飞秒激光与生物细胞作用机理及应用

飞秒激光是自1960年第一台激光器诞生以来,过去20年间由激光科学发展起来的最强有力的新工具之一。飞秒激光由于脉冲持续时间短、瞬时功率大、聚焦尺寸小的特点,使得其在超快、超强和超精细领域有着广阔的应用前景。其中最重要的一个方向是飞秒激光在生物细胞方面的应用。细胞是生命活动的基本单位。所有的病源微观上都体现在细胞中细胞器的工作,所以用飞秒激光作用在病体的细胞器上达到治疗的目的,是一个很有前景的领域。由于生物大分子和水几乎不吸收近红外光,故应用近红外飞秒激光对细胞进行手术,同时可在不损伤细胞活性的前提下对细胞进行实验。这种激光手术技术已被用于对细胞内结构进行切割和蚀除。介绍了该技术在细胞领域中的一些应用,如纳米手术、基因转染和染色体切割等;还介绍了飞秒激光技术与生物细胞中主要细胞器的祛除的原理、飞秒激光细胞操作与手术系统和实验中荧光成像、多光子成像显微镜等手段。     

新型准分子激光角膜屈光矫正系统

小光斑高速飞点扫描的准分子激光系统以其具有的治疗时间短、矫正精度高、角膜切削表面极其光滑,易于集成波前像差技术实现“个体化切削”方案等优点,成为眼科屈光矫正激光手术发展趋势。本文从理论基础、光学系统等方面综述了具有自主知识产权的飞点扫描式激光系统,此系统已经在临床动物实验和盲眼实验中取得了非常好的效果。     

声光偏转器对飞秒激光的光斑畸变分析

分析了声光偏转器(acousto-optical deflector,AOD)对飞秒脉冲激光光斑的空间畸变效应。研究表明:由于飞秒激光在锁模情况下具有较宽的光谱带宽,经过AOD后的一级衍射光斑带有严重的角色散,导致光斑形状发生畸变,影响成像的空间分辨率。实验测试了一维AOD器件在不同声波频率下的光斑形状,获得了光斑尺寸的放大倍数,验证了AOD对飞秒激光的光斑畸变效应。     

准分子激光双面式切削原位角膜磨镶术的研究进展

准分子激光双面式切削原位角膜磨镶术(Both-sided LASIK,BSL)是准分子激光原位角膜磨镶术(laser in situ keratomileusis, LASIK)的改良,BSL将部分激光切削分布在角膜瓣基质面,因而减少了对角膜基质床的切削,最大限度的保留了角膜基质床的剩余厚度,为降低术后角膜膨出提供可能,对屈光度相对偏高和/或角膜相对偏薄的患者,尽量增加手术的安全性,并为LASIK术后屈光回退的增强手术提供了一种新的方法。本文对近年BSL的研究进展作一综述。     

飞秒激光在白内障手术中的应用新进展

飞秒激光辅助的白内障手术是目前最热门的白内障手术之一。即在计算机系统引导下利用飞秒激光进行透明角膜切口的制作,晶状体核裂解和晶状体前囊膜的切开,明显降低了传统超声乳化手术中的并发症,具有十分广阔的临床应用前景。然而飞秒激光辅助的白内障手术目前仍然处于临床应用初级阶段,其昂贵的价格是影响其研究和应用的主要原因,手术安全性及远期屈光效果仍然需要长期的随访观察。本文根据文献资料,就飞秒激光在白内障手术中的优势及临床应用的局限性进行综述。     

阳离子胶体金标记活细胞阴离子位点的双光子荧光成像及其应用

使用阳离子胶体金标记中国仓鼠卵巢细胞(CHO-K1)的阴离子位点,并采用双光子荧光显微成像和荧光寿命成像技术记录活细胞的阴离子场分布.阳离子胶体金是纳米量级金微粒与多聚L-赖氨酸的结合物,金纳米微粒在超短激光脉冲的照射下可以产生高度局域化的光热效应.当飞秒激光脉冲聚焦在细胞膜上标记的金纳米微粒时会产生这种纳米尺度的微光热效应,并在不影响细胞活性的前提下暂时提高细胞膜的通透性.基于这种效应,使用聚焦的飞秒激光脉冲三维扫描照射CHO-K1细胞,将分子质量为10ku的荧光探针大分子异硫氰酸荧光素葡聚糖(fluorescein isothioeyanate-dextran, FITC-D)递送到CHO-K1细胞的内部,并用双光子荧光图像记录其递送的过程.使用流式细胞仪分析不同实验条件下FITC-D的转导率和细胞死亡率的关系.     

基于有限元方法的角膜组织热分析研究

生物医学与虚拟现实技术、计算机仿真技术的结合是现代科学与技术的一个重要发展趋势。为了解决角膜热成形术中的预测性及可控制性问题,本文针对角膜的特性进行医学虚拟研究,分析其变形机理;利用有限元方法,模拟了激光作用于眼角膜时的温度场分布情况,给出了不同参数下的激光作用时角膜的温度场分布曲线和穿透深度的比较;仿真结果表明,激光能量对角膜组织穿透深度影响很大,为角膜热成形术的研究提供了定性的基础,在减少实验费用、提高手术的预测性方面有非常重要的价值。     

光透明剂对兔子动脉血管壁二次谐波成像的影响

为了了解光透明剂对生物组织二次谐波成像的影响,利用二次谐波成像术(SHG) 对动脉血管组织(兔颈部动脉血管壁外膜层,含有丰富的胶原纤维)经无水甘油处理后的光透明效果进行研究.实验结果表明,经无水甘油处理后的动脉血管壁外膜层胶原纤维的二次谐波成像的成像深度和成像对比度均得到明显的改善;动脉血管组织的衰减系数降低了50 %左右,而成像深度由35 μm 提高到75 μm .这说明甘油溶液具有提高生物组织二次谐波成像深度和对比度的光透明效应.由于组织的浑浊特性使其对可见光和近红外波长具有很强的散射效应,基于激光的治疗和诊断技术受到很大限制,使用光透明剂提高组织内部的折射率匹配从而降低散射效应的方法有望在生物医学领域得到广泛应用.     

综述: 用于神经活动观测的随机扫描双光子显微成像

双光子荧光显微镜是神经科学研究中的重要观测仪器,但是现有的商品化仪器受限于较低的成像速度,难以满足脑功能研究中毫秒量级神经信号检测的需要．基于声光偏转器的快速随机扫描双光子显微成像技术,有望在保持信噪比的同时提高观测速度．本文综述了这一研究的最新进展,从飞秒激光经过角色散器件后的时空演化理论、声光偏转器的色散补偿方法、随机扫描成像仪器及仪器应用到神经成像时钙信号的识别方法四个方面分别进行介绍,最后分析了随机扫描双光子显微成像技术的发展趋势．这项技术的系统深入研究将为神经活动观测提供一种全新的方法,推动脑科学研究的发展．     

飞秒激光辅助全板层角膜移植术治疗圆锥角膜临床观察

目的:评价飞秒激光辅助全板层角膜移植治疗圆锥角膜患者早期临床效果。方法:回顾性分析。15例17眼圆锥角膜患者均采用飞秒激光辅助联合Anwar大气泡技术暴露后弹力层的角膜移植手术。术前17只眼均测量裸眼视力(Uncorrected visual acuity,UCVA)、最佳矫正视力(Best corrected visual acuity,BCVA)、角膜内皮细胞计数(endothelial cell density,ECD)、角膜中央平均厚度、角膜曲率1(K1)、角膜曲率2(K2)、角膜地形图角膜散光度数和眼压(intraocular pressure,IOP)。所有患者随访时间为术后第1周、第1月、第2月和第3月。结果:到第3月时,UCVA和BCVA均有明显提高。测量中央角膜中央厚度为(493.0±46.80)μm;角膜曲率已接近正常水平,K1和K2平均值分别为(44.56±4.86)D和(40.22±3.60)D,以上数据与术前相比,差异均具有统计学意义(P0.001)。散光值下降至(4.57±3.60)D(P=0.185,P0.05)。角膜内皮细胞丢失率为14.3%。术后眼压均正常。结论:飞秒激光辅助全板层角膜移植治疗圆锥角膜患者早期临床效果明显,具有精确性、安全性和可预测性。     

Second- and third-harmonic generation microscopies for the structural imaging of intact tissues

One principal advantage of multiphoton excitation microscopy is that it preserves its three-dimensional micrometer resolution when imaging inside light-scattering samples. For that reason two-photon-excited fluorescence microscopy has become an invaluable tool for cellular imaging in intact tissue, with applications in many fields of physiology. This success has driven increasing interest in other forms of nonlinear microscopy that can provide additional information on cells and tissues, such as second- (SHG) and third- (THG) harmonic generation microscopies. In recent years, significant progress has been made in understanding the contrast mechanisms of these recent methodologies, and high-resolution imaging based on intrinsic sources of signal has been demonstrated in cells and tissues. Harmonic generation exhibits structural rather than chemical specificity and can be obtained from a variety of non-fluorescent samples. SHG is observed specifically in dense, non-centrosymmetric arrangements of polarizable molecules, such as collagen fibrils, myofilaments, and polarized microtubule bundles. SHG imaging is therefore emerging as a novel approach for studying processes such as the physiopathological remodelling of the collagen matrix and myofibrillogenesis in intact tissue. THG does not require a non-centrosymmetric system ; however no signal can be obtained from a homogeneous medium. THG imaging therefore provides maps of sub-micrometer heterogeneities (interfaces, inclusions) in unstained samples, and can be used as a general purpose structural imaging tool. Recent studies showed that this technique can be used to image embryo development in small organisms and to characterize the accumulation of large lipid bodies in specialized cells. SHG and THG microscopy both rely on femtosecond laser technology and are easily combined with two-photon microscopy.     

Femtosecond Laser Axotomy in Caenorhabditis elegans and Collateral Damage Assessment Using a Combination of Linear and Nonlinear Imaging Techniques

In this work highly localized femtosecond laser ablation is used to dissect single axons within a living Caenorhabditis elegans (C. elegans). We present a multimodal imaging methodology for the assessment of the collateral damage induced by the laser. This relies on the observation of the tissues surrounding the targeted region using a combination of different high resolution microscopy modalities. We present the use of Second Harmonic Generation (SHG) and Polarization Sensitive SHG (PSHG) to determine damage in the neighbor muscle cells. All the above is done using a single instrument: multimodal microscopy setup that allows simultaneous imaging in the linear and non-linear regimes and femtosecond-laser ablation.     

Interest of second harmonic generation imaging for diagnosis in thick and opaque tissue

In articular hyaline cartilage, chondrocytes are surrounded by an extracellular matrix which is mainly composed by collagen and proteoglycanes. Pathological specimens show a partial or complete degradation of this matrix. Therefore, it could be interesting to know how mechanical or biochemical constraints applied to cartilage specimens induce modifications of the cartilage network. Multiphoton technology combined to Second Harmonic Generation (SHG) enables to image cartilage specimens in a non-invasive mode with high resolution at deep penetration. By placing a band pass filter in front of the transmitted light detector, SHG signal with frequency doubled can be isolated for a new contrast imaging. SHG (second harmonic generation) is a diffusion process generated from organized structures and does not need any fluorescent staining. Due to their non-centrosymetric structure, collagen fibrilles present a high second-order non-linear susceptibility and thus give rise to a strong SHG signal when exposed to high enough electric fields produced by a focal point of a femtosecond pulsed laser (multiphoton microscopy). As the extracellular matrix of cartilage is in part constituted by collagen fibers, it can be imaged with this contrast tool. The intensity of SHG signals strongly depends on the organization of collagen fibers. Thus a modification of the extracellular matrix in terms of 3D-organization of collagen induced by mechanical stress can be shown with this contrast tool.     

Structural Characterization of Edematous Corneas by Forward and Backward Second Harmonic Generation Imaging

The purpose of this study was to image and quantify the structural changes of corneal edema by second harmonic generation (SHG) microscopy. Bovine cornea was used as an experimental model to characterize structural alterations in edematous corneas. Forward SHG and backward SHG signals were simultaneously collected from normal and edematous bovine corneas to reveal the morphological differences between them. In edematous cornea, both an uneven expansion in the lamellar interspacing and an increased lamellar thickness in the posterior stroma (depth > 200 μm) were identified, whereas the anterior stroma, composed of interwoven collagen architecture, remained unaffected. Our findings of heterogeneous structural alteration at the microscopic scale in edematous corneas suggest that the strength of collagen cross-linking is heterogeneous in the corneal stroma. In addition, we found that qualitative backward SHG collagen fiber imaging and depth-dependent signal decay can be used to detect and diagnose corneal edema. Our work demonstrates that SHG imaging can provide morphological information for the investigation of corneal edema biophysics, and may be applied in the evaluation of advancing corneal edema in vivo.     

Hyperglycemia-Induced Abnormalities in Rat and Human Corneas: The Potential of Second Harmonic Generation Microscopy

Background

Second Harmonic Generation (SHG) microscopy recently appeared as an efficient optical imaging technique to probe unstained collagen-rich tissues like cornea. Moreover, corneal remodeling occurs in many diseases and precise characterization requires overcoming the limitations of conventional techniques. In this work, we focus on diabetes, which affects hundreds of million people worldwide and most often leads to diabetic retinopathy, with no early diagnostic tool. This study then aims to establish the potential of SHG microscopy for in situ detection and characterization of hyperglycemia-induced abnormalities in the Descemet’s membrane, in the posterior cornea.

Methodology/Principal Findings

We studied corneas from age-matched control and Goto-Kakizaki rats, a spontaneous model of type 2 diabetes, and corneas from human donors with type 2 diabetes and without any diabetes. SHG imaging was compared to confocal microscopy, to histology characterization using conventional staining and transmitted light microscopy and to transmission electron microscopy. SHG imaging revealed collagen deposits in the Descemet’s membrane of unstained corneas in a unique way compared to these gold standard techniques in ophthalmology. It provided background-free images of the three-dimensional interwoven distribution of the collagen deposits, with improved contrast compared to confocal microscopy. It also provided structural capability in intact corneas because of its high specificity to fibrillar collagen, with substantially larger field of view than transmission electron microscopy. Moreover, in vivo SHG imaging was demonstrated in Goto-Kakizaki rats.

Conclusions/Significance

Our study shows unambiguously the high potential of SHG microscopy for three-dimensional characterization of structural abnormalities in unstained corneas. Furthermore, our demonstration of in vivo SHG imaging opens the way to long-term dynamical studies. This method should be easily generalized to other structural remodeling of the cornea and SHG microscopy should prove to be invaluable for in vivo corneal pathological studies.     

Perfectly registered multiphoton and reflectance confocal video rate imaging of in vivo human skin

We present a multimodal in vivo skin imaging instrument that is capable of simultaneously acquiring multiphoton and reflectance confocal images at up to 27 frames per second with 256 × 256 pixel resolution without the use of exogenous contrast agents. A single femtosecond laser excitation source is used for all channels ensuring perfect image registration between the two‐photon fluorescence (TPF), second harmonic generation (SHG), and reflectance confocal microscopy (RCM) images. Images and videos acquired with the system show that the three imaging channels provide complementary information in in vivo human skin measurements. In the epidermis, cell boundaries are clearly seen in the RCM channel, while cytoplasm is better seen in the TPF imaging channel, whereas in the dermis, SHG and TPF channels show collagen bundles and elastin fibers, respectively. The demonstrated fast imaging speed and multimodal imaging capabilities of this MPM/RCM instrument are essential features for future clinical application of this technique. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

High-resolution nonlinear optical imaging of live cells by second harmonic generation

By adapting a laser scanning microscope with a titanium sapphire femtosecond pulsed laser and transmission optics, we are able to produce live cell images based on the nonlinear optical phenomenon of second harmonic generation (SHG). Second harmonic imaging (SHIM) is an ideal method for probing membranes of living cells because it offers the high resolution of nonlinear optical microscopy with the potential for near-total avoidance of photobleaching and phototoxicity. The technique has been implemented on three cell lines labeled with membrane-staining dyes that have large nonlinear optical coefficients. The images can be obtained within physiologically relevant time scales. Both achiral and chiral dyes were used to compare image formation for the case of single- and double-leaflet staining, and it was found that chirality plays a significant role in the mechanism of contrast generation. It is also shown that SHIM is highly sensitive to membrane potential, with a depolarization of 25 mV resulting in an approximately twofold loss of signal intensity.     

Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections

We performed second harmonic generation (SHG) imaging of collagen in rat-tendon cryosections, using femtosecond laser scanning confocal microscopy, both in backscattering and transmission geometries. SHG transmission images of collagen fibers were spatially resolved due to a coherent, directional SHG component. This effect was enhanced with the use of an index-matching fluid (n(i) = 1.52). The average SHG intensity oscillated with wavelength in the backscattered geometry (isotropic SHG component), whereas the spectral profile was consistent with quasi-phase-matching conditions in transmission geometry (forward propagating, coherent SHG component) around 440 nm (lambda(p) = 880 nm). Collagen type I from bovine Achilles tendon was imaged for SHG in the backscattered geometry and its first-order effective nonlinear coefficient was determined (|d(eff)| approximately 0.085(+/-0.025)x10(-12)mV(-1)) by comparison to samples of inorganic materials with known effective nonlinear coefficients (LiNbO3 and LiIO3). The SHG spectral response of collagen type I from bovine Achilles tendon matched that of the rat-tendon cryosections in backscattered geometry. Collagen types I, II, and VI powders (nonfibrous) did not show any detectable SHG, indicating a lack of noncentrosymmetric crystalline structure at the molecular level. The various stages of collagen thermal denaturation were investigated in rat-tendon cryosections using SHG and bright-field imaging. Thermal denaturation resulted in the gradual destruction of the SHG signal.     

Three-Dimensional High-Resolution Second-Harmonic Generation Imaging of Endogenous Structural Proteins in Biological Tissues

We find that several key endogenous protein structures give rise to intense second-harmonic generation (SHG)—nonabsorptive frequency doubling of an excitation laser line. Second-harmonic imaging microscopy (SHIM) on a laser-scanning system proves, therefore, to be a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of live cell and tissue architecture. Unlike fluorescence, SHG suffers no inherent photobleaching or toxicity and does not require exogenous labels. Unlike polarization microscopy, SHIM provides intrinsic confocality and deep sectioning in complex tissues. In this study, we demonstrate the clarity of SHIM optical sectioning within unfixed, unstained thick specimens. SHIM and two-photon excited fluorescence (TPEF) were combined in a dual-mode nonlinear microscopy to elucidate the molecular sources of SHG in live cells and tissues. SHG arose not only from coiled-coil complexes within connective tissues and muscle thick filaments, but also from microtubule arrays within interphase and mitotic cells. Both polarization dependence and a local symmetry cancellation effect of SHG allowed the signal from species generating the second harmonic to be decoded, by ratiometric correlation with TPEF, to yield information on local structure below optical resolution. The physical origin of SHG within these tissues is addressed and is attributed to the laser interaction with dipolar protein structures that is enhanced by the intrinsic chirality of the protein helices.     

Cover Picture: J. Biophotonics 10/2012

Pixel‐resolution mapping of collagen fiber spatial orientation in bovine leg tendon (upper row), chicken skin (middle row) and chicken leg tendon (bottom row) was achieved using polarization‐resolved SHG microscopy. Shown in the left column are SHG intensity images acquired by circularly polarized femtosecond laser. In addition, maps of fiber azimuthal angles are shown in the middle column. Finally, SHG image at different depths for bovine tendon (right column, upper panel) and fiber elevation angle maps for chicken skin and chicken leg tendon are shown in right column. Individual image size: 120 × 120 mm². (Picture: V. A. Hovhannisyan et al., pp. 768–776 in this issue)