原位細(xì)胞3D切割成像平臺(tái)-CellSurgeon

應(yīng)用領(lǐng)域

■  染色體切割

■  亞細(xì)胞器的實(shí)時(shí)觀(guān)測(cè)切割

■  原位組織的單細(xì)胞分離

■  薄組織的顯微切割

■  基于激光的光轉(zhuǎn)染技術(shù)

CellSurgeon切割原理

CellSurgeon將近紅外超短脈沖激光器耦合到顯微鏡中，并利用高數(shù)值孔徑物鏡聚焦超短激光脈沖，僅在最小的聚焦體積內(nèi)產(chǎn)生高強(qiáng)度能量引起多光子吸收，然后以非常精確的方式在活細(xì)胞中實(shí)現(xiàn)亞細(xì)胞水平的細(xì)胞結(jié)構(gòu)可視化操作。由于幾乎沒(méi)有熱能或機(jī)械能傳遞，靠近激光束緊焦點(diǎn)的細(xì)胞結(jié)構(gòu)依舊保持完好無(wú)損。

CellSurgeon的切割方式

為何選用CellSurgeon？

     ■  多光子實(shí)時(shí)成像追蹤

     ■  的3D切割

     ■  無(wú)需前處理即可直接切割

     ■  直接的原位切割

     ■  活細(xì)胞或組織均可直接切割

     ■  大限度保存生物信息的完整

     ■  能夠兼容多種型號(hào)的顯微鏡

基本參數(shù)

■  激光：飛秒近紅外激光，單波長(zhǎng)或可變

■  掃描器：雙獨(dú)立掃描鏡

■  掃描精度：700 X 700 ~ 300 X 100

■  最大分辨率：700 X 700（1,43 f/s）

■  最大掃描速度：300 X 100（10 f/s）

■  切割模式：不同波長(zhǎng)的2D或3D精準(zhǔn)手動(dòng)或自動(dòng)切割

■  控制器：驅(qū)動(dòng)所有機(jī)動(dòng)單元：顯微鏡、掃描器、 z驅(qū)動(dòng)器、掃描臺(tái)以及所有相關(guān)配件

數(shù)據(jù)測(cè)試

■  動(dòng)脈激光切割和成像

30 fps超短激光脈沖對(duì)小鼠血管的損傷

體內(nèi)激光誘導(dǎo)血栓的三維重建，采用FITC-葡聚糖染色雙光子成像監(jiān)測(cè)激光損傷后血栓的形成情況

■  肌動(dòng)蛋白絲的切割

用飛秒激光切割肌動(dòng)蛋白細(xì)絲

■  有絲分裂紡錘體的亞細(xì)胞解剖

GBP標(biāo)記的有絲分裂紡錘體，光漂白(A)和切割消融(B)

■  細(xì)胞器消融

不同功率激光對(duì)核的消融，激光消融前(A)和后(B)

線(xiàn)粒體消融，激光消融前(左)和后(右)

■  從細(xì)胞到組織的動(dòng)態(tài)觀(guān)測(cè)與切割

CellSurgeon能夠勝任各種類(lèi)型的切割任務(wù)，無(wú)論是的染色質(zhì)還是活體組織，它都能很好地勝任。

該設(shè)備可以兼容多種型號(hào)的顯微鏡，并且支持顯微操作針等配件，能夠在切割后實(shí)現(xiàn)對(duì)切割部分的轉(zhuǎn)移。

從細(xì)胞團(tuán)中切除的細(xì)胞并用微毛細(xì)管將提取細(xì)胞切出

白蟻的組織切割

小鼠活體血管切割

 ■  基于激光的原位細(xì)胞轉(zhuǎn)染

無(wú)論是電轉(zhuǎn)還是脂質(zhì)體都需要先將細(xì)胞懸浮才能夠進(jìn)行入轉(zhuǎn)染，但是Cellsurgeon能夠在原位對(duì)細(xì)胞進(jìn)行光穿孔實(shí)現(xiàn)細(xì)胞的轉(zhuǎn)染，這種技術(shù)對(duì)于研究原位的細(xì)胞轉(zhuǎn)染有著重大意義。

使用CellSurgeon對(duì)ZMTH3細(xì)胞進(jìn)行轉(zhuǎn)染pEGFP-C1、pEGFP-HMGA2、pEGFP-HMGB1經(jīng)過(guò)48小時(shí)的圖像

發(fā)表文章

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3. Géraud FORESTIER, Jonathan CORTESE, Sylvia M. BARDET, Maxime BAUDOUIN, Kévin JANOT, Voahirana RATSIMBAZAFY, Marie-Laure PERRIN, Jérémy MOUNIER, Claude COUQUET, Catherine YARDIN, Yan LARRAGNEGUY, Flavie SOUHAUT, Romain CHAUVET, Alexis BELGACEM, Sonia BRISCHOUX, Julien MAGNE, Charbel MOUNAYER, Faraj TERRO, Aymeric ROUCHAUD. (2023) “Comparison of Arterial Wall Integration of different Flow Diverters in rabbits” the CICAFLOW study Journal of Neuroradiology, In press. 

4. Donath, S?ren, Leon Angerstein, Lara Gentemann, Dominik Müller, Anna E. Seidler, Christian Jesinghaus, André Bleich, Alexander Heisterkamp, Manuela Buettner, and Stefan Kalies. (2022). “Investigation of Colonic Regeneration via Precise Damage Application Using Femtosecond Laser-Based Nanosurgery” Cells 11, no. 7: 1143. https://doi.org/10.3390/cells11071143 

5. Müller, Dominik, S?ren Donath, Emanuel G. Brückner, Santoshi Biswanath Devadas, Fiene Daniel, Lara Gentemann, Robert Zweigerdt, Alexander Heisterkamp, and Stefan M.K. Kalies. (2021). “How Localized Z-Disc Damage Affects Force Generation and Gene Expression in Cardiomyocytes” Bioengineering 8, no. 12: 213. https://doi.org/10.3390/bioengineering8120213 

6. Müller D, Klamt T, Gentemann L, Heisterkamp A, Kalies SMK (2021) Evaluation of laser induced sarcomere micro-damage: Role of damage extent and location in cardiomyocytes. PLoS ONE 16(6): e0252346. https://doi.org/10.1371/journal.pone.0252346

7. Bouyer M; Garot C; Machillot P; Vollaire J; Fitzpatrick V; Morand S; Boutonnat J; Josserand V; Bettega G; Picart C (2021) 3D-printed scaffold combined to 2D osteoinductive coatings to repair a critical-size mandibular bone defect Materials Today Bio 11 100113 

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11. Neuerburg C, Mittlmeier L M, Keppler A M, Westphal I, Glass ?, Saller M M, Herlyn P K E, Richter H, B?cker W, Schieker M, Aszodi A, Fischer D C (2019) Growth factor-mediated augmentation of long bones: evaluation of a BMP-7 loaded thermoresponsive hydrogel in a murine femoral intramedullary injection model. Journal of Orthopaedic Surgery and Research 14 297 

12. Kunert-Keil C, Richter H, Zeidler-Rentzsch I, Bleeker I, Gredes T (2019) Histological comparison between laser microtome sections and ground specimens of implant-containing tissues. Annals of Anatomy 222 153–157 

13. Gabler C, Sa? JO, Gierschner S, Lindner T, Bader R, Tischer T (2018) In Vivo Evaluation of Different Collagen Scaffolds in an Achilles Tendon Defect Model. BioMed Research International 208

14.    Wolkers W, Vásquez-Rivera A, Oldenhof H, Dipresa D, Goecke T, Kouvaka  A, Will F, Haverich A, Korossis S, Hilfiker A (2018) Use of sucrose to diminish pore formation in freeze-dried heart valves. Scientific Reports 8 12982 

15. Albers J, Markus MA, Alves F, Dullin C (2018) X-ray based virtual histology allows guided sectioning of heavy ion stained murine lungs for histological analysis. Scientific Reports 8(1) 7712

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17.    Pobloth AM, Checa S, Razi H, Petersen A, Weaver JC, Schmidt-Bleek K, Windolf M, Tatai Aá, Roth CP, Schaser KD, Duda GN, Schwabe P (2018) Mechanobiologically optimized 3D titanium-mesh scaffolds enhance bone regeneration in critical segmental defects in sheep. Science Translational Medicine 10 423

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20. Kowtharapu BS, Marfurt C, Hovakimyan M, Will F, Richter H, Wree A, Stachs O, Guthoff RF (2017) Femtosecond laser cutting of human corneas for the subbasal nerve plexus evaluation. Journal of Microscopy 265(1) 21–26

21. Will F, Richter H (2015) Laser-based Preparation of Biological Tissue. Laser Technik Journal 12(5) 44-47

22. Richter H, Ratliff J, Will F, Stolze B (2015) Time- and material saving laser microtomy for hard tissue and implants. European Cells and Materials 29 Suppl.2 4

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

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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

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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

部分用戶(hù)單位：

Bayer HealthCare, Cardiovascular Research

Leibniz University Hannover, Institute of Biophysics

Leibniz University Hannover, Institute for Quantum Optics-1，-2

University of Rostock, Division of Medicine Clinic III, Hematology, Oncology and Palliative Medicine

Institute for Bioprocessing and Analytical Measurement Techniques (iba)

mfd Diagnostics GmbH 

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