双光子3D组织切割成像系统-TissueSurgeon
双光子3D组织切割成像系统-TissueSurgeon

双光子3D组织切割成像系统-TissueSurgeon

——OCT图像引导的组织和材料的非接触精确切割,更适合切小鼠胫骨等小鼠骨骼系统,牙齿的激光切片设备


德国LLS ROWIAK公司的TissueSurgeon是一款专门设计的快速、方便、灵活的组织切片机设备。该设备使用高速高能激光系统,能够对样品实施如同外科手术般精准的非接触式切割。其独特的多光子切割技术有别于目前市场上的任何产品,能够从样品中的任意位置开始,直接在指定的样品部位直接进行切割并且不会对样品部位造成灼伤。

双光子3D组织切割成像系统-TissueSurgeon是一种多用途切片制备仪器,可以精确和无接触地切割生物样品、生物材料及其他材料。基于飞秒激光技术,Tissuesurgeon可用于二维/三维组织和材料的切片、结构化或温和提取。设备克服了传统机械切割的限制,对硬组织、植入组织或难以切割的材料也能轻松应对。

应用领域


■  骨科方面,尤其是非脱钙硬组织和种植体界面研究

■  心脏病学和心血管研究与医学,尤其是软组织与生物材料和支架,钙化斑块研究

■  再生医学与组织工程学,尤其是植入物、支架等研究

■  口腔、面部和牙科医学,尤其是非脱钙硬组织,金属、陶瓷或聚合物植入物研究

■  耳鼻喉相关研究,如耳蜗、耳蜗植入物等

■  从小鼠到大型动物模型的临床前研究等


为何选用TissueSurgeon?


■  样本损失小:几乎连续切片非脱钙硬组织,无需大深度磨片,材料损失小;

■  难切割样本:硬组织、软组织、软硬结合组织切割,甚至脆弱的样本(如耳蜗)切割;

■  适合界面研究:种植体组织界面组织学(如牙钉、心血管支架、支架);

■  无接触切割:无接触激光切割组织可避免挤压、划伤或裂纹等;

■  用3D切片方法可以沿着牙钉种植体-组织界面对特定部位的样本进行定向、温和的分离;

■  切割过程不会污染、灼伤或机械力损伤样品,可用于生物化学分析的无污染和无接触样品的制备;

■  用于组织工程的生物材料切割(如支架、聚四氟乙烯、水凝胶);

■  组织,基质和材料的3D微结构切割;

■  薄切片厚度:硬组织切片10 µm;

■  切片速度:≥1 mm²/s;

■  光源类型:红外飞秒脉冲激光;

■  光学相干断层扫描(OCT)引导切割,可以测量样品尺寸和层厚,并能够高效定位到病变部位,直接对病变部位进行切割,大大提高了切割效率。



TissueSurgeon产品升级!激光组织学的新维度:超大尺寸和可调节


可以实现最大6.6 cm样本切割


SizeSlide SizeSample Size
Extra Large76 x 102 mm (3 inch x 4 inch)up to 66 x 66 mm
Double Standard76 x 52 mm (3 inch x 2 inch)up to 42 x 42 mm
Standard76 x 26 mm (3 inch x 1 inch)up to 32 x 20 mm





■  TissueSurgeon可视化切片系统,实现边看边切


对于病理等多种研究来说,涉及到组织切片, 困难的部分莫过于寻找病变部位。 相比一个完整组织来说, 有时候研究者所关注的部分仅仅是其中变异的一小部分组织的形态而已。 但是对于传统切片手段来说, 缺乏一种有效的手段来定位这个区域, 因此往往需要投入大量人力和物力去多次制样,大量切片来寻找这个部位。 TissueSurgeon 自身集成了适合深层组织细胞成像的光学相干断层扫描(OCT)成像功能, 帮助您直接定位到 ROI 区域。 让切片变得可视化, 实现更加精准和可控的切片,为研究者更加迅速直观的找到病变位置,大大提高了研究效率。


大鼠膝关节的OCT成像


大鼠膝关节的OCT 3D重构



对含有金属钉的骨骼进行成OCT成像,并引导切片




■  原位细胞3D切割成像技术基于青鳉胚胎组织的单细胞提取


单细胞的原位组织提取一直以来都是一项十分困难的工作,这主要受制于组织之间连接致密难以消化,而机械力往往很难精确地将单个细胞与组织完整的分离。激光切割具有传统切割技术所难以匹及的切割精度,是目前一种比较理想的切割手段,因此围绕激光切割技术的相关显微产品也孕育而生,并在科研领域中越来越受到关注。但是激光切割也有其局限性,首先显微激光切割往往要从表面开始,无法对深层组织进行切割;另一方面激光的光源往往采用紫外激光光源,这种类型的光源很容易造成组织灼伤,从而影响切割下来样品的品质,因此激光切割的应用发展也受到了诸多限制。


如今ROWIAK公司推出的一款全新的单细胞分离系统有望解决这一难题。它采用了近红外双光子激光切割技术,在保留了激光切割精度优势的同时,采用近红外波长的激光从而避免了激光切中对组织灼烧的问题。因此能够实现精准的原位组织中的单个细胞的分离。


  

双光子3D组织切割成像系统TissueSurgeon


 

发育中的青鳉胚胎


 

青鳉是一种成熟的模式生物,常用于分析发育和发育过程中的细胞信号神经生物学研究。其中使用表达荧光蛋白的转基因胚胎是一种揭示胚胎发育的良好方法。随着基因技术的发展,研究者们越来越多地开始关注这些标记细胞中转录组中的信息。虽然单细胞测序技术发展迅速,但是从组织中获得单细胞的手段却十分有限。目前几乎没有手段能够直接在组织的原位上快速获取一个细胞,但是基于ROWIAK双光子切割技术,研究者成功地在这方面取得了一些进展。


青鳉胚胎中感知神经中表达mcherry的细胞成像


研究者为了研究青鳉感觉神经分泌细胞细胞群中特定表达m-cherry的转基因细胞的内部遗传信息,将ROWIAK双光子3D组织切割成像系统与传统的显微操作系统进行结合,成功实现了对目标细胞的原位分离。


研究者首先利用双光子3D组织切割成像系统对青鳉胚胎中的mcherry细胞进行了定位,然后根据其细胞群的形态设定了切割部位,随后系统根据预先设定的范围进行切割。待切割完成后使用玻璃微管移液器将目标的细胞部位直接取出,即获得了目标组织区域。这种方法能够在不破坏样品原位信息的情况下将感兴趣的部位直接精准的分离,这对于揭示生物体的基因表达情况具有着深远的意义。


从青鳉胚胎中分离特定表达mcherry的细胞团

 

参考文献:
Wittbrodt, J. et al. Medaka — a model organism from the Far East. Nature Reviews Genetics 3, 53-64.
Yamamoto, T. (ed.) MEDAKA (Killifish): Biology and strains. Yamamoto, T. (ed.) Keigaku Pub. Co., Tokyo, 1975, pp.365.
Kristin Tessmar-Raible et al.Removal of fluorescently-labeled sensory-neurosecretory cells from forebrain of transgenic Medaka embryos, focusonmicroscop. 2011.



染色(缩写)

染色

图像

描述

ABFR

阿尔新蓝-核固红


狗,唾液腺:

核仁:红色

微酸粘蛋白:蓝色


ABFR

阿尔新蓝-核固红


大鼠股骨(未脱钙):

软骨细胞外基质:蓝色


CF

纤维蛋白-卡斯塔莱斯


兔血管:

纤维蛋白:亮红色

血小板:灰到深蓝色

胶原:亮蓝色

肌肉:红色

红细胞:明黄色


EVG

Elastica Van Gieson染色


兔,带支架血管:

核:褐色

结缔组织:黄色

弹性纤维:紫色

肌肉:红色

Plasma:红色


EO

伊红


狗爪(未脱钙):

骨细胞,荧光


HE

苏木精和伊红


带支架兔冠状动脉:

核:蓝色

其余组织:红色


LL

Levai-Laczko染色


羊骨连接处(未脱钙):

核:violett-blue

细胞质:蓝色

红细胞:深蓝色

软骨基质:亮蓝色

骨基质:鲜红色

类骨质:紫色

纤维:蓝紫色

McN

McNeil Tetra Chrome染色


狗胫骨(未脱钙):

骨:粉红色/红色

细胞和细胞核:蓝色

软骨:紫色

结缔组织:红/粉红色


MG

Masson Goldner Trichchrome with light green and anilin blue染色


小鼠股骨(未脱钙),生长板:

骨:绿色

类骨质:橙色

软骨:粉红色

肌肉纤维:红色

胶原蛋白:绿色

细胞质:粉红色

核:棕色


MP

Movat Pentachrome染色


狗爪(未脱钙):

核仁:蓝-黑色

肌肉组织:红色

基质:蓝色

胶原:蛋白:黄色

软骨::蓝-绿色

弹性纤维:黑色

骨:黄-红色


Nissl

尼氏染色法


人脑:

核和尼氏体:

红紫罗兰色/紫罗兰色

细胞质和其他组织:

亮蓝色到亮紫罗兰色


Sirius

天狼星红


人主动脉斑块:

纤维组织:红色


 

SRS

Sanderson Rapid Stain染色


鼠下颌骨(未脱钙):

骨和细胞核:蓝色


SRS   + VG

Sanderson Rapid Stain + van Gieson染色


大鼠股骨(未脱钙),生长板:

骨:粉红色

骨髓细胞:蓝色到紫色

生长板软骨:红色


VEL

Verhoeffs Elastica染色

兔,带支架血管:

弹性纤维:黑色

其余组织:红色


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中国人民解放军军事医学科学院


University of Iowa Carver College of Medicine


HAWK University of Applied Sciences and Arts


German Heart Centre of the Technical University Munich


Georgia Institute of Technology, School of Chemistry and Biochemistry


Rostock University Medical Center, Department of Ophthalmology-1,-2


Rostock University Medical Center, Experimental Pediatrics Group-3


Queen Mary University of London


University of Gothenburg, BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy-1


University of Gothenburg, Department of Clinical Chemistry and Transfusion-2


alizée pathology, LLC (now: StageBio)


Ratliff Histology Consultants, LLC



小鼠脑切割


切割