This work was supported by NIH grants R56DK084321 (AC), R01DK084321 (AC), R01DK111538 (AC), R01DK113093 (AC), and R21ES025673 (AC), and by the BEST/2015/043 grant (Conseller&#237;a de Educaci&#243;, cultura i esport, Generalitat valenciana, Valencia, Spain) (EO). Authors thank the SENT team at the Universidad Cat&#243;lica de Valencia San Vicente M&#225;rtir, Valencia, Spain and Alberto Hernandez at Centro de Investigaci&#243;n Pr&#237;ncipe Felipe, Valencia, Spain for their help with video filming and editing.

The Institutional Animal Care and Use Committee (IACUC) of the University of Miami approved all the experiments according to IACUC guidelines.
1. Isolation and Trimming of Newborn Thymi
<ol>
	<li>Prepare all reagents and instruments by autoclaving or other methods, ensuring sterile conditions.</li>
	<li>To minimize contaminations, perform all surgical procedures under a laminar flow hood.</li>
	<li>Prior to euthanizing donor mice, fill a 60 mm sterile dish with sterile prechilled 1x phosphate-buffered saline (PBS, pH 7.4) and place it on ice. It will be used for rinsing and storing the excised thymi from donor mice prior to the transplantation.</li>
	<li>Proceed to euthanize newborn donor mice by decapitation, in accordance with ethical guidelines.</li>
	<li>Place the mouse on sterile absorbent paper towels in a dorsal upright position and spray, and later wipe, the mouse abdomen with 70% ethanol.</li>
	<li>Expose the thoracic cavity by making a superficial V-shaped incision at the level of the lower abdomen and cut the skin with a pair of straight 10 cm dissecting scissors following a ventral midline to leave an opening of about 0.5-1&#160;cm at the chest level. Fold the skin over each side of the chest to expose the thoracic cavity.</li>
	<li>Make two deeper 0.5-1&#160;cm lateral incisions through the diaphragm and ribcage with the same type of scissors to access the superior mediastinum in the anterior thoracic cavity. The thymus should appear as two pale lobes right above the heart.</li>
	<li>Place a set of curved forceps underneath the thymus and pull vertically to extract the complete organ. Prevent the foldback of the ribcage with a pair of forceps. If needed, use fine forceps to carefully tear apart the connective tissue surrounding the organ without disrupting the capsule before extracting the organ. 
	NOTE: This step is facilitated by using a dissecting scope.</li>
	<li>Submerge the isolated thymus into cold sterile 1x PBS (pH 7.4) previously displayed into a 60 mm sterile dish, and cut the connective isthmus with a scalpel to separate the thymic lobes. Remove any debris from the dish without harming the capsule.To minimize the time thymi are exposed, trim thyme lobes right before implant. 
	NOTE: Each isolated newborn thymus will typically render six segments of about 1 mm wide for a maximum of three host mice when receiving implants in both eyes.</li>
	<li>Repeat Steps 1.4-1.9 for each donor mouse. To minimize the time thymi are exposed, isolate one thymus at a time.</li>
</ol>
2. Thymus Implantation into the Anterior Chamber of the Eye
<ol>
	<li>Prior to the transplantation, tag and weigh each recipient mouse.</li>
	<li>Use a dose between 1-2% of isofluorane vapors to anesthetize the recipient mouse. Ensure proper anesthetization by the absence of reflex following toe pinch before starting the surgical procedure.</li>
	<li>Proceed with thymus segments transplant as follows:
	<ol>
		<li>Place the mouse in a side lateral recumbent position so that one eye is facing up directly exposed to the lens of the dissecting scope.</li>
		<li>Excise one of the isolated thymic lobes lying in the cold 1x PBS (pH 7.4)-filled 60 mm dish into pieces with up to 1 mm width for implant. Follow a zig-zag pattern to ensure that each segment contains thymic cortex and medulla. Use vannas scissors for trimming according to guidelines provided in Figure 1A. 
		NOTE: Trimming of the thymus should be done right before implant to optimize tissue engraftment.</li>
		<li>Starting in the base of the cornea corresponding to the surgical area, introduce the tip of a 40 mm G18 needle to make a small incision into external cornea layers so that the dissecting scissors tip can be introduced.</li>
		<li>Make a 5-10 mm flank incision directly around the base of the cornea using vannas scissors while holding the cornea opening firmly to prevent resealing. Use forceps with flat ends to avoid tissue damage.</li>
		<li>Grasp the cut corneal epithelium and expose the opening by holding the cornea with a pair of flat-ended forceps while pushing a thymic segment through the opening. Wet the eye with sterile 1x PBS (pH 7.4) or artificial tears as needed to prevent tissue desiccation until the manipulation is complete.</li>
		<li>Press softly on the eye surface to slide the introduced tissue segment to a lateral position with respect to the pupil to preserve eye&#180;s function. Ensure that the graft lies at a location opposite to the eye opening to prevent posterior interference with imaging by hazing.</li>
		<li>With the aid of flat forceps, press firmly, for about 3-5 s, the two sides of the corneal opening against each other to promote self-sealing. The surgery does not require any stapling or sewing.</li>
	</ol>
	</li>
	<li>If the transplants are performed on both eyes, turn the mouse over the opposite lateral side to directly expose the second eye to the dissecting microscope lens and repeat Steps 2.3.2-2.3.7.</li>
	<li>After the surgery is completed, return the mouse to an empty cage prewarmed with a heat lamp.</li>
	<li>Make sure that each transplanted mouse regains complete mobility and consciousness before placing it into a cage shared with other animals. This usually takes place within 1 h after the surgery.</li>
	<li>Upon transplant, treat the mice with pain killers as needed, and provide the mice with acetaminophen at a concentration of 1.6 mg/mL in the drinking water.</li>
	<li>Repeat Steps 2.2-2.9 for each recipient mouse.</li>
	<li>Monitor post-operative animal activity as well as the appearance of the implanted eyes to detect potential health complications.</li>
</ol>
3. Confocal Imaging of Implanted Thymi using 3D Single Photon Fluorescence Confocal Microscopy
<ol>
	<li>Anesthetize the recipient mouse using a dose between 1-2% of isofluorane vapors. Ensure proper anesthetization by the absence of reflex following toe pinch before starting the imaging procedure</li>
	<li>Place the mouse on a fixed stage microscope platform in a side lateral recumbent position so that one eye is facing up. A heat pad is placed on the microscope platform to ensure constant body temperature of mice during recordings. 
	NOTE: See Supplementary Figure 1 for details.</li>
	<li>Insert the head of the mouse into a stereotaxic headholder and adjust the knob to restrain the mouse head on a lateral side position allowing direct access of the microscope objective to the eye holding the thymus graft.</li>
	<li>Place the mouse snout into a gas mask to keep the animal anesthetized throughout procedure. This allows the adjustment of isofluorane vapors as needed.</li>
	<li>To facilitate the stabilization of the eye during recordings while avoiding the retraction of the eyelids, pull back the eyelids while holding the eye at the corneal margin with a pair of tweezers that have their tips covered by a polythene tube which are attached to a UST-2 solid universal joint. This arrangement permits a steady fixation of the head and eye and provides flexibility without disruption of blood flow in the eye, as previously described22. 
	NOTE: Supplementary Figures 1B, C show the complete assembly.</li>
	<li>Add a few drops of sterile saline or artificial tears as the immersion liquid between the cornea and the lens, before placing the microscope objective on the mouse eye. 
	NOTE:Additional drops are dispensed on need throughout the procedure to keep the microscope path and prevent eye drying.</li>
	<li>First use low magnification (5X) lens to locate the thymus in the microscope field. Then switch to higher resolution (10, 20 and 40X) water immersion dipping objectives with long working distance. Avoid LSM photodamage and bleaching of the thymic implant by applying minimal laser power and reduce scanning time as much as possible. This is achieved by using the resonant scanner of the microscope. 
	NOTE: The confocal microscope we use is equipped with resonant scanning mirrors capable of gathering images at 25 frames/s. Other brands have their own microscopes with resonant scanners.</li>
	<li>Select the acquisition mode using the microscope software and start the resonant scanner mode. Then choose the XYZT imaging mode and configure the acquisition settings as follows:
	<ol>
		<li>Turn the Argon laser on and adjust the power to 30% for fluorescence excitation.</li>
		<li>Choose an excitation laser line and set the acousto-optical beam splitter (AOBS) control for different emission wavelengths. In addition, to detect backscatter and delineate tissue structure, use reflection detection simultaneously. 
		NOTE: Upon selecting a set of colors for excitation (GFP and RFP), the AOBS is automatically programmed to direct these excitation lines onto the specimen and transmit the emission between them. For instance, for GFP and RFP, we use narrow reflection bands for excitation light around 488 nm and 561 nm, respectively. This leaves broad bands for the collection of emitted fluorescence photons, thus reducing the need for laser power and acquisition time. In reflection mode, the AOBS is used as a 50/50 beam splitter to image reflected light in any wavelength away from the ones used for fluorescence emission detection.</li>
		<li>Collect the emission at selected wavelength, then select a resolution of 512&#215;512 pixels and start live imaging by pressing the Live button, adjusting the gain levels as needed (typical gain is around 600 V).</li>
		<li>Define the beginning and end of the z-stack by focusing on the top of the thymic implant and select Begin, then move to the last plane that can be focused in the implanted thymus and select End. Use a z-step size of 5 &#181;m. The software will automatically calculate the number of confocal planes.</li>
		<li>Choose the time interval for acquisition of each z-stack (Typically 1.5 to 2 seconds) and select the option Acquire until stopped for continuous imaging.</li>
	</ol>
	</li>
	<li>Press the Start button to initialize.</li>
	<li>Image the grafts repeatedly at different times from the same animal by repeating Steps 3.1-3.9. 
	NOTE: If the animal holds grafts on both eyes, recordings from either eye can be taken by repeating Steps 3.2-3.9&#160;after switching the upright position side of the mouse head.</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+lymphoid+tissues.">Evolution of lymphoid tissues.</a> Trends in Immunology. 33, 315-321 (2012).</li><li>Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Journey+through+the+thymus:+stromal+guides+for+T-cell+development+and+selection.">Journey through the thymus: stromal guides for T-cell development and selection.</a> Nature Reviews Immunology. 6 (2), 127-135 (2006).</li><li>Dzhagalov, I., Phee, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+find+your+way+through+the+thymus:+a+practical+guide+for+aspiring+T+cells.">How to find your way through the thymus: a practical guide for aspiring T cells.</a> Cellular and Molecular Life Sciences. 69 (5), 663-682 (2012).</li><li>James, K. 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The authors have nothing to disclose.

Due to the importance of the T-cell maturation process for individual immune competency4 and the presumed impact of precursor cell dynamics on mature T-cells produced by the thymus2,3, extensive efforts have been invested to develop alternatives to the classical fixed tissue snapshot approach.
Although tissue slices and other explants are clearly superior in reproducing tissue architecture than monolayers or aggregate thymic organ cultures14,15, the absence of a connection to the vasculature limits their use to study the steps of entry or TSP (translocation of progenitors across blood vessel endothelial cells barrier) and exit (translocation of mature T-cells across blood vessel endothelial cells barrier) from the organ. As it is hypothesized that the dynamics of T-cell contacts with other cells, including thymic stromal cells, and their motility across microenvironments within the cortex and the medulla determine their differentiation status and the process of subpopulation selection (positive and negative)2,3,4,5, and since these processes may depend on TSP and mature T-cell egress events, a model that includes the connection to the individual&#180;s blood flow system would be desirable.
Intradermal and kidney capsule implants of the thymus allow for this upgrade17,18. However, they constitute invasive methods with imposed limitations to imaging by TPLSM due to the deep position of the implant within the body or the presence of opaque layers of tissue above the graft. The adaptation of the mouse anterior eye chamber, previously used as a &#34;window&#34;21,22 to longitudinally monitor the performance of several tissues, as a site to track T-cells within a vascularized thymus provides several advantages versus other transplantation strategies. On the one side, the surgery is simpler because no sutures are required, leading to faster recovery of host animals. On the other side, the observation of implants does not require an additional surgery as it happens for kidney capsule implants, allowing for repeated recordings from the same animal. In addition, the particular anatomical characteristics of the eye allow to easily modulate regulatory inputs on the implanted tissue not only systemically but also locally. As previously described, with this approach, substances can be applied onto the eye topically or injected into the anterior eye chamber. It also represents an easy option to introduce substances or cells directly into the thymus (i.e., progenitors or maturation intermediates labeled ex vivo and adoptively transferred), which has traditionally been a highly complex procedure due to the thymus anatomic location24,25. Furthermore, the perfusion of the anterior chamber permits the exchange of the aqueous humor or loading of the graft by diffusion with fluorescent indicators, as previously shown for other type of implants26.
The approach that more faithfully allows the study of thymus physiology is provided by the intravital imaging of transgenic medaka fish expressing fluorescently labeled lymphocyte markers. The transparency of medaka during development and throughout life in many medaka strains makes this organism advantageous for monitoring cellular dynamics in vivo21. The low number of motile thymocytes observed in medaka (29%)18 in reference to mouse&#180;s (95%)13, in addition to differences in motility rates between these two organisms, however, suggests that the cold blood smallest vertebrate with an adaptive immune system may be limited at revealing particular mechanistic kinetics of higher vertebrate T-cell maturation events.
One limitation of the use of the anterior chamber of the mouse eye as the site for thymus transplantation is space restriction. Long-term studies might be, therefore, compromised; particularly for newborn or embryonic tissues with enlarged growth potential. In this sense, we must state that although newborn implants presented with apparent tissue expansion after the first week post-implant in the mouse eye (data not shown), the enlargement did not seem to put the integrity of the eye at risk up to two months post-implant. Nevertheless, it should be of interest to evaluate whether the tissue environment of the implant (eye chamber, kidney capsule or dermis) influences implant&#180;s growth and/or vascularization before the adequacy of the approach can be established for defined applications.
The large incision required on the cornea to allow the passage of the thymic fragment to the anterior eye chamber leads occasionally to some hazing at this level (data not shown), most likely related to certain degree of fibrosis associated to the healing process. Attention is demanded on Step 2.3.6 of the protocol to ensure that if hazing occurs will minimally interfere with the downstream imaging steps. As the incision is made at a lateral level, neither the implant nor the hazing should significantly affect animals sight performance.
The need for tissue trimming to make the thymus fit into the eye chamber appears to be a disadvantage if compared to other anatomic sites that do not require altering the anatomic integrity of the organ. Nevertheless, the kidney capsule approach has shown that the fragments of the thymus containing, both, cortex and medulla obtained by similar dissection guidelines to those shown in Figure 1, become fully functional as they can reconstitute T-cell mediated immunity in immunodeficient thymeless hosts18. This argues in favor of intact functional traits of this type of thymus segments. Also, the protocol described here stresses that the isolated thymus must be kept in cold (Step 1.9) and only trimmed right before implant (Step 2.3.2) to minimize cold ischemia and tissue exposure towards optimizing tissue engraftment.
An additional potential limitation of the approach resides in the composition differences between the aqueous humor in the anterior chamber of the eye and blood plasma, issue that needs to be kept in mind when interpreting observations made at this anatomic location. Previous studies with other tissues, however, have not reported any obvious effects on normal graft function by the local eye environment22. Although the experience with the model presented here is restricted to a few individuals (N = 6 and N = 3 for recipients &#38; donors, respectively) at present, we did not observe any major alterations associated to implant site in the case of thymic implants in any of the individuals either.
The protocol presented here constitutes a proof-of principle for the study of intravital longitudinal imaging of thymocyte dynamics by implanting thymic tissue into the anterior chamber of the mouse eye. Due to the selection of immunocompetent recipients, the system did not allow questioning the systemic function of the graft. Future studies may build on this approach by choosing different donors and/or recipients and/or by subjecting the hosts to irradiation and bone marrow (BM) transplantation according to particular study designs. For example, the choice of SCID (severe combined immunodeficiency) mice as recipients eliminates the interference of endogenous T-cells, and the choice of transgenic mice donors (thymic implant and/or BM) presenting T-cell markers, DC markers, thymic stromal cell-markers, etc., individually or in combinations should permit direct in vivo tracking of cellular subpopulations. Alternatively, subpopulations of cells in the anterior eye chamber of mice could be tracked, as previously described, by in vivo fluorescence cytolabeling26.
One major advantage of the model presented here is that it allows for studying progenitor and mature T-cells translocation from and towards blood vessels. Thus, it should be relevant to determine if the rapidly acquired vascularization of the thymus after being implanted in the anterior eye chamber recapitulates the endogenous vasculature. In favor of this possibility, it should be mentioned that the neovascularization in the anterior mouse chamber seems to be dictated by the graft18,20 rather than by the recipient and therefore the connections to the iris vasculature should preserve the integrity of endogenous thymus cortico-medullary junction (CMJ) domain.
An additional advantage comes from the fact that the system allows the evaluation of two independent implants (two anterior eye chambers) on identical individual background (a single host). Studies requiring combinations of tissues may also find it advantageous to use this system.
As the thymus plays an important role in autoimmune and immune deficient pathologies, this approach could be used to address a myriad of potential questions. In particular, the system should allow macroscopic in addition to intra-organ imaging for the progression of thymus involution. It may also serve to study transplantation tolerance events and immune dysfunctions related to infections. It is likely that the fine dissection of mechanisms governing thymocyte maturation will provide new clues for the design of additional therapeutic strategies targeting developing T cells.
Transplantation of the thymus to the anterior chamber of the eye takes less than 45 min, including neonatal thymus isolation and trimming for each host individual with implants in either eye. In vivo imaging recordings require 1 to 5 h, depending on monitored features.

Intrathymic T-cell differentiation and T-cell subpopulation selection constitute key processes for the development and maintenance of cell-mediated immunity in vertebrates1. This process involves a complex sequence of tightly organized events including the recruitment of progenitors from bloodstream, cell proliferation and migration, differential expression of membrane proteins, and massive programmed cell death for subsets selection. The result is the release of mature T-cells reactive to an ample spectrum of foreign antigens while displaying minimized responses to self-peptides, which end-up colonizing the peripheral lymphoid organs of the individual2,3. Aberrant thymocyte selection of the &#945;&#946;TCR repertoire leads to autoimmune disease or immune imbalance4 that mainly derive from&#160;defects during the processes of negative or positive precursor selection, respectively.
Directional migration of thymocytes across the thymus is intrinsic to all stages of T-cell maturation and it is envisaged as a series of simultaneous, or sequential multiple stimuli, including chemokines, adhesive, and de-adhesive extracellular matrix (ECM) protein interactions3,5. The study of fixed tissues has rendered critical information regarding the patterns of expression for thymocyte migratory cues in defined thymic microenvironments5,6, while ex vivo studies has revealed two prevalent migratory behaviors of thymocytes in two histologically distinct areas of the organ: slow stochastic movements in the cortex and fast, confined motility in the medulla7,8,9,10,11,12,13. Increased migratory rates correlate with thymic positive selection13 and negative selection is associated with crawling behavior supporting the hypothesis that the kinetics of the journey through the thymus determines proper maturation of thymocytes. Despite their relevance, the topology of thymocyte-stromal cell interactions and the dynamics of thymocyte motility across organ microenvironments during T-cell maturation remain ill-defined.
Most ex vivo studies performed to date include fetal or reaggregate thymic organ cultures14,15, tissue slices or intact thymic lobe explants where thymocyte movements are visualized by two-photon laser scanning microscopy (TPLSM)8, an intravital imaging technique with a restricted maximum working distance and imaging depth of 1 mm in accordance with the tissue examined16. In contrast to the laborious thymic organ cultures which depend on extended incubation times to form 3D-structures, both, the thymic slice technique and the intact thymic lobe approach permit controlled introduction of particular subsets of pre-labeled thymocytes into a native tissue architecture environment. However, since blood flow is absent in these models, they are clearly limited for studying the recruitment process of thymus settling progenitors (TSPs) to the thymus parenchyma or the dynamics of thymic egression of mature T-cells.
In vivo models for the study of thymic T-cell maturation physiology in mice include the grafts of fragments or entire organ lobes placed either inside the kidney capsule17 or intradermally18. Although these options showed their utility to interrogate systemic functional engraftment of the tissue, the position of thymic grafts deep within the animal or covered by layers of opaque tissue restricts their use for in vivo examination of implants by TPLSM.
The anterior chamber of the eye provides an easily accessible space for direct monitoring of any grafted tissue by virtue of the transparency of corneal layers. Of advantage, the base of the chamber formed by the iris is rich in blood vessels and autonomic nerve endings, enabling rapid revascularization and reinnervation of the grafts19,20. Dr. Caicedo has successfully used this anatomical space for the maintenance and longitudinal study of pancreatic islets in the past21. Here, we show that this strategy not only constitutes a valid approach to study thymocytes&#39; dynamics within the native organ structure, but also uniquely permits to extend the in vivo longitudinal recordings to the study of progenitor recruitment and mature T-cell egression steps in mouse.

<table><tbody><tr><td>Isofluorane vaporizer w/isofluorane</td><td>Kent Scientific Corp</td><td>VetFlo-1215</td><td></td></tr><tr><td>Dissecting scope w/light source</td><td>Zeiss</td><td>Stemi 305</td><td></td></tr><tr><td>Fine dissection forceps</td><td>WPI</td><td>500455</td><td></td></tr><tr><td>Medium dissection forceps</td><td>WPI</td><td>501252</td><td></td></tr><tr><td>Curved tip fine dissection forceps</td><td>WPI</td><td>15917</td><td></td></tr><tr><td>Vannas scissors</td><td>WPI</td><td>503371</td><td></td></tr><tr><td>Dissecting scissors</td><td>WPI</td><td>503243</td><td></td></tr><tr><td>Scalpel</td><td>WPI</td><td>500353</td><td></td></tr><tr><td>40 mm 18G needles</td><td>BD</td><td>304622</td><td></td></tr><tr><td>Disposable transfer pipette</td><td>Thermofisher</td><td>201C</td><td></td></tr><tr><td>Heat pad and heat lamp</td><td>Kent Scientific Corp</td><td>Infrarred</td><td></td></tr><tr><td>Ethanol 70%</td><td>VWR</td><td>83,813,360</td><td></td></tr><tr><td>60 mm sterile dish</td><td>SIGMA</td><td>CLS430166</td><td></td></tr><tr><td>Sterile 1x PBS pH(7,4)</td><td>Thermofisher</td><td>10010023</td><td></td></tr><tr><td>Sterile wipes</td><td>Kimberly-Clark</td><td>LD004</td><td></td></tr><tr><td>Drugs for pain management</td><td>Sigma-Aldrich</td><td>A3035-1VL</td><td></td></tr><tr><td>Saline solution or Viscotears</td><td>Novartis</td><td>N/A</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>MZ FLIII</td><td></td></tr><tr><td>Head-holding adapter</td><td>Narishige</td><td>SG-4N-S</td><td></td></tr><tr><td>Gas mask</td><td>Narishige</td><td>GM-4_S</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica</td><td>TCS SP5 II</td><td></td></tr><tr><td>Laminar flow hood</td><td>Telstar</td><td>BIO IIA</td><td></td></tr></tbody></table>

real time in vivo tracking of thymocytes in the anterior chamber of the eye by laser scanning microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes&#180; dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Thymus from newborn mice were isolated from B6.Cg-Tg(CAG-DsRed*MST)1Nagy/Jas mice as described in this protocol (Steps 1.1-1.9). In these transgenic mice, the chicken beta actin promoter directs the expression of the red fluorescent protein variant DsRed. MST under the influence of the cytomegalovirus (CMV) immediate early enhancer facilitating the tracking of implants.
To prevent tissue rejection, isogenic individuals with genotype: C57BL/6-Tg(UBC-GFP)30Scha/J were selected as hosts. In these transgenic mice, the human ubiquitin C promoter directs the expression of enhanced green fluorescent protein (GFP) in all tissues allowing for a clear differentiation between the recipient and donor tissue. The action of the human ubiquitin C promoter leads to differential GFP expression patterns in certain hematopoietic cell types. In particular, T cells present a 2-fold higher GFP expression levels than CD19+B220+ B cells or other peripheral blood cells which may facilitate their identification by quantitative sensitive techniques such as flow cytometry, without any needs for additional labeling. Furthermore, fluorescent expression in these mice is observed in adult animals, as well as in all embryonic stages and it is uniform within a cell type lineage granting marker stability.
The pattern for thymus trimming shown in Figure 1A follows previous descriptions of functional thymus fragments implanted in the kidney capsula23. This trimming procedure allows for a reduction of the total volume of tissue to be implanted while preserving the functional histological basic units of the organ (cortex, medulla and cortico-medullary junction (CMJ) blood vessels). The notable difference in the size between newborn thymus (Figure 1A, left) and thymus from one-week old mice (Figure 1A, right) should be taken into account for trimming, as the space available to fit implants in the anterior chamber of the mouse eye is restricted. The results presented here include the implants obtained from newborns only. Figure 1B shows a macroscopic image of a thymic segment implanted in the anterior chamber of a GFP mouse eye, accommodated to preserve animal vision. Histological details of the implant in reference to the cornea (GFP tissue adjacent to RFP implant) can be observed in the 3D reconstruction of confocal images in Supplementary Video 1. The engraftment to the iris is shown in Video 1 and Figure 2.
Figure 1C and 1D respectively illustrate bright field and fluorescence images of the same area which may serve for a double-macroscopic follow up of tissue growth and involution in continuation studies. Figure 1C also illustrates the vascularization of the implanted tissue which should uniquely allow for studying thymus physiology dependent on organ blood supply along time.
The vascularization of the inserted thymic fragments occurred within 72h post-implant. The rapid vascularization process is in good agreement with the large amount of blood vessels found in the iris and the richness of the thymus in cytokine production. These characteristics enable fast engraftment of inserted thymi. The vascularization of implants is illustrated in Video 2, showing the engraftment of thymic blood vessels with the iris&#180; of the host individual (GFP tissue adjacent to RFP implant), therefore, confirming that the model presented here, in fact, allows for noninvasive continuous monitorization of vascularized thymus at the microscopic level. The use of LSM to visualize endogenous thymus or thymus previously implanted in different anatomic locations is restricted by tissue opacity and tissue thickness of over 1 mm. The anterior chamber of the eye is considered as a &#34;window&#34; to the organism facilitating the observation of implanted tissues at the microscopic level.
Here, it is shown that the model developed by our group allows the visualization of transmigration of the cells from the blood torrent (Video 2) into the implanted thymus (presumably progenitors); for the tracking of the contacts of GFP progenitor cells incoming into the thymic implant with epithelial and stromal thymic resident cells (RFP-labeled) during differentiation and selection processes (Videos 3 and 4) and, also, for the egress of cells (presumably mature T-cells) into blood vessels (Video 5).
The approach we describe thus represents the &#34;proof of principle&#34;, based on recordings taken between 72 h and one month post-implant from six individuals transplanted with the segments from three independent newborn donors, for an animal model that permits in vivo real time tracking of thymocyte maturation in a vascularized thymic fragment by noninvasive procedures along time, and as such, the protocol might need further refinements. This model could be further worked out making use of transgenic animals harboring labeled thymocyte molecular markers, for example, as fluorescent fusion products, thus permitting direct visualization of T-cell maturation intermediates. The system may help resolving controversies and respond incognitas linked to thymocyte differentiation and selection events among other.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58236/58236fig1.jpg" /> 
Figure 1. Sectioning of donor newborn thymic lobes and transplant into the anterior chamber of mice eye. (A) Newborn (left) and one week post-natal (right) thymi images. Dash lines on newborn thymus represent guides to prepare sections for transplantation into the anterior eye chamber of mice to estimate the maximum number of transplants from one donor. (B) Representative image showing newly implanted thymus. (C) Representative image of grafted, vascularized thymus 2 weeks post-implant. (D) Fluorescence image of thymus graft shown on (C). Scale bar is shown (1 mm). <a href="https://www.jove.com/files/ftp_upload/58236/58236fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58236/58236fig2.jpg" /> 
Figure 2. Confocal image showing the detail of thymus segment (red) engraftment in the iris (green). Image obtained with a microscope, using the following settings: 488 and 561 nm excitation/500 and 575 nm emission wavelengths, 512x512 pixel resolution and 40X dipping objective with long working distance. Scale bar is shown (40 &#181;m). <a href="https://www.jove.com/files/ftp_upload/58236/58236fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 1" src="/files/ftp_upload/58236/58236supfig1.jpg" /> 
Supplementary Figure 1. Setup for confocal imaging of thymic graft using 3D single photon fluorescence laser confocal microscopy. (A) Heatpad, stereotaxic headholder and gas mask details. (B) Set up of the mouse holding the thymus graft for LSM. <a href="https://www.jove.com/files/ftp_upload/58236/58236supfig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 1" src="/files/ftp_upload/58236/58236video1.jpg" /> 
Video 1. In vivo imaging of thymus implants in the anterior chamber of the mouse eye. <a href="https://www.jove.com/files/ftp_upload/58236/Video_1.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Video 2" src="/files/ftp_upload/58236/58236video2.jpg" /> 
Video 2. Recruitment of presumable thymus settling progenitors (TSP) from the blood stream (GFP) into the thymic parenchyma (RFP). <a href="https://www.jove.com/files/ftp_upload/58236/Video_2.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Video 3" src="/files/ftp_upload/58236/58236video3.jpg" /> 
Video 3. Stochastic movement of thymocytes (early thymic progenitors or ETPs) in the cortex. <a href="https://www.jove.com/files/ftp_upload/58236/Video_3.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Video 4" src="/files/ftp_upload/58236/58236video4.jpg" /> 
Video 4. Interaction of thymocytes (GFP) with thymic stromal cells (RFP) either cTECs (cortical thymic epithelial cells) or mTECs (medullary thymic epithelial cells). <a href="https://www.jove.com/files/ftp_upload/58236/Video_4.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Video 5" src="/files/ftp_upload/58236/58236video5.jpg" /> 
Video 5. Real time egress imaging of potential mature T-cells. <a href="https://www.jove.com/files/ftp_upload/58236/Video_5.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Supplementary Video" src="/files/ftp_upload/58236/58236svideo1.jpg" /> 
Supplementary Video 1. 3D-in vivo imaging of thymus implants in the anterior chamber of the mouse eye. <a href="https://www.jove.com/files/ftp_upload/58236/Supplementary_Video_1.avi">Please click here to download this file.</a>

Watch this Scientific Journal Video about Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy at JoVE.com

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

real-time-vivo-tracking-thymocytes-anterior-chamber-eye-laser

Universidad San Sebastian

Research

JoVE Journal

Biology

6.7K Views.  Universidad Católica de Valencia San Vicente Mártir. The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

Video: Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

Cancer Research

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Immunology and Infection

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

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.

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.

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

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Medicine

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.

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

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Neuroscience

Real-Time Analysis of Resident T-cell Migration in Different Tumor Regions Using Confocal Microscopy

Source: Peranzoni, Elisa, et al. <a href="https://app.jove.com/v/55709">Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy.</a> J. Vis. Exp. (2017).
This video describes the imaging of resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

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Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

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