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原位細胞3D切割成像平臺-CellSurgeon

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德國LLS ROWIAK公司推出的CellSurgeon是一款JZ、非接觸的3D納米激光活細胞顯微成像切割系統。它獨具特色的多光子切割技術,能夠從細胞內或組織內的任意點開始切割,實現真正意義上的定點操作。并且CellSurgeon還配有MPM成像模塊,能夠實現實時的熒光標記或無標記成像,JZ定位所需操作的部位和實時觀測細胞動態變化。

詳細介紹


原位細胞3D切割成像平臺-CellSurgeon


20210222-1428514782.jpg


       德國LLS ROWIAK公司推出的CellSurgeon是一款JZ、非接觸的3D納米激光活細胞顯微成像切割系統。它獨具特色的多光子切割技術,能夠從細胞內或組織內的任意點開始切割,實現真正意義上的定點操作。并且CellSurgeon還配有MPM成像模塊,能夠實現實時的熒光標記或無標記成像,JZ定位所需操作的部位和實時觀測細胞動態變化。通過CellSurgeon研究者能夠進行實時JZ的活細胞、組織操作和觀測,幫助研究者更好的研究原位細胞的生理活性。


應用領域

■  染色體切割

■  亞細胞器的實時觀測切割

■  原位組織的單細胞分離

■  薄組織的顯微切割

■  基于激光的光轉染技術


CellSurgeon切割原理


CellSurgeon將近紅外超短脈沖激光器耦合到顯微鏡中,并利用高數值孔徑物鏡聚焦超短激光脈沖,僅在小的聚焦體積內產生高強度能量引起多光子吸收,然后以非常精確的方式在活細胞中實現亞細胞水平的細胞結構可視化操作。由于幾乎沒有熱能或機械能傳遞,靠近激光束緊焦點的細胞結構依舊保持完好無損。

?

CellSurgeon的切割方式



雙光子切割 VS 單光子切割

可從組織中的任意部位開始切割


CellSurgeon的切割方式


為何選用CellSurgeon?

     ■  多光子實時成像追蹤

     ■  JZ的3D切割

     ■  無需前處理即可直接切割

     ■  直接的原位切割

     ■  活細胞或組織均可直接切割

     ■  大限度保存生物信息的完整

     ■  能夠兼容多種型號的顯微鏡

基本參數


■  激光:飛秒近紅外激光,單波長或可變

■  掃描器:雙獨立掃描鏡

■  掃描精度:700 X 700 ~ 300 X 100

■   Max大分辨率:700 X 700(1,43 f/s)

■  Max大掃描速度:300 X 100(10 f/s)

■  切割模式:不同波長的2D或3D手動或自動切割

■  控制器:驅動所有機動單元:顯微鏡、掃描器、 z驅動器、掃描臺以及所有相關配件


數據測試


30 fps超短激光脈沖對小鼠血管的損傷


體內激光誘導血栓的三維重建,采用FITC-葡聚糖染色雙光子成像監測激光損傷后血栓的形成情況


■  肌動蛋白絲的切割

用飛秒激光切割肌動蛋白細絲


■  有絲分裂紡錘體的亞細胞解剖

GBP標記的有絲分裂紡錘體,光漂白(A)和切割消融(B)


■  細胞器消融

不同功率激光對核的消融,激光消融前(A)和后(B)


線粒體消融,激光消融前(左)和后(右)


■  從細胞到組織的動態觀測與切割


CellSurgeon能夠勝任各種類型的切割任務,無論是的染色質還是活體組織,它都能很好地勝任。

該設備可以兼容多種型號的顯微鏡,并且支持顯微操作針等配件,能夠在切割后實現對切割部分的轉移。


從細胞團中切除的細胞并用微毛細管將提取細胞切出


固定的CHO的Alexa488標記的毒傘素切割


 

活U2OS細胞的FP635標記的肌動蛋白的切割


活GM-7373牛主動脈內皮細胞的

MitoTracker Orange ?的單線粒體消融實驗

 

 

活GM-7373牛主動脈內皮細胞誘導凋亡實驗




人發絲切割


 染色質切割


激光介導的細胞轉染



白蟻的組織切割


小鼠活體血管切割



 ■  基于激光的原位細胞轉染

 

無論是電轉還是脂質體都需要先將細胞懸浮才能夠進行入轉染,但是Cellsurgeon能夠在原位對細胞進行光穿孔實現細胞的轉染,這種技術對于研究原位的細胞轉染有著重大意義。


使用CellSurgeon對ZMTH3細胞進行轉染pEGFP-C1、pEGFP-HMGA2、pEGFP-HMGB1經過48小時的圖像


■  動脈激光切割和成像

30 fps超短激光脈沖對小鼠血管的損傷


體內激光誘導血栓的三維重建,采用FITC-葡聚糖染色雙光子成像監測激光損傷后血栓的形成情況


■  肌動蛋白絲的切割

用飛秒激光切割肌動蛋白細絲


■  有絲分裂紡錘體的亞細胞解剖

GBP標記的有絲分裂紡錘體,光漂白(A)和切割消融(B)


■  細胞器消融

不同功率激光對核的消融,激光消融前(A)和后(B)


線粒體消融,激光消融前(左)和后(右)


■  從細胞到組織的動態觀測與切割


CellSurgeon能夠勝任各種類型的切割任務,無論是的染色質還是活體組織,它都能很好地勝任。

該設備可以兼容多種型號的顯微鏡,并且支持顯微操作針等配件,能夠在切割后實現對切割部分的轉移。


從細胞團中切除的細胞并用微毛細管將提取細胞切出


固定的CHO的Alexa488標記的毒傘素切割


 

活U2OS細胞的FP635標記的肌動蛋白的切割


活GM-7373牛主動脈內皮細胞的

MitoTracker Orange ?的單線粒體消融實驗

 

 

活GM-7373牛主動脈內皮細胞誘導凋亡實驗




人發絲切割


 染色質切割


激光介導的細胞轉染



白蟻的組織切割


小鼠活體血管切割



 ■  基于激光的原位細胞轉染

 

無論是電轉還是脂質體都需要先將細胞懸浮才能夠進行入轉染,但是Cellsurgeon能夠在原位對細胞進行光穿孔實現細胞的轉染,這種技術對于研究原位的細胞轉染有著重大意義。


使用CellSurgeon對ZMTH3細胞進行轉染pEGFP-C1、pEGFP-HMGA2、pEGFP-HMGB1經過48小時的圖像


發表文章

1. Nolte, P.; Brettmacher M.; Gr?ger, C. J.; Gellhaus, T.; Svetlove A.; Schilling, A. F.; Alves, A.; Ru?mann, C.; Dullin, C.; (2023) Spatial correlation of 2D hard?tissue histology with 3D microCT scans through 3D printed phantoms Sci Rep 13, 18479 

2.  Kevin Janot, Grégoire Boulouis, Géraud Forestier, Fouzi Bala, Jonathan Cortese, Zoltán Szatmáry, Sylvia M. Bardet, Maxime Baudouin, Marie-Laure Perrin, Jérémy Mounier, Claude Couquet, Catherine Yardin, Guillaume Segonds, Nicolas Dubois, Alexandra Martinez, Pierre-Louis Lesage, Yong-Hong Ding, Ramanathan Kadirvel , Daying Dai, Charbel Mounayer, Faraj Terro, Aymeric Rouchaud. (2023) WEB shape modifications: “angiography–histopathology correlations in rabbits” J NeuroIntervent Surg 2023;0:1–7. 

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 

8. Verhaegen C, Kautbally S, Zapareto D C, Brusa D, Courtoy G, Aydin S, Bouzin C, Oury C, Bertrand L, Jacques P J, Beauloye C, Horman S, Kefer J (2020) Early thrombogenicity of coronary stents: comparison of bioresorbable polymer sirolimus-eluting and bare metal stents in an aortic rat model. Am J Cardiovasc Dis. 10(2):72-83 

9. Zeller-Plumhoff B, Malicha C, Krüger D, Campbella G, Wiesea B, Galli S, Wennerberg A, Willumeit-R?mer R, Wieland F (2020) Analysis of the bone ultrastructure around biodegradable Mg–x Gd implants using small angle X-ray scattering and X-ray diffraction Acta Biomaterialia 101 637–645

10. Rousselle S D , Wicks J R, Tabb B C, Tellez A, O’Brien M (2019) Histology Strategies for Medical Implants and Interventional Device Studies Toxicologic Pathology Vol. 47(3) 235-249 

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

16. Boyde A (2018) Evaluation of laser ablation microtomy for correlative microscopy of hard tissues. Journal of Microscopy 271(8) 1-14

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

18. Joner M, Nicol P, Rai H, Richter H, Foin N, Ng J, Cuesta J, Rivero F, Serrano R, Alfonso F (2018) Very Late Scaffold Thrombosis: Insights from Optical Coherence Tomography and Histopathology. EuroIntervention 13(18)

19. Boyde A, Staines KA, Javaheri B, Millan JL, Pitsillides AA, Farquharson C (2017) A distinctive patchy osteomalacia characterises Phospho1 deficient mice. Journal of Anatomy 231 298-308

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

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




用戶單位

部分用戶單位:

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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