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 "Funda&ccedil;&atilde;o para Ci&ecirc;ncia e Tecnologia" (FCT, Portugal), grant # PTDC/EBB-BIO/115514/2009.

1. 동물 준비 중요 사항 : BM 세포 정지와 thymic transplantations는 무균 조건에서 수행되었다. thymic 이식은 우리의 동물 시설 내에있는 수술 방에서 수행하는 동안 BM 정지는, 우리의 세포 배양 실의 후드 안쪽 준비했다. 이러한 무균 조건을 유지하고 보장하기 위해, 우리는이 장소에서 우리의 동영상을 녹화할 수 없습니다되었습니다. 그러나 모든 수술 자료는 autoclaved되었으며 수술 벤치는 이전에 다음 ID로 Virkon (Pharmacal 연구소 연구소 주식 회사)와 70 % 에탄올로 세척했다. 모든 절차는 기관 동물 케어에 의해 승인위원회 (IACUC)를 사용하여 그들은 유럽의 실험실 동물 과학 협회 (FELASA) 지시어의 연맹과 계약에있었습니다. 승인 ID 번호는 AO10/2010입니다. 모든 이미지 실험은 터미널 성형 수술과 동물은 이미지의 끝에 바로 다음 euthanized되었습니다인수. 다음과 같은 세부적인 절차는 다음과 같습니다 <ol><li> 비추다 (γ - irradiator 포함) 뼈 marrows (BM) 내부의 내생 조혈 엽 성의 전구 물질을 파괴 900 래드와 8주 오래된 누드 마우스. 당신은 정맥 주사를 더욱 BM reconstitution에 대한 기증자 BM 세포를 준비하기 전까지 조사 후, 자신의 새장에 동물을 반환합니다. 이 BM 전송은 조사 후 1 H을 수행했다. </li><li> BM 기증자 동물을 분리합니다. 한 동물의 뒷다리 사지 Tibias과 대퇴골은 일반적으로 최대 3받는 동물 reconstitute 충분히 있습니다. CO 2와 안락사 후, 두 다리를 제거한 후 뼈가에서 피부와 근육을 제거하십시오. </li><li> 각 뼈의 폐쇄 끝을 잘라하고 BM을 제거하기 위해 PBS (또는 RPMI) 2 ML과 유봉을 사용하여, PBS (또는 RPMI)로 플러시 또는 절구에 모두를 부술. </li><li> 활발한 흡인 usi의 반복에 의한 단일 셀 중단에 BM 구조를 방해NG P1000의 피펫. 또는, 당신은 또한 21G 주사 바늘을 통해 여러 번 BM을 전달할 수 있습니다. 원심 분리기 (400g, 5 분, RT)은 PBS로 세포를 다시 중지, 그리고 누드 - 조사받는 사람으로 정맥 주입. 우리의 실험에서 우리는 규제 T 세포 (Tregs)는 GFP 1 표현됩니다 Foxp3 - KI GFP / GFP 마우스에서 BM 사용됩니다. </li><li> 비 BM - 재구성 동물들이 이상 십사일 면할 수 없다는 이후 기증자 BM의 수락, 이전 후 첫 일 이내에 발생합니다. 그러나, 하나는 모두 BM 구획의 완전 회복을 허용하도록 4~6주 기다려야한다. Bactrim은 (Roche는) 세균 감염의 위험을 감소하기 위해 조사 후 첫 주 동안 물 (2 MG / ML)에 추가되었습니다. </li><li> 배아 thymus 이식은 이미 2 설명했다. 간단히, isogenic 생쥐의 쌍을 사육 시작하고 매일 (성교의 증거) 연결해 있는지 확인합니다. 별도의 연결의 여자와 공동 pregn 하루에 15을 2 - 안락사ancy (플러그의 관찰은 임신 1 일입니다.) 배아와 thymuses을 제거합니다. 이전까지 차가운 PBS에서 배아 thymuses를 놓습니다. </li><li> thymuses 이식, ketamin (120 마우스 중량 μg / g) / xylazine (마우스 중량의 16 μg / g)과 마취 BM -받는 누드 마우스를 수행하고 그들이 얻을 ° C까지 37 가열 패드 위에 그들을 유지하려면 무의식. </li><li> 수술 thymuses의 이식을 수행할 수있는 신장을 노출. 먼저 ChloraPrep (Carefusion 주식 회사)와 70 % 에탄올로 피부를 씻어. 그런 다음 백 측면 바디 동물의 부분 2mm 우는 지난 흉부 리브의 피부에 상처 1cm합니다. </li><li> 복막 구멍을 잘라 이런 곳에의 신장을 가져옵니다. 메스 블레이드와 신장 캡슐의 작은 피상적인 상처를 확인합니다. 매우 얇은 팁을와 집게를 사용하면받는 마우스의 신장 캡슐 밑에 주머니를 만들고이 주머니에 4 thymic 엽 (叶)까지 추가할 수 있습니다. </li><li> 신장 B를 넣고<html> 그 장소에 ACK, 하나 또는 두 개의 바늘로 복막 구멍을 봉합하고 같은 치료 방법을 사용하여 마우스 피부를 닫습니다. 또는 봉합 수술은 접착제를 사용하여 수행할 수 있습니다. </li><li> subcutaneously 바로 동물의 고통을 피할들이 마취에서 회복하기 위해 시작 때까지 가열 패드에있는 동물을 유지하기 위해 수술을 마친 후 (5 밀리그램 / kg에서 Butorphanol) 진통제 주사. 생쥐는 실밥을 제거 케이지 짝을을 방지하기 위해 이식 후 처음 3 일 동안 개별적으로 갇힌 수 있습니다. 일주 후 바늘을 제거합니다. </li><li> 누드 마우스는 T 세포를 필요가 없습니다. 따라서 혈액에 CD4 + CD8 + T 또는 세포의 비율을 모니터링하여 thymus 그래프트 동의 평가할 수 있습니다. 일단 주당, 이주 thymus 이식 후 동물 피가 및 안티 - CD4와 CD8 단클론 항 항체 (MAbs)로 혈액을 얼룩. CD4시 : CD8 비율이 약 2시 1분이며, 이식 thymus는 (그림 1) 완벽하게 작동합니다. </li></ol>&quot;&gt; 2. Intravital 이미지 수집 미리 필요로하고 그들을 버려 것입니다 모든 재료를 준비합니다. <ol><li> 37 ketamin / xylazine (항목 1.5에서 설명한 동일한 복용)와 동물 마취 및 난방 패드의 상단에 넣어 ° C. 혈액 흐름이 미래의 시각화를 허용 자에게 Rhodamine B isothiocyanate - Dextran (PBS에서 20 밀리그램 / ML) 100 μl를 주입. </li><li> 이전 항목 1.6에서 설명하는 프로토콜에 따라 이식 thymus를 포함하는 신장을 폭로. </li><li> 원래 위치로 복귀에서 신장을 방지하기 위해, 바늘로 피부 절개의 측면 측면을 닫습니다. </li><li> 그것이 촉촉한 유지하는 기관의 상단에 PBS - 절어 코튼 조각을 넣으십시오. </li><li> 동물 홀더 단계 (그림 2A)의 상단에있는 가열 패드를 넣어. </li><li> 동물 홀더, 장기 접속 (그림 2B)에서 가열 패드의 상단에있는 동물을 넣어. </li><li> 클램프 m로 양쪽에서 오르간을 핀치얇은 알루미늄 호일의 ADE (그림 2C). </li><li> 동물 홀더를 닫고 가까이 가능한 귀하의 조직 (그림 2D) 가기 히터 프로브를 놓습니다. 이 히터 프로브는 지속적으로 장기 온도를 측정하고 37 ° C.에 보관, 전기 전류를 조정하는 히터에이 정보를 전송 </li><li> PBS - 절어 목화를 제거하고 ° C 귀하의 준비 위에 30 낮은 용융 아가로 오스를 (PBS 2 %) 추가합니다. </li><li> 상단 히터 (그림 2E)로 전체 준비를 커버. 이 히터의 조리개에서 목표 (그림 2 층)에서 아가로 오스를 분리 coverglass있다. 마우스 마취를 모니터링하고 반 선량 부스트마다 40 분을 주입. 이미지 수집 후, CO 2와 함께 동물을 안락사. </li></ol> 참고 : 알루미늄 호일 클립 및 상단 히터의 사진은 그림 3에서 제공됩니다. 3. 두 광자 영상 획득 우리는 직립 &quot;프레리 울티마 XY&quot;두 광자 현미경을 사용. 우리 시스템은 티를 갖춘 : 사파이어 레이저, 동시 최대 4 채널 인수를위한 네 부류의 일급 PMTs 및 20x 물 침지 목표. <ol><li> 티 켜고 : 사파이어 레이저와 전체 현미경 시스템. </li><li> 원하는 파장에 따라 이색성 거울과 필터 세트를 추가합니다. 우리의 경우 우리는 565 nm의 이색성 거울을 포함하는 올림푸스 - BX2 채도 홀더, 하나 50분의 525 nm의 필터 (Foxp3 - GFP 신호에 대해) 한 50분의 595 nm의 필터 (혈관 안에서 Dextran - Rhodamine - B 신호에 사용 ). 사용되는 레이저 파장 범위는 880-900 nm의 사이되었습니다. </li><li> , 현미경으로 동물 홀더를 추가 연결하여 물로, 상단 히터 구멍에 물을 추가, 모든 히터에 조심스럽게 낮은 목표를 설정하고, 선박 또는 일부 GFP - Tregs에 중점을두고 있습니다. </li><li> 현미경의 X와 Y, Z 외부 컨트롤은 빠르게 영역을 찾을 수있는 조직을 조사<html> 관심. </li><li> 색상 분리를 최적화하고 배경을 통해 충분한 신호를 달성하는 데 필요한 레이저의 양을 최소화하기 위해 PMTs에 게인을 조정합니다. 당신의 조직 사진 손상을 방지하려면 레이저의 최소 금액을 사용해보십시오. </li><li> 관심의 영역이있는되면, 시간이 경과 이미징을 시작합니다. 우리의 경우 전형적인 5D에서 (X, Y, Z, T, 색상) 수집 프로토콜은 4.0 μm의 Z - 단계, X와 140 μm의의 Y 길이로 나누어 50 μm의 깊이있는 조직 볼륨의 연속 이미지를 인수로 구성되어 있습니다 각. 각 볼륨은 약 30 초이 취득 걸립니다. 따라서, 60 인수는 이미지 수집 중 약 30 분을 수행합니다. </li><li> 기관 서버로 데이터를 전송 및 다중 차원 렌더링과 셀 추적을 수행하는 ImageJ, Imaris 또는 Volocity 소프트웨어를 사용합니다. </li></ol> 4. 대표 결과 <html> g1.jpg &quot;/&gt; 그림 1. . 배아 thymus 접수 및 기능 BM 복구 후, 배아 thymuses는 BM 재구성 동물 이식했고, 이주 thymus 이식 후,이 생쥐는 안티 - CD4 또는 방지 CD8와 얼룩에 의한 T 세포 subtypes의 비율을 모니터 한 번 주당 출혈이 있었다 MAbs. 우리는 thymus가 성공적으로 CD4와 동물의 수락으로 간주 : 1.5-2.0:1 주변 CD8 비율 (A). 그 기능을 확인하려면, 우리는 몇 가지 thymus - 이식 동물을 희생하고 WT 생쥐 (B)와 다른 thymocyte subpopulations의 비율을 비교했다. subpopulations (안티 CD25 및 안티 CD44 MAbs와 얼룩에 의해), 이중 긍정적인 (DP; CD4 + CD8 + thymocytes), (- - CD8 thymocytes CD4 DN)과 단일 긍정적인 (SP) CD4 이중 부정의 비율 + 나 CD8 + thymocytes이 동물 사이에 유사한했다. ig2.jpg &quot;/&gt; 그림 2. 동물 홀더 어셈블리가. 깊게 anesthetized 후, 동물 가열 패드 위에 넣고, 이전에 동물 홀더 단계의 상단 (A)에 장착. 이식 thymus를 포함하는 신장은 (B)까지 직면하고 있습니다. 알루미늄 호일 클램프는 정교한 그 자리에 유지하기 위해 전체 신장 (C)를 pinches. 그림. 1C 삽입 상세 클램프를 보여줍니다. 동물 소유자의 상단 부분은 장소 (D)에 들어가게된다. PBS - 절어 목화는 따뜻한 낮은 용융 아가로 오스로 교​​체 기관의 최상위에서 제거하고, 상단 히터는 (E) 추가됩니다. 마지막으로, 온 회중은 여기 현미경으로 전송 목적 (F)으로 표시됩니다. <img alt="그림 3" src="/files/ftp_upload/3504/3504fig3.jpg" /> 그림 3. 홀더 부분의 세부 사항.이 사진은 알루미늄 호일 클램프를 (A)는 신장 (B)를 개최 보여줍니다. 이식 thymus가있는 지역도 있습니다. 이것은 약 영역을 나타냅니다thymus 캡슐을 잘라 배아 thymus 넣어 주머니를 만들 수 있습니다. 상단 히터 coverglass (C)는 자사의 하단 부분 (D)에 실리콘 접착제로 고정되었습니다. 영화 1. 산소 및 온도의 생리 수준 (37 ° C)이 전체 과정 동안 유지되었습니다 thymus 내부 Foxp3 - GFP + thymocytes의 Intravital 이미징. Tregs의 혈액 흐름과 움직임을합니다. 이러한 속도는 측정 및 게시된 데이터와 비교할 수 있습니다. <a href="/files/ftp_upload/3504/3504movie1.avi">동영상을 보려면 여기를 누르십시오.</a> 영화 2. 이미지가 30 취득했다 thymus 내부 Tregs의 Intravital 이미징 ° C. 혈액 흐름의 유지에도 불구하고 움직임과 세포의 둥근 모양의 부재가. 참고 <a href="/files/ftp_upload/3504/3504movie2.avi">동영상을 보려면 여기를 누르십시오.</a>

<ol>
	<li>Bettelli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+developmental+pathways+for+the+generation+of+pathogenic+effector+TH17+and+regulatory+T+cells.">Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells.</a> Nature. 441, 235-238 (2006).</li><li>Ramsdell, F., Z&#250;&#241;iga-Pfl&#252;cker, J. C., Takahama, Y., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+systems+for+the+study+of+T+cell+development:+fetal+thymus+organ+culture+and+OP9-DL1+cell+coculture.">In vitro systems for the study of T cell development: fetal thymus organ culture and OP9-DL1 cell coculture.</a> Current protocols in immunology. , (2006).</li><li>Tadokoro, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+T+cells+inhibit+stable+contacts+between+CD4++T+cells+and+dendritic+cells+in+vivo.">Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo.</a> J. Exp. Med. 203, 505-511 (2006).</li><li>Mempel, T. R., Henrickson, S. E., Von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+priming+by+dendritic+cells+in+lymph+nodes+occurs+in+three+distinct+phases.">T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases.</a> Nature. 427, 154-159 (2004).</li><li>Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+single+cell+dynamics+of+CD4++T+cell+activation+by+dendritic+cells+in+lymph+nodes.">Imaging the single cell dynamics of CD4+ T cell activation by dendritic cells in lymph nodes.</a> J. Exp. Med. 200, 847-856 (2004).</li><li>Shakhar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+T+cell-dendritic+cell+interactions+precede+the+development+of+both+tolerance+and+immunity+in+vivo.">Stable T cell-dendritic cell interactions precede the development of both tolerance and immunity in vivo.</a> Nat. Immunol. 6, 707-714 (2005).</li><li>Hugues, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+T+cell+dynamics+in+lymph+nodes+during+the+induction+of+tolerance+and+immunity.">Distinct T cell dynamics in lymph nodes during the induction of tolerance and immunity.</a> 5, 1235-1242 (2004).</li><li>Tang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+regulatory+T+cell+control+of+autoimmune+responses+in+nonobese+diabetic+mice.">Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice.</a> Nat. Immunol. 7, 83-92 (2006).</li><li>Le Borgne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+negative+selection+on+thymocyte+migration+in+the+medulla.">The impact of negative selection on thymocyte migration in the medulla.</a> Nature immunology. 10, 823-830 (2009).</li><li>Baez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+cremaster+muscle+preparation+for+the+study+of+blood+vessels+by+in+vivo+microscopy.">An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.</a> Microvascular research. 5, 384-394 (1973).</li><li>Wang, B., Zinselmeyer, B. H., McDole, J. R., Gieselman, P. A., Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+Imaging+of+Leukocyte+Homing+and+Migration+in+vivo.">Non-invasive Imaging of Leukocyte Homing and Migration in vivo.</a> J. Vis. Exp. (46), e2062-e2062 (2010).</li><li>Barretto, R. P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+imaging+of+disease+progression+in+deep+brain+areas+using+fluorescence+microendoscopy.">Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy.</a> Nature medicine. 17, 223-228 (2011).</li></ol>

우리는 공개 아무것도 없어.

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 &#x03B3;&delta;, 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.

<table border="1">
<thead>
<tr>
<td>Name of the reagent</td>
<td>Company</td>
<td>Catalogue number</td>
<td>Comments</td>
</tr>
</thead>
<tbody>
<tr>
<td>Rhodamine B ishothiocyanate-Dextran</td>
<td>Sigma-Aldrich</td>
<td>R9379</td>
<td>prepare stock at 20 mg/ml</td>
</tr>
<tr>
<td>Two-photon microscope</td>
<td>Prairie Technologies Inc.</td>
<td>Prairie Ultima X-Y</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ti:Sapphire laser</td>
<td>Coherent, Inc.</td>
<td>Chameleon Ultra Family</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>20x/1.00 NA immersion objective</td>
<td>Olympus Inc.</td>
<td>XLUMPLFLN 20XW</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Holder (Filters/Dichroic)</td>
<td>Chroma Technology Corp.</td>
<td>91018 BX2 (U-MF2)</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>525 nm/50 filter</td>
<td>Chroma Technology Corp.</td>
<td>ET525/50m</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>595 nm/50 filter</td>
<td>Chroma Technology Corp.</td>
<td>ET595/50m</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>565 nm dichroic</td>
<td>Chroma Technology Corp.</td>
<td>565dcxr</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Imaris software</td>
<td>Bitplane AG Inc.</td>
<td>Imaris</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Volocity</td>
<td>PerkinElmer Inc.</td>
<td>Volocity</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>ImageJ</td>
<td>NIH, USA</td>
<td>ImageJ</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>

intravital imaging of the mouse thymus using 2-photon microscopy

두 광자 현미경 (TPM)은 조직과 장기 깊숙한 지역에서 이미지 수집을 제공합니다. 새로운 stereotactic 도구 및 수술 절차의 개발과 함께, TPM은 장기 내부의 &#39;틈새 시장&#39;을 파악하고, 살아있는 동물의 세포 &quot;행동&quot;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 &quot;stereotactic&quot;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 &quot;자연 - 발생 Tregs&quot;(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.

Watch this Scientific Journal Video about Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy at JoVE.com

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

2 - 광자 현미경을 사용하여 마우스 Thymus의 Intravital 영상

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

intravital-imaging-of-the-mouse-thymus-using-2-photon-microscopy

Research

JoVE Journal

Immunology and Infection

16.9K 조회수.  Instituto Gulbenkian de Ciência. 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.

비디오: Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

사타구니 림프 노드의 Intravital 현미경

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

연구

면역학 및 감염병학

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

2 광자 현미경을 사용하여 감염된 폐의 Neutrophils에 의해 Phagocytosis와 NET - 형성의 직접 관측

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

비장의 Intravital 현미경 : 기생충 이동성 및 혈류의 정량 분석

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

마우스 오금 림프절의 Intravital 이미징

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

어린 싹 센터의 높은 해결의 intravital 스트라이프 - 조명 현미경

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

다음 간 생체 내에 현미경 이미징<em&gt; 슈만 편모충</em&gt; 감염 : 간 혈류의 평가

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

CXCR6.Gfp 기자 마우스를 사용하여 건강하고 병에 걸린 간에서 면역 세포의 장기 생체 내에 다중 광자 현미경 이미징

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

분석<em&gt; 예 르시 니아의 enterocolitica</em숙주 세포 내로&gt; 이펙터 전좌 베타 - 락타 마제 이펙터의 용해 사용

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

실시간으로 마취 마우스에 생체 내에 현미경으로 장간막 혈관에서 호중구와 단핵구 이미징

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

Intravital Imaging of Neutrophil Priming Using IL-1&amp;#946; Promoter-driven DsRed Reporter Mice

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

IL-1β의 발기인 기반을 DsRed 기자 마우스를 사용 호중구 초벌의 생체 내에 영상

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...