2 - 광자 현미경을 사용하여 마우스 Thymus의 Intravital 영상
Summary
We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of “niches” within the thymus where T cell development occurs.
Abstract
두 광자 현미경 (TPM)은 조직과 장기 깊숙한 지역에서 이미지 수집을 제공합니다. 새로운 stereotactic 도구 및 수술 절차의 개발과 함께, TPM은 장기 내부의 '틈새 시장'을 파악하고, 살아있는 동물의 세포 "행동"을 문서화하는 강력한 기술이됩니다. intravital 이미징이 가장 장기 내부의 실제 세포 동작과 유사한 정보를 제공하고 있지만, 더 힘드는 및 기술적 대안 전직 생체내 이미징 인수보다 필요한 장비 / 절차의 측면에서 까다로운 둘 수 있습니다. 따라서, 우리는 수술과 우리가 thymus 내에서 Foxp3의 움직임 + 세포를 수행하실 수 있습니다 소설 "stereotactic"장기 보유자에 대해 설명합니다.
Foxp3은 규제 T 세포 (Tregs)의 생성에 대한 마스터 조절기입니다. 예 : 또한, 이러한 세포들은 원산지에 따라 분류하실 수 있습니다. thymus – 차별화된 Tregs는 O로 "자연 – 발생 Tregs"(nTregs)를 부릅니다peripherally – 변환 Tregs (pTregs)에 pposed. 연구의 상당 금액이 T 세포의 표현형 및 생리학에 관한 문헌에서보고되었습니다 있지만, 거의 자신의 다른 세포와 생체내 상호 작용에 대한 알려져있다. 이 결핍은 관찰을 허용 것이 기술의 부재로 인해 수 있습니다. 이 문서에서 설명하는 프로토콜은 이러한 상황에 대한 구제 수단을 제공합니다.
우리 프로토콜들은 thymic 상피 세포 등 일부 상피 세포의 차별을 떨어뜨리고 DNA 순서에 엄격 돌연변이 때문에 내생 thymus 부족 누드 마우스를 사용하여 구성되어 있습니다. 누드 마우스는 감마 조사되었으며 Foxp3 – KI GFP / GFP 마우스에서 뼈 marrows (BM)와 재구성. BM 복구 (6 주) 후, 각각의 동물은 신장 캡슐 내부의 배아 thymus 이식을 받았습니다. thymus 수용 (6 주) 후, 동물 anesthetized 있었다, 신장을 포함하는이식 thymus는 노출의 장기 보유자에 고정하고, TPM에 의해 생체내 이미징에 대한 생리적 조건 하에서 보관되었습니다. 우리는 규제 T 세포의 생성에 약물의 영향을 연구하기 위해이 방법을 사용하고 있습니다.
Protocol
Discussion
In this paper we demonstrated the procedures for two-photon imaging of thymocytes inside a living animal. We also described some parameters that one should carefully control, such as the continuation of blood flow and the maintenance of organ temperature during the imaging procedures. Nonetheless, despite careful efforts to keep the organ stable, motion artifacts such as “organ drifting” can occur. Posterior image correction can be performed by the development of algorithms specifically designed for this purpose. Further image analysis could also be the source of new protocols development which seeks to minimize errors.
The thymus is the organ where all T cells are produced and, therefore, it is the organ where immunologists interested in understanding the generation of γδ, CD4, or CD8 T cells will focus their attention. Most studies concerning T cells are based upon differences in the numbers and/or stability of these cells after different in vitro/in vivo manipulations. However, only after the in vivo visualization we could observe the interaction between cells of the immune system involved in maintaining homeostasis3-7. Therefore, the in vivo observation of thymocytes is probably one of the most important missing information to better understand T cell biology. Intravital TPM provides a detailed picture of T cell movements and interactions and we demonstrate here how it can be used for detailed thymocyte studies. However, every technique has its limitations. While intravital imaging acquisition is the most accurate system for reflecting cells behavior inside the body, it is also true that explanted image acquisition of organs is less laborious and has been used to collect important information about the immune system8,9. Moreover, one cannot deny intravital imaging methods require surgery to expose tissues and blood vessels in anesthetized animals, which per se could cause an alteration in the whole organ physiology10. Nevertheless, there are non-invasively methods that abolish the artifacts caused by the surgical procedure11 and new methods are being developed that better prepare in advance the animals to be used12. Therefore, new surgical procedures and tolls will minimize or bypass actual limitations of intravital imaging acquisition and become more and more accessible to the scientific community.
We have demonstrated that the method we have described is feasible and it reports all in vivo systemic manipulations, such drug administration, that we have used. Thus, we suggest the use of this method together with ex vivo techniques already available in order to complement and strengthen further studies concerning thymocytes development.
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We would like to thank Dr. David Olivieri for critical review of this manuscript, Dr. Nuno Moreno for the logistic help to build our animal holder and heating pads and Dr. Vijay K. Kuchroo for the kind donation of Foxp3-KIgfp/gfp mice. This work is supported by “Fundação para Ciência e Tecnologia” (FCT, Portugal), grant # PTDC/EBB-BIO/115514/2009.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Rhodamine B ishothiocyanate-Dextran | Sigma-Aldrich | R9379 | prepare stock at 20 mg/ml |
| Two-photon microscope | Prairie Technologies Inc. | Prairie Ultima X-Y | |
| Ti:Sapphire laser | Coherent, Inc. | Chameleon Ultra Family | |
| 20x/1.00 NA immersion objective | Olympus Inc. | XLUMPLFLN 20XW | |
| Holder (Filters/Dichroic) | Chroma Technology Corp. | 91018 BX2 (U-MF2) | |
| 525 nm/50 filter | Chroma Technology Corp. | ET525/50m | |
| 595 nm/50 filter | Chroma Technology Corp. | ET595/50m | |
| 565 nm dichroic | Chroma Technology Corp. | 565dcxr | |
| Imaris software | Bitplane AG Inc. | Imaris | |
| Volocity | PerkinElmer Inc. | Volocity | |
| ImageJ | NIH, USA | ImageJ |
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