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>

<ol>
	<li>Boehm, T., Hess, I., Swann, J. 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. D., Jenkinson, W. E. &. a. m. p. ;., Anderson, G. <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+egress+from+the+thymus:+Should+I+stay+or+should+I+go?.">T-cell egress from the thymus: Should I stay or should I go?.</a> Journal of Leukocyte Biology. , (2018).</li><li>Savino, W., Mendes-Da-Cruz, D. A., Smaniotto, S., Silva-Monteiro, E., Villa-Verde, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+governing+thymocyte+migration:+combined+role+of+chemokines+and+extracellular+matrix.">Molecular mechanisms governing thymocyte migration: combined role of chemokines and extracellular matrix.</a> Journal of Leukocyte Biology. 75 (6), 951-961 (2004).</li><li>Petrie, H. T., Z&#250;&#241;iga-Pfl&#252;cker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zoned+out:+functional+mapping+of+stromal+signaling+microenvironments+in+the+thymus.">Zoned out: functional mapping of stromal signaling microenvironments in the thymus.</a> Annual Review of Immunology. 25, 649-679 (2007).</li><li>Bousso, P., Bhakta, N. R., Lewis, R. S., Robey, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+thymocyte-stromal+cell+interactions+visualized+by+two-photon+microscopy.">Dynamics of thymocyte-stromal cell interactions visualized by two-photon microscopy.</a> Science. 296, 1876-1880 (2002).</li><li>Ladi, E., Herzmark, P., Robey, 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+situ+imaging+of+the+mouse+thymus+using+2-photon+microscopy.">In situ imaging of the mouse thymus using 2-photon microscopy.</a> Journal of Visualized Experiments. (11), e652 (2008).</li><li>Bhakta, N. R., Oh, D. Y., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+oscillations+regulate+thymocyte+motility+during+positive+selection+in+the+three-dimensional+thymic+environment.">Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment.</a> Nature Immunology. 6, 143-151 (2005).</li><li>Ehrlich, L. I., Oh, D. Y., Weissman, I. L., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+chemotaxis+and+substrate+restriction+to+segregation+of+immature+and+mature+thymocytes.">Differential contribution of chemotaxis and substrate restriction to segregation of immature and mature thymocytes.</a> Immunity. 31, 986-998 (2009).</li><li>Le Borgne, M., Ladi, E., Dzhagalov, I., Herzmark, P., Liao, Y. F., Chakraborty, A. K., et al. <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>Sanos, S. L., Nowak, J., Fallet, M., Bajenoff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cell+networks+regulate+thymocyte+migration+and+dendritic+cell+behavior+in+the+thymus.">Stromal cell networks regulate thymocyte migration and dendritic cell behavior in the thymus.</a> Journal of Immunology. 186, 2835-2841 (2011).</li><li>Witt, C. M., Raychaudhuri, S., Schaefer, B., Chakraborty, A. K., Robey, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+migration+of+positively+selected+thymocytes+visualized+in+real+time.">Directed migration of positively selected thymocytes visualized in real time.</a> PLoS Biology. 3 (6), e160 (2005).</li><li>Ramsdell, F., Z&#250;&#241;iga-Pfl&#252;cker, J. C., 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=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>White, A., Jenkinson, E., Anderson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaggregate+thymus+cultures.">Reaggregate thymus cultures.</a> Journal of Visualized Experiments. (18), e905 (2008).</li><li>Dunn, K. W., Sutton, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+studies+in+living+animals+using+multiphoton+microscopy.">Functional studies in living animals using multiphoton microscopy.</a> ILAR Journal. 49, 66-77 (2008).</li><li>Caetano, S. S., Teixeira, T., 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=Intravital+imaging+of+the+mouse+thymus+using+2-photon+Microscopy.">Intravital imaging of the mouse thymus using 2-photon Microscopy.</a> Journal of Visualized Experiments. (59), e3504 (2012).</li><li>Li, J., Iwanami, N., Hoa, V. Q., Furutani-Seiki, M., 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=Noninvasive+intravital+imaging+of+thymocyte+dynamics+in+medaka.">Noninvasive intravital imaging of thymocyte dynamics in medaka.</a> Journal of Immunology. 179 (3), 1605-1615 (2007).</li><li>Adeghate, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-graft+circulation+and+vascular+morphology+in+pancreatic+tissue+transplants+in+rats.">Host-graft circulation and vascular morphology in pancreatic tissue transplants in rats.</a> Anatomical Record. 251, 448-459 (1998).</li><li>Adeghate, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+tissue+grafts+are+reinnervated+by+neuro-peptidergic+and+cholinergic+nerves+within+five+days+of+transplantation.">Pancreatic tissue grafts are reinnervated by neuro-peptidergic and cholinergic nerves within five days of transplantation.</a> Transplant Immunology. 10 (1), 73-80 (2002).</li><li>Speier, S., Nyqvist, D., K&#246;hler, M., Caicedo, A., Leibiger, I. B., Berggren, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+high-resolution+in+vivo+imaging+of+cell+biology+in+the+anterior+chamber+of+the+mouse+eye.">Noninvasive high-resolution in vivo imaging of cell biology in the anterior chamber of the mouse eye.</a> Nature Protocols. 3 (8), 1278-1286 (2008).</li><li>Speier, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+in+vivo+imaging+of+pancreatic+islet+cell+biology.">Noninvasive in vivo imaging of pancreatic islet cell biology.</a> Nature Medicine. 14 (5), 574-578 (2008).</li><li>Morillon, Y. M., Manzoor, F., Wang, B., Tisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+transplantation+of+different+aged+murine+thymic+grafts.">Isolation and transplantation of different aged murine thymic grafts.</a> Journal of Visualized Experiments. 99 (99), (2015).</li><li>Liu, L. L., Du, X. M., Wang, Z., Wu, B. J., Jin, M., Xin, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+intrathymic+injection+technique+for+mice.">A simplified intrathymic injection technique for mice.</a> Biotechnic &amp; Histochemestry. 87 (2), 140-147 (2012).</li><li>Manna, S., Bhandoola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrathymic+Injection.">Intrathymic Injection.</a> Methods in Molecular Biology. 1323, 203-209 (2016).</li><li>Abdulreda, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution,+noninvasive+longitudinal+live+imaging+of+immune+responses.">High-resolution, noninvasive longitudinal live imaging of immune responses.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (31), 12863-12868 (2011).</li></ol>

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy