Laboratory of Immune Regulation, Instituto Gulbenkian de Ciência
Caetano, S. S., Teixeira, T., Tadokoro, C. E. Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy. J. Vis. Exp. (59), e3504, doi:10.3791/3504 (2012).
Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.
1. Animal preparation
Important notes: BM cell suspensions and thymic transplantations were performed in aseptic conditions. The BM suspensions were prepared inside the hoods of our cell culture room, while the thymic transplantation was performed in the surgical room located within our animal facility. In order to keep and guarantee these aseptic conditions, we were not allowed to record our video in these places. However, all the surgical materials were autoclaved and the surgical bench was previously cleaned with Virkon (Pharmacal Research Laboratories Inc.) and 70% ethanol. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and they were in agreement with the Federation of European Laboratory Animal Science Associations (FELASA) directives. The approval ID number is AO10/2010. All the image experiments were terminal procedures and the animals were euthanized immediately following the end of image acquisition.
The detailed procedure is as follows:
2. Intravital image acquisition
Prepare all the materials you will need in advance and put them aside.
Note: pictures of the aluminum foil clip and the top heater are presented in Figure 3.
3. Two-photon imaging acquisition
We used an upright "Prairie Ultima X-Y" two-photon microscope. Our system is equipped with a Ti:Sapphire laser, four top PMTs for simultaneous up to 4 channel acquisitions and a 20x water immersion objective.
4. Representative Results
Figure 1. Embryonic thymus acceptance and function. After BM recover, embryonic thymuses were transplanted to BM reconstituted animals; two weeks after thymus transplantation, these mice were bled once per week to monitor the percentages of T cell subtypes by staining with anti-CD4 or anti-CD8 MAbs. We considered the thymus was successfully accepted in animals with a CD4:CD8 ratio around 1.5-2.0:1 (A). To confirm its functionality, we sacrificed some thymus-transplanted animals and compared the percentage of different thymocyte subpopulations with WT mice (B). The percentages of double-negative (DN; CD4-CD8- thymocytes) subpopulations (by staining with anti-CD25 and anti-CD44 MAbs), double-positive (DP; CD4+CD8+ thymocytes) and single-positive (SP) CD4+ or CD8+ thymocytes were similar between these animals.
Figure 2. Animal holder assembly. After deeply anesthetized, the animal is put on top of a heating pad, previously mounted on top of the animal holder stage (A). The kidney containing the transplanted thymus is facing up (B). An aluminum foil clamp delicately pinches the whole kidney (C) to keep it in place. Fig. 1C insert shows in detail the clamp. The top part of the animal holder is put in place (D). The PBS-soaked cotton is removed from the top of the organ, replaced by warm low-melting agarose, and the top heater is added (E). Finally, the whole assembly is transferred to the microscope, here represented by the objective (F).
Figure 3. Details of holder parts. These pictures show the aluminum foil clamp (A) holding the kidney (B). Note also the region where the transplanted thymus is located. This approximately indicates the region to cut the thymus capsule and make the pocket to put the embryonic thymus. The top heater coverglass (C) was fixed with silicone glue to its bottom part (D).
Movie 1. Intravital imaging of Foxp3-GFP+ thymocytes inside the thymus where the physiological levels of oxygenation and temperature (37°C) were kept during the whole process. Note the blood flow and the movement of Tregs. These speeds can be measured and compared with published data. Click here to view the movie.
Movie 2. Intravital imaging of Tregs inside a thymus where the images were acquired at 30°C. Note the absence of movement and the round shape of the cells despite the maintenance of blood flow. Click here to view the movie.
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.
We have nothing to disclose.
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.
|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|