(1): 126 -132. l [2]VOLODYM YR N, BOAZ N. A Two photon and Second harmonic Microscope [J]. Methods, 2003, 30: 3 -15.

相干拉曼散射显微术 (Coherent Raman Scattering Microscopy)

早期的光学显微镜 v Anton Van Leeuwenhoek(1632 -1723) § is considered the father of microscopy because of the advances he made in microscope design and use. § was the first to see and describe bacteria (1674). v Characteristics §通过透射光照明来进行“明场”观察； §利用样品对光吸收能力的不同来产生成像的衬度； §为了区分许多样本精细结构, 发展了许多染色剂，以提高衬度便于观察。

对比度增强显微镜 v Frits Zernike(1888 -1966) § was a Dutch-born German mathematician and physicist who discovered the phase contrast phenomenon and won a Nobel Prize in 1953. v Characteristics § 透明样本的折射率分布会改变透射光波前的相位分布 § 相位的差异转化为振幅的变化 § 非标记显微术的重大突破 v 微分干涉显微术 § 透明物体的立体感

荧光显微术 v 特点 § 特异性成像是了解生命活动细节的关键 § 荧光发色团提供化学衬度 § 适合生命科学和物质科学中复杂体系的观察 v 荧光标记 § 荧光染料 § 荧光探针 § 荧光蛋白 v 缺陷 § 不可避免地对所研究体系造成影响 § 电子能级间的跃迁, 样本容易受到激发光的损伤 § 细胞内的小分子和脂类等很难或者无法被荧光标记 Lichtman J W, Conchello J. Fluorescence microscopy. Nature Methods, 2005, 2(12): 910– 919

非线性光学显微成像技术种类 v双光子激发荧光、二次谐波产生、相干拉曼散射等特点 v高灵敏度、高空间分辨率 § 非线性光学过程 § 高数值孔径的物镜将光束聚焦才能获得高功率密度 § 成像焦斑比激发光斑小，从而突破经典衍射极限 v近红外光激发 § 穿透深度大 § 光损伤小 v三维成像层析能力应用 v研究厚组织中的分子和细胞

无标记（Label Free）显微成像 v 种类 § 受激辐射、多光子吸收、红外吸收、自发拉曼散射 § 受激拉曼散射（SRS）、相干反斯托克斯拉曼散射（CARS） v 无标记的非线性光学显微成像技术 § § 基于拉曼散射的光学显微成像方法探测目标分子特定的振动、转动来提供成像所需的衬度活体(in vivo)成像分子跃迁至虚能级，不涉及电子激发态，不存在光漂白应用 v 脂类等不易被标记的物质 v 生物体内特定小分子物质如药物等 v 生物大分子如核酸、蛋白质等

各种显微成像术的比较

拉曼散射 v 散射过程散射光波长是否变化 § 弹性散射：米氏散射、瑞利散射 § 非弹性散射：布里渊散射、拉曼散射 v 1928年印度物理学家拉曼发现（1930年获诺贝尔奖） § 不同于荧光现象，不吸收激发光 § 虚的上能级概念 v 特点 § 斯托克斯线与反斯托克斯线在瑞利散射线两侧呈对称分布 § 拉曼谱线的频移只与分子的振动或转动能级有关 § 拉曼光谱具有很好的化学特异性, 从拉曼光谱的峰位信息就可以推导化学键或者官能团的组成、含量及其周围的微观环境 § 通常情况下，斯托克斯光强度远大于反斯托克斯光强度。温度升高，斯托克斯光强度变化不大，而反斯托克斯光的信号强度迅速增加 Raman CV. A change of wavelength in light scattering. Nature, 1928, 121: 619– 619

拉曼散射并不是每一种振动模式都具有拉曼活性，拉曼散射产生于振动中极化率的变化。以CH 2 为例，CH 2 有四种本征平面振动模式，包含键长和键角的变化，如上图所示，这四种 CH 2 平面振动模式都会带来极化率的变化，所以都具有拉曼活性。

相干反斯托克斯拉曼散射（CARS） v 1965年福特汽车公司的两位科学家发现了CARS 现象 v 1982年美国海军研究实验室的科学家们将 CARS 技术与光学显微镜结合, 作为衬度来成像 v 1999年美国太平洋西北国家实验室谢晓亮研究组 § 新的激光控制技术，将激发光装置布局从非共性转为共性 § 很大程度上简化了CARS显微成像系统，逐步走向实用 §当三者满足共振条件ΩR=ωP-ωS与相位匹配条件 k. AS=2 k. P±k. S时, 将激发出频率为 ωAS = 2ωP-ωS 的反斯托克斯光

CARS §可靠性与可操作性最高的 CARS 显微镜架构 §由商品化的共聚焦或者多光子显微镜经过简单的改装或配置来实现对外部引入激光的扫描以及CARS信号的收集 §CARS 显微镜装置的关键在于泵浦光和斯托克斯光的成功引入和同步： • • http: //bernstein. harvard. edu/pages/About. Prof. Xie. html 两束光空间上完全重合：通过显微镜外适当的光路调节实现两束光的脉冲序列在时间上的同步：利用光学参量振荡器(Optical Parameter Oscillator, OPO)作为波长调谐的关键装置

CARS v 谢晓亮研究组在 CARS 显微学上的开创性作使生物学家可以更精确地观测脂类分子的体内分布与代谢过程 § § § 利用CARS的高灵敏度，成功观察到了单个磷脂双层膜的图像借助CARS信号的偏振敏感性，观察到了磷脂分子取向的不同不同分子的特征振动频率，观察到磷脂膜内的相分离利用CARS显微镜的高灵敏度，实现高速的活细胞成像, 观察细胞内脂滴的分布与动态行为通过对富含脂肪的组织样品进行碳氢键伸缩振动的CARS成像, 可以重建活体样本上组织的三维显微结构利用 CARS 优异的化学成像特性, 还可以从活细胞的动态脂肪代谢成像上来理解鱼油的生理学功能与医学价值 v 美国普度大学的程继新研究组实验室通过 CARS 完成了离体和活体脂肪代谢的记录。 v 在活体实验中, 实验鼠的一部分肠道通过手术引到体外, 贴附在玻璃板上以利于显微观察, 而后用橄榄油对鼠进行灌胃处理, 分别记录不同时刻细胞质内脂滴的大小和数量。 http: //www. chem. purdue. edu/jcheng/

CARS §图中可以看出, 细胞内的脂滴很快出现了数量和尺寸上的增加(图 (a), (d))； §在一段时间内会持续增加(图 (b), (e)) ； §过了峰值后脂类的聚集开始消退(图 (c), (f))； §清晰地从形态上反映了动态过程。 Cheng JX. et al. A dynamic, cytoplasmic triacylglycerol pool in enterocytes revealed by ex vivo and in vivo coherent anti-Stokes Raman scattering imaging. J Lipid Res, 2009, 50(6): 1080– 1089

CARS §(g)、(h)是在大范围内多次拍摄拼接重构出的鼠脑切片图像；(h)图是(g)中白色小方框内的图像； §(i)视频帧率下乳鼠红细胞流过毛细血管的记录, 截取一帧； §(j)对(i)中白色箭头所指的毛细血管位置进行图像序列分析, 得到这一点细胞流经情况随时间的变化。 Evans CL, Xu XY, Kesari S, Xie XS, Wong STC, Young GS. Chemically-selective imaging of brain structures with CARS microscopy. Opt Express, 2007, 15(19): 12076– 12087

CARS Tissue Imaging Images of a hairless mouse ear (B) Sebaceous glands at ~30 m from skin surface. (A) Stratum corneum(角质层) with bright signals from the lamellar lipid(层状脂质) intercellular space that surrounds the polygonal corneocytes(角化细胞). Bright punctuated dots are ducts of sebaceous glands(皮脂腺管道). (C) Individual cells of the gland compartment can be recognized, with nuclei visible(细胞核可见) as dark holes (arrow). (D) Adipocytes of the dermis(真皮层脂肪细胞) at ~70 m from skin surface.

CARS Tissue Imaging Images of a hairless mouse ear (E) Adipocytes of the subcutaneous layer(皮下层)at a depth of ~100 m. (F) 2 D projection of 60 depth-resolved slices separated by 2 m. Panels to the right and under F show the yz and xz cross sections taken at the white lines, respectively.

CARS Offers Chemical Selectivity A combined sequential CARS and two-photon fluorescence tissue image The CARS signal is colored blue, and the two-photon fluorescence is colored red. The Raman shift is set to 2, 845 cm-1, with the 816. 7 nm pump Beam driving two-photon fluorescence excitation of the injected Di. D dye. The sebaceous glands can be seen within the branched and looped capillary(毛细血管)network.

CARS Offers Chemical Selectivity Diffusion of mineral oil through mouse epidermis(表皮) (A) Externally applied mineral oil penetrates the stratum corneum (角质层) through the lipid clefts(脂质裂口) between Corneocytes(角化细胞). Image was taken 20 m below the surface 15 min after application of oil

CARS Offers Chemical Selectivity Diffusion of mineral oil through mouse epidermis(表皮) (B) The same area is shown 5 min later. Brighter signal indicates a higher oil concentration caused by time-dependent diffusion, which can be clearly seen during the 5 -min time window.

受激拉曼散射(SRS) v 1962年E. J. Woodbury和 W. K. Ng首先在红宝石中观察到了SRS 现象。 v 2008年谢晓亮研究组首先将此应用于显微成像, 研制出SRS显微术。 § 泵浦光发生了受激拉曼损失(Stimulated Raman Loss, SRL) ΔIp, 导致强度降低； § 同时斯托克斯光发生了受激拉曼增益 (Stimulated Raman Gain, SRG) ΔIs, 强度升高。 §在泵浦光将能量转移给斯托克斯光的过程中, 同时将一部分能量转移到样品的振动能级上面, 分子的振动能级布居发生改变. 通过一定的技术手段来检测SRL或者SRG, 即可作为成像的对比度来源。

SRL microscopy setup. Stokes beam is provided by a 1064 nm pulsed lasers and the pump beam by a synchr-onously pumped optical parametric oscillator. The intensity of the Stokes beam is modulated with an acoustic-optic modulator at radiofrequency. Pump- and Stokes beams are overlapped in time and space and aligned into a state-of-the-art laser– scanning microscope. In forward and epi-direction, the Stokes beam is blocked with a filter and the pump beam detected with a large-area photodiode (PD). The SRL signal is extracted with a lock-in amplifier that detects at the same frequency of the modulation of the Stokes beam. v 相比CARS ，SRS主要增加了：一个斯托克斯光路上的调制器与信号后的锁相放大器

受激拉曼散射(SRS) v SRS 显微术无需标记，没有非共振背景的干扰, 同时信号强度与浓度呈线性关系，这些要素大大简化了图像数据的处理与定量分析，可以获得更多信息。 v 2008年谢晓亮研究组首次利用 SRS 显微镜在实验中观察了组织中无标记的药物输运过程。常用的荧光标记方法往往会导致所标记上的荧光基团比小分子药物还大，影响其转运过程, 通过 SRS 实现无标记的化学成像则避免了这个问题。 v 利用 SRS 显微术进行生物质转化成生物燃料过程的动态监测，搭建了一套双色 SRS 显微系统用于同时监测两种不同化学物质的动态图像，利用一台高功率的脉冲激光光源同时泵浦两套 OPO系统，获取不同波长的激光作为不同物质的泵浦光，而基频的1064 nm激光则作为共同的斯托克斯光。玉米秸秆中木质素与纤维素含量的SRS图像。 (a) 木质素在反应开始前的分布; (b) 纤维素在开始前的分布; (c, d) 反应开始53分钟后, 木质素和纤维素各自的含量, 可以看到木质素明显减少而纤维素含量基本不变; (e) 表示了维管束不同区域的木质素反应速率, 以(a)(c)为时间起讫点, 在单指数衰减拟合下的结果; (f~i) 对应(e)图中各点(绿色, 分别为f-韧皮部, g-导管, h-木纤维, i-无细胞的空白对照处)木质素在一段时间内的含量变化。 Xie XS. et al. . Label-free, real-time monitoring of biomass processing with stimulated raman scattering microscopy. Angew Chem Int Ed, 2010, 49(32): 5476– 5479

SRS tissue imaging of fresh mouse skin. For the acquisition of this image stack in mouse ear, SRL image contrast was tuned into the CH 2 -stretching vibration. As such, lipid rich structures of the skin were highlighted. From top (beginning of the movie) to bottom (end of the movie): • Polygonal intercellular space of the stratum corneum（角质层的多边形细胞间隙） • Viable epidermis with hair follicle（毛囊中活表皮） • Sebaceous gland（皮脂腺） The image stack was taken on fresh tissue, without any preparation or labeling.

总结 v CARS和 SRS 显微术都是基于相干拉曼散射现象的无标记光学成像技术, 两者具有很多相似点。 § 通过探测化学键的振动, CARS 和 SRS 显微术能够以特定化学成分的浓度作为衬度成像； § 利用近红外区域的激光光源, 相干拉曼散射在穿透深度上优于单光子荧光, 有利于组织水平和活体模式生物的成像； § 非线性的强度依赖本征地提供三维成像能力, 分辨率可以与多光子显微镜比拟。

Multimodal nonlinear optical microscopy

Catalog v 1. Introduction v 2. NLO modalities v 3. Strategies in biological and biomedical science v 4. Example

1. Introduction One platform, many insights!

A multimodal NLO microscope, which integrates multiple imaging modalities on the same platform, allows visualization of different endogenous and exogenous structuresin cells and tissues. Coupling of nonlinear optics and scanning microscopy has generated a panel of imaging tools for biology and materials research. The nonlinear optical (NLO) microscopy family can be categorized into one-beam and two-beam modalities. The one-beam modality includes multiphoton fluorescence , second-harmonic generation (SHG) and third-harmonic generation microscopy. The two-beam modality includes coherent anti. Stokes Raman scattering (CARS), four-wave mixing (FWM) , stimulated Raman scattering (SRS) , pump-probe , and hotothermal microscopy. Because each NLO imaging modality is sensitive to specific molecules or structures, multimodal NLO imaging capitalizes the potential of NLO microscopy for studies of complex tissue samples.

Advantage of Multimodal nonlinear optical microscopy NLO imaging is fast and can be applied on normal histology sections. The method avoids all staining procedures and has the potential for in-vivo imaging. Easy to use multimodal non-linear systems, suited for clinical use.

2. NLO modalities Over the last several decade, so many biological NLO microscopy techniques have been developed including: two-photon excited fluorescence(TPEF) Coherent anti-Stokes Raman scattering (CARS) second harmonic generation(SGH) third harmonic generation (THG) four-wave mixing (FWM) stimulated Raman scattering (SRS) pump-probe（泵浦探测光谱） photothermal(光热) microscopy

Among these techniques, coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) are prominent.

TPEF provides images with good signal/noise ratio of molecular（分子） autofluorescence signals in living tissues by stimulating native fluorophores. TPEF have the capabilities for label-free morphological imaging. In biological samples the strongest source of SHG is fibrillar collagen（纤维胶原蛋白）. CARS is on the structure and composition of imaging.

3. Strategies in biological and biomedical science Despite considerable scientific gains in cancer diagnosis and treatment, cancer remains to threaten human life. Current clinically used imaging methods for cancer diagnosis include X-ray, CT, MRI, and OCT. One of many fields in which NLO microscopy has proven to be useful is cancer research. These techniques have recently emerged（应运而生）as a valuable tool nondestructive（无损的）, chronic（长期的） imaging of living tumors. The combination of different image analysis approaches described in this work may represent a powerful combination of tools to investigate collagen organization and remodeling of extracellular（细胞外） matrix in carcinogenesis(癌症) processes.

Because lipid-rich cell membranes and lipid-poor cell nuclei generate substantial contrast in CARS imaging, we have used CARS microscopy to visualize（形象地） tumor cells with submicrometer resolution（纳米级的分辨） in a label-free manner (Fig). Moreover, simultaneous CARS and SFG imaging enables label-free visualization of cell arrangement and collagen type-I fibrils organization. (a)Emission spectra of SFG signals from collagen fibrils (blue), TPEF signals from Hoechst 33342 -labeled nuclei(green), and CARS signals from lipid droplets (red).

(b)CARS/SFG images of rat mammary adenoma. Abundant wavy collagen type-I fibrils (green) form a smooth organized outer contour (arrow heads) to concentrically wrap around the localized tumor mass (red), whose signals come from lipid-rich cell membranes（细胞膜） and lipid droplets（脂肪滴）.

Ratio between collagen and elastic tissue (SAAID) The SAAID value is a measure of the ratio between collagen and elastic tissue（弹性蛋白组织）. As the stroma is composed primarily of collagen and elastic fibers, it allows the use of nonlinear optical signals to discriminate between altered connective tissue regions in the surrounding tumor area.

TACS This parameter is frequently used to determine the collagen fibers orientation at the tumor—stroma boundary.

FFT has proven to be a good method to assign the degree of image organization.

4. Example v Non-linear optical microscopy of kidney tumours

The multimodal non-linear imaging system used to image the renal tumours was integrated in house, with a commercial laser scanning microscope for multiphoton microscopy and two near infrared fiber lasers（近红光光纤激光器）. The frequency difference between the two lasers was set in order to excite the CARS signal from the symmetric stretching of CH 2 groups (“pump” laser emiting at 780 nm and “Stokes” laser at 1005 nm). CARS, TPEF (in the range 500– 550 nm) and SHG signals were simultaneously（同时地） acquired and combined to build an RGB image (red channel: CARS, green channel: TPEF, blue channel: SHG). A 32/0, 85 NA objective was used; as the scanning field of the high NA objective is small, the pictures were obtained by stitching of many smallertiles.

Figure shows NLO multimodal images of a representative sample and the histological H&E staining of a non-consecutive section for comparison. (A); multimodal NLO image (B); H&E (C) amd multimodal NLO image (D) of the border between tumour and normal tissue; H&E (E) and multimodal NLO (F) high magnification image of the tumour. In all NLO images: red channel=CARS, green channel= TPEF, blue channel=SHG.

H&E staining (A); multimodal NLO image (B); H&E staining and multimodal NLO zoom-in images of tumour tissue (C and D), connective tissue (E and F) and necrosis (G and H). In all NLO images: red channel=CARS, green channel=TPEF, blue channel =SHG.

The quantity of information contained in the NLO images is superior to H&E staining stains specifically cell nuclei and unspecifically intra- and extra-cellular tissue proteins. Multimodal NLO microscopy visualises the tissue morphology through the tissue composition: intracellular and extracellular lipids are imaged by CARS, while extracellular matrix proteins, such as collagen and elastin, are imaged by SHG and TPEF respectively. Multiple immunohistochemical staining can providemore specific information.

[1]Watson J M, Marion S L, Rice P F, et al. In vivo time-serial multimodality optical imaging in a mouse model of ovarian tumorigenesis[J]. Cancer biology & therapy, 2014, 15(1): 42. [2]Adur J, Carvalho H F, Cesar C L, et al. Nonlinear Optical Microscopy Signal Processing Strategies in Cancer[J]. Cancer informatics, 2014, 13: 67. [3]http: //onlinelibrary. wiley. com/doi/10. 1002/lpor. 201000027/abstr act [4]https: //lcmrc. colorado. edu/facilities/multimodal_nonlinear_micros copy. html [5]Jepsen P U, Cooke D G, Koch M. Terahertz spectroscopy and imaging – Modern techniques and applications[J]. Laser & Photonics Reviews, 2012, 6(3).

v多模态非线性光学显微系统 报告人 王若曦 吕卓明 余建 曾佑君 v 王若曦 共焦显微技术 Confocal

v多模态非线性光学显微系统报告人王若曦吕卓明余建曾佑君 v 王若曦共焦显微技术 Confocal