雙光子3D組織切割成像系統(tǒng)-TissueSurgeon
——OCT圖像引導(dǎo)的組織和材料的非接觸精確切割,更適合切小鼠脛骨等小鼠骨骼系統(tǒng),牙齒的激光切片設(shè)備
| 德國(guó)LLS ROWIAK公司的TissueSurgeon是一款專(zhuān)門(mén)設(shè)計(jì)的快速、方便、靈活的組織切片機(jī)設(shè)備。該設(shè)備使用高速高能激光系統(tǒng),能夠?qū)悠穼?shí)施如同外科手術(shù)般精準(zhǔn)的非接觸式切割。其獨(dú)特的多光子切割技術(shù)有別于目前市場(chǎng)上的任何產(chǎn)品,能夠從樣品中的任意位置開(kāi)始,直接在指定的樣品部位直接進(jìn)行切割并且不會(huì)對(duì)樣品部位造成灼傷。 雙光子3D組織切割成像系統(tǒng)-TissueSurgeon是一種多用途切片制備儀器,可以精確和無(wú)接觸地切割生物樣品、生物材料及其他材料?;陲w秒激光技術(shù),Tissuesurgeon可用于二維/三維組織和材料的切片、結(jié)構(gòu)化或溫和提取。設(shè)備克服了傳統(tǒng)機(jī)械切割的限制,對(duì)硬組織、植入組織或難以切割的材料也能輕松應(yīng)對(duì)。 |
應(yīng)用領(lǐng)域
■ 骨科方面,尤其是非脫鈣硬組織和種植體界面研究
■ 心臟病學(xué)和心血管研究與醫(yī)學(xué),尤其是軟組織與生物材料和支架,鈣化斑塊研究
■ 再生醫(yī)學(xué)與組織工程學(xué),尤其是植入物、支架等研究
■ 口腔、面部和牙科醫(yī)學(xué),尤其是非脫鈣硬組織,金屬、陶瓷或聚合物植入物研究
■ 耳鼻喉相關(guān)研究,如耳蝸、耳蝸植入物等
■ 從小鼠到大型動(dòng)物模型的臨床前研究等
為何選用TissueSurgeon?
■ 樣本損失?。簬缀踹B續(xù)切片非脫鈣硬組織,無(wú)需大深度磨片,材料損失小;
■ 難切割樣本:硬組織、軟組織、軟硬結(jié)合組織切割,甚至脆弱的樣本(如耳蝸)切割;
■ 適合界面研究:種植體組織界面組織學(xué)(如牙釘、心血管支架、支架);
■ 無(wú)接觸切割:無(wú)接觸激光切割組織可避免擠壓、劃傷或裂紋等;
■ 用3D切片方法可以沿著牙釘種植體-組織界面對(duì)特定部位的樣本進(jìn)行定向、溫和的分離;
■ 切割過(guò)程不會(huì)污染、灼傷或機(jī)械力損傷樣品,可用于生物化學(xué)分析的無(wú)污染和無(wú)接觸樣品的制備;
■ 用于組織工程的生物材料切割(如支架、聚四氟乙烯、水凝膠);
■ 組織,基質(zhì)和材料的3D微結(jié)構(gòu)切割;
■ 薄切片厚度:硬組織切片10 μm;
■ 切片速度:≥1 mm2/s;
■ 光源類(lèi)型:紅外飛秒脈沖激光;
■ 光學(xué)相干斷層掃描(OCT)引導(dǎo)切割,可以測(cè)量樣品尺寸和層厚,并能夠定位到病變部位,直接對(duì)病變部位進(jìn)行切割,大大提高了切割效率。
設(shè)備參數(shù)
TissueSurgeon產(chǎn)品升級(jí)!激光組織學(xué)的新維度:超大尺寸和可調(diào)節(jié)
可以實(shí)現(xiàn)最大6.6 cm樣本切割
| Size | Slide Size | Sample Size |
| Extra Large | 76 x 102 mm (3 inch x 4 inch) | up to 66 x 66 mm |
| Double Standard | 76 x 52 mm (3 inch x 2 inch) | up to 42 x 42 mm |
| Standard | 76 x 26 mm (3 inch x 1 inch) | up to 32 x 20 mm |
應(yīng)用案例
■ TissueSurgeon可視化切片系統(tǒng),實(shí)現(xiàn)邊看邊切
對(duì)于病理等多種研究來(lái)書(shū),涉及到組織切片的內(nèi)容, 困難的部分莫過(guò)于尋找病變部位。 相比一個(gè)完整組織來(lái)說(shuō), 有時(shí)候研究者所關(guān)注的部分僅僅是其中變異的一小部分組織的形態(tài)而已。 但是對(duì)于傳統(tǒng)切片手段來(lái)說(shuō), 缺乏一種有效的手段來(lái)定位這個(gè)區(qū)域, 因此往往需要投入大量人力和物力去多次制樣,大量切片來(lái)尋找這個(gè)部位。 TissueSurgeon 自身集成了適合深層組織細(xì)胞成像的光學(xué)相干斷層掃描(OCT)成像功能, 幫助您直接定位到 ROI 區(qū)域。 讓切片變得可視化, 實(shí)現(xiàn)更加和可控的切片。為研究者更加迅速直觀的找到病變位置,大大提高了研究效率。
大鼠膝關(guān)節(jié)的OCT成像 | 大鼠膝關(guān)節(jié)的OCT 3D重構(gòu) |
對(duì)含有金屬釘?shù)墓趋肋M(jìn)行成OCT成像,并引導(dǎo)切片
■ 原位細(xì)胞3D切割成像技術(shù)基于青鳉胚胎組織的單細(xì)胞提取
單細(xì)胞的原位組織提取一直以來(lái)都是一項(xiàng)十分困難的工作,這主要受制于組織之間連接致密難以消化,而機(jī)械力往往很難地將單個(gè)細(xì)胞與組織完整的分離。激光切割具有傳統(tǒng)切割技術(shù)所難以匹及的切割精度,是目前一種比較理想的切割手段,因此圍繞激光切割技術(shù)的相關(guān)顯微產(chǎn)品也孕育而生,并在科研領(lǐng)域中越來(lái)越受到關(guān)注。但是激光切割也有其局限性,先顯微激光切割往往要從表面開(kāi)始,無(wú)法對(duì)深層組織進(jìn)行切割;另一方面激光的光源往往采用紫外激光光源,這種類(lèi)型的光源很容易造成組織灼傷,從而影響切割下來(lái)樣品的品質(zhì),因此激光切割的應(yīng)用發(fā)展也受到了諸多限制。
如今ROWIAK公司推出的一款全新的單細(xì)胞分離系統(tǒng)有望解決這一難題。它采用了近紅外雙光子激光切割技術(shù),在保留了激光切割精度優(yōu)勢(shì)的同時(shí),采用近紅外波長(zhǎng)的激光從而避免了激光切中對(duì)組織灼燒的問(wèn)題。因此能夠?qū)崿F(xiàn)的原位組織中的單個(gè)細(xì)胞的分離。
雙光子3D組織切割成像系統(tǒng)TissueSurgeon | 發(fā)育中的青鳉胚胎 |
青鳉是一種成熟的模式生物,常用于分析發(fā)育和發(fā)育過(guò)程中的細(xì)胞信號(hào)神經(jīng)生物學(xué)研究。其中使用表達(dá)熒光蛋白的轉(zhuǎn)基因胚胎是一種揭示胚胎發(fā)育的良好方法。隨著基因技術(shù)的發(fā)展,研究者們?cè)絹?lái)越多地開(kāi)始關(guān)注這些標(biāo)記細(xì)胞中轉(zhuǎn)錄組中的信息。雖然單細(xì)胞測(cè)序技術(shù)發(fā)展迅速,但是從組織中獲得單細(xì)胞的手段卻十分有限。目前幾乎沒(méi)有手段能夠直接在組織的原位上快速獲取一個(gè)細(xì)胞,但是基于ROWIAK雙光子切割技術(shù),研究者成功地在這方面取得了一些進(jìn)展。
青鳉胚胎中感知神經(jīng)中表達(dá)mcherry的細(xì)胞成像
研究者為了研究青鳉感覺(jué)神經(jīng)分泌細(xì)胞細(xì)胞群中特定表達(dá)m-cherry的轉(zhuǎn)基因細(xì)胞的內(nèi)部遺傳信息,將ROWIAK雙光子3D組織切割成像系統(tǒng)與傳統(tǒng)的顯微操作系統(tǒng)進(jìn)行結(jié)合,成功實(shí)現(xiàn)了對(duì)目標(biāo)細(xì)胞的原位分離。
研究者先利用雙光子3D組織切割成像系統(tǒng)對(duì)青鳉胚胎中的mcherry細(xì)胞進(jìn)行了定位,然后根據(jù)其細(xì)胞群的形態(tài)設(shè)定了切割部位,隨后系統(tǒng)根據(jù)預(yù)先設(shè)定的范圍進(jìn)行切割。待切割完成后使用玻璃微管移液器將目標(biāo)的細(xì)胞部位直接取出,即獲得了目標(biāo)組織區(qū)域。這種方法能夠在不破壞樣品原位信息的情況下將感興趣的部位直接的分離,這對(duì)于揭示生物體的基因表達(dá)情況具有著深遠(yuǎn)的意義。
從青鳉胚胎中分離特定表達(dá)mcherry的細(xì)胞團(tuán)
參考文獻(xiàn):
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.
測(cè)試數(shù)據(jù)
染色(縮寫(xiě)) | 染色 | 圖像 | 描述 |
ABFR | 阿爾新藍(lán)-核固紅 | 狗,唾液腺: 核仁:紅色 微酸粘蛋白:藍(lán)色 | |
ABFR | 阿爾新藍(lán)-核固紅 | 大鼠股骨(未脫鈣): 軟骨細(xì)胞外基質(zhì):藍(lán)色 | |
CF | 纖維蛋白-卡斯塔萊斯 | 兔血管: 纖維蛋白:亮紅色 血小板:灰到深藍(lán)色 膠原:亮藍(lán)色 肌肉:紅色 紅細(xì)胞:明黃色 | |
EVG | Elastica Van Gieson染色 | 兔,帶支架血管: 核:褐色 結(jié)締組織:黃色 彈性纖維:紫色 肌肉:紅色 Plasma:紅色 | |
EO | 伊紅 | 狗爪(未脫鈣): 骨細(xì)胞,熒光 | |
HE | 蘇木精和伊紅 | 帶支架兔冠狀動(dòng)脈: 核:藍(lán)色 其余組織:紅色 | |
LL | Levai-Laczko染色 | 羊骨連接處(未脫鈣): 核:violett-blue 細(xì)胞質(zhì):藍(lán)色 紅細(xì)胞:深藍(lán)色 軟骨基質(zhì):亮藍(lán)色 骨基質(zhì):鮮紅色 類(lèi)骨質(zhì):紫色 纖維:藍(lán)紫色 | |
McN | McNeil Tetra Chrome染色 | 狗脛骨(未脫鈣): 骨:粉紅色/紅色 細(xì)胞和細(xì)胞核:藍(lán)色 軟骨:紫色 結(jié)締組織:紅/粉紅色 | |
MG | Masson Goldner Trichchrome with light green and anilin blue染色 | 小鼠股骨(未脫鈣),生長(zhǎng)板: 骨:綠色 類(lèi)骨質(zhì):橙色 軟骨:粉紅色 肌肉纖維:紅色 膠原蛋白:綠色 細(xì)胞質(zhì):粉紅色 核:棕色 | |
MP | Movat Pentachrome染色 | 狗爪(未脫鈣): 核仁:藍(lán)-黑色 肌肉組織:紅色 基質(zhì):藍(lán)色 膠原:蛋白:黃色 軟骨::藍(lán)-綠色 彈性纖維:黑色 骨:黃-紅色 | |
Nissl | 尼氏染色法 | 人腦: 核和尼氏體: 紅紫羅蘭色/紫羅蘭色 細(xì)胞質(zhì)和其他組織: 亮藍(lán)色到亮紫羅蘭色 | |
Sirius | 天狼星紅 | 人主動(dòng)脈斑塊: 纖維組織:紅色 | |
SRS | Sanderson Rapid Stain染色 | 鼠下頜骨(未脫鈣): 骨和細(xì)胞核:藍(lán)色 | |
SRS + VG | Sanderson Rapid Stain + van Gieson染色 | 大鼠股骨(未脫鈣),生長(zhǎng)板: 骨:粉紅色 骨髓細(xì)胞:藍(lán)色到紫色 生長(zhǎng)板軟骨:紅色 | |
VEL | Verhoeffs Elastica染色 | 兔,帶支架血管: 彈性纖維:黑色 其余組織:紅色 |
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23. Richter H, Ramirez Ojeda DF, Will F (2014) Lasergesteuerte Probenpr?paration von Hartgeweben und Biomaterialien. BIOspektrum 05 14
24. Bourassa D, Gleber S-C, Vogt S, Yi H, Will F, Richter H, Shin CH, Fahrni CJ (2014) 3D Imaging of Transition Metals in the Zebrafish Embryo by X-ray Fluorescence Microtomography. Metallomics 6 1648-1655
25. Schimek K, Busek M, Brincker S, Groth B, Hoffmann S, Lauster R, Lindner G, Lorenz A, Menzel U, Sonntag F, Walles H, Marx U, Horland R. (2013) Integrating biological vasculature into a multi-organ-chip microsystem. Lab Chip 13 3588-3598
26. Richter H, Ratliff J (2012) A Non-Contact Method of Sectioning Cardiovascular Arteries Containing Metallic Stents Using Laser Technology. J Histotechnol 35 (4) 205
27. Richter H, Lubatschowski H, Will F (2011) Laser in Medizin & Biologie: Laser-Mikrotomie mit ultrakurzen Pulsen – Neue Perspektiven für die Gewebe- und Biomaterialbearbeitung. Biophotonik 09 50-52
28. Lubatschowski H, Will F, Przemeck S, Richter H (2011) Laser Microtomy. Handbook of Biophotonics Vol. 2: Photonics for Health Care Wiley-VCH 151-157
29. Kermani O, Will F, Massow O, Oberheide U, Lubatschowski H (2010) Control of Femtosecond Thin-flap LASIK Using OCT in Human Donor Eyes. Journal of Refractive Surgery 26(1) 57-61
30. Baumgart J, Bintig W, Ngezahayo A, Lubatschowski H, Heisterkamp A (2010) Fs-laser-induced Ca2+ concentration change during membrane perforation for cell transfection. Optics Express 18 (3) 2219
31. Kermani O, Will F, Massow O, Oberheide U, Lubatschowski H. (2009) Echtzeitsteuerung einer Femtosekundenlaser Sub-Bowman-Keratomileusis an humanen Spenderaugen mittels optischer Koh?renztomographie. Klin Monatsbl Augenheilkd 226 965-969
32. Kütemeyer K, Baumgart J, Lubatschowski L, Heisterkamp A (2009) Repetition rate dependency of low density plasma effects during femtosecond-laser-based surgery of biological tissue. Appl. Phys. B 97(3) 695
33. Baumgart J, Kuetemeyer K, Bintig W, Ngezahayo A, Ertmer W, Lubatschowski H, Heisterkamp A (2009) Repetition rate dependency of reactive oxygen species formation during femtosecond laser-based cell surgery. J Biomed Opt 14(5) 054040
34. Kermani O, Will F, Lubatschowski H (2008) Real-Time Optical Coherence Tomography-Guided Femtosecond Laser Sub-Bowman Keratomileusis on Human Donor Eyes. Am J Ophthalmol 146 42–45.
35. Kermani O (2008) ?Sehendes Skalpell” schon heute realisierbar. Ophthalmologische Nachrichten 09 (Kongressausgabe)
36. Baumgart J, Bintig W, Ngezahayo A, Willenbrock S, Murua Escobar H, Ertmer W, Lubatschowski H, Heisterkamp A (2008) Quantified femtosecond laser based opto-perforation of living GFSHR-17 and MTH53a cells. Opt. Express 16(5) 3021-3031
37. Baumgart J, Kuetemeyer K, Bintig W, Ngezahayo A, Ertmer W, Lubatschowski H, Heisterkamp A (2008) Investigation of reactive oxygen species in living cells during femtosecond laser based cell surgery. Proc. SPIE Optical Interactions with Tissue and Cells XIX Vol 6854
38. Heisterkamp A, Baumgart J, Maxwell IZ, Ngezahayo A, Mazur E, Lubatschowski H (2007) Fs-Laser Scissors for Photobleaching, Ablation in Fixed Samples and Living Cells, and Studies of Cell Mechanics. Laser Manipulation of Cells and Tissues; Elsevier Inc. 293-307
39. Will F, Block T, Menne P, Lubatschowski H (2007) Laser Microtome: all optical preparation of thin tissue samples. Proceedings of SPIE 6460 646007-1
40. Lubatschowski H (2007) Laser Microtomy – Opening a new Feasibility for Tissue Preparation. Optic & Photonic WILEY-VCH 49 – 51
41. Menne P (2007) Microtomy with Femtosecond Lasers. Biophotonics International; Laurin Publishing Co. Inc. May 2007 35 – 37
用戶(hù)單位
中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)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 |
