We thank Dr. Etsuo Susaki for their valuable help and recommendations regarding tissue-clearing and immunostaining protocols. Also, we are grateful to M. Kandasamy from the CTEGD Biomedical Microscopy Core for technical support using LSFM and confocal imaging. We also thank all the members of Tarleton Research Group for helpful suggestions throughout this study.

This study was carried out in strict accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and Association for Assessment and Accreditation of Laboratory Animal Care accreditation guidelines. The Animal Use Protocol (control of T. cruzi infection in mice-A2021 04-011-Y1-A0) was approved by the University of Georgia Institutional Animal Care and Use Committee. B6.C+A2:A44g-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J and C57BL/6J-Tg(Cd8a*-cre)B8Asin/J mice (female, 1-2 months old) were used for the present study. The mice were obtained from commercial sources (see Table of Materials).
1. Infection, perfusion, and dissection
<ol>
	<li>Intraperitoneally infect mice with tissue culture-derived trypomastigotes of Colombiana (DTU TcI) or Brazil (DTU TcV) T. cruzi strain expressing tdTomato or GFP fluorescent proteins, respectively. The infection dose could range from 50,000 to 200,000 trypomastigotes diluted in 100 &#181;L of 1x Phosphate-Buffered Saline (PBS). 
	NOTE: Specific details about the generation of reporter parasites and mouse models of infection are available in Canavaci et al.29 and Bustamante et al.30.</li>
	<li>Euthanize the mice by carbon dioxide inhalation at a flow rate of 3-7 L/min. As soon as the animals stop showing any pedal reflex, make a longitudinal incision through the skin from the abdomen towards the sternum. Then cut the body wall from the abdomen and continue through the ribs on each side of the thorax until the sternum can be lifted away, exposing the heart.</li>
	<li>As described in Figure 1, make a 2.5 mm incision in the heart&#39;s right auricle and collect the draining blood using a 1 mL micropipette tip.</li>
	<li>Insert a butterfly needle (connected to one end of the perfusion system) into the apex of the left ventricle until it reaches the ascending aorta. Use gel-based glue (see Table of Materials) to seal the inlet hole around the needle and maintain the needle in position during perfusions.</li>
	<li>Perfuse the mice30 with 50 mL of cold Heparin-PBS (pH 7.4, 10 U/mL of Heparin) or until the fluid that comes out of the mouse toward the collection tray is clear of blood.</li>
	<li>Perfuse with 50 mL of cold 4% (w/v) paraformaldehyde (PFA) (pH 7.4) in PBS. 
	NOTE: PFA tissue fixation is likely one of the critical steps of the protocol, especially for maintaining epitope structures and subsequent immunodetection. PFA degrades over time, so it must be freshly prepared. The pH of the solution is also important to avoid over-fixation, which could lead to poor clearing of tissues. 
	CAUTION: PFA is moderately toxic by skin contact. Acute exposure is also highly irritating to the nose, eyes, and throat. Long-term exposure to low levels in the air or skin may cause skin irritation such as dermatitis and itching, and asthma-like respiratory problems. Wear face and eye protection and do not breathe dust, gas, mist, fumes, or vapors.
	<ol>
		<li>Following the PFA perfusion, perfuse the mouse with 100 mL of CUBIC-P buffer to reach the clarification levels mentioned in step 1.5.</li>
		<li>To prepare CUBIC-P buffer, dissolve 10% N-butyldiethanolamine, 5% Triton X-100, and 5% 1-N-Methylimidazole in double-distilled water or use the commercial cocktails (see Table of Materials). 
		NOTE: Steps 1.6.1.-1.6.2. are recommended only for highly pigmented organs such as the heart and kidney since they require a pre-clearing step to obtain increased levels of transparency. After using CUBIC-P, dissect the organs and proceed directly to step 2: Tissue clearing.</li>
	</ol>
	</li>
	<li>Dissect the tissue samples/organs30 to be imaged and post-fix them in 4% (w/v) PFA in PBS (~10 mL/whole organ) overnight (ON) at 4 &#176;C with gentle shaking (no more than 5 x g) in 50 mL conical tubes. 
	NOTE: All the incubations from hereafter must be performed on tubes laying horizontally at 20-30 &#176;C protected from light.</li>
	<li>Wash the sample in 10 mL of PBS (supplemented with 0.05% sodium azide (NaN3) for 3 h (three times) at room temperature (RT) with gentle shaking (5 x g). 
	&#8203;NOTE: Tissues can be frozen by incubating in 10 mL of 30% sucrose in PBS with gentle shaking (5 x g) at 4 &#176;C ON in 50 mL conical tubes. After tissues sink to the bottom of the tube, embed them in the OCT compound and keep them at -80 &#176;C. Thaw at RT until the OCT compound completely melts, then wash in PBS (~10 mL/whole organ) ON at 4 &#176;C with gentle shaking (5 x g) in 50 mL conical tubes. Proceed to step 2.</li>
</ol>
2. Tissue clearing
NOTE: All the tissue clearings performed in this work were done using CUBIC protocol I22. Three different cocktails were used: CUBIC-P for delipidation and rapid decolorization during perfusions, CUBIC-L for delipidation and decolorization, and CUBIC-R for RI matching. DNA staining and immunostainings were performed using CUBIC-HV 1 3D nuclear staining kit and CUBIC-HV 1 3D immunostaining kit, respectively (see Table of Materials).
<ol>
	<li>Immerse individual organs in 10 mL of 50% water-diluted CUBIC-L (see Table of Materials) with gentle shaking (5 x g) at RT (ON) in 50 mL conical tubes. Keep tubes flat on the shaking plate. 
	NOTE: To avoid tissue damage, organs are maintained in the same tube, and solutions are collected using a vacuum system. To prepare CUBIC-L, dissolve 10% N-butyldiethanolamine and 10% Triton X-100 in double-distilled water using the commercial cocktails (see Table of Materials). 
	CAUTION: CUBIC-L causes serious eye damage.Wear eye and face protection. Dispose to an approved waste disposal plant.</li>
	<li>Immerse sample in 10 mL of 100% CUBIC-L for 6 days (refreshing the solution on day 3). 
	NOTE: At the end of this incubation period, the tissues must be almost completely transparent.</li>
	<li>Wash the transparent organs with PBS (supplemented with 0.05% NaN3) for 2 h (three times) at 37 &#176;C with gentle shaking (5 x g). Transfer tissues to a new 50 mL conical tube with each wash to remove Triton X-100. 
	NOTE: As described in Figure 2B, if the goal of the experiment is to visualize endogenously-expressed reporter proteins from transgenic T. cruzi parasites or mice (Figure 2C-F), skip steps 3, 4, and 5 and continue directly to step 6.</li>
</ol>
3. DNA staining
<ol>
	<li>Dilute the commercially available nucleic acid dye (see Table of Materials) in 5 mL of staining buffer (included in the kit) at 1/2,500 dilution.</li>
	<li>Immerse tissue in the nuclear dye solution and incubate at 37 &#176;C with gentle rotation for 5 days using 15 mL conical tubes in a standing position.</li>
	<li>Wash with 15 mL of 3D nuclear staining wash buffer (included in the kit) for 2 h (three times) at RT with gentle shaking (5 x g). 
	&#8203;NOTE: Other DNA dyes can be used in these concentrations and incubation times: DAPI (included in the kit): 1/200, 5 days incubation; BOBO-1: 1/400, 5 days incubation; Propidium Iodide (PI) (included in the kit): 1/100, 3 days incubation; RedDot2: 1/250, 3 days incubation. If the specific aim of the experiment is to visualize both endogenously-expressed reporter proteins and use nuclear stains, skip steps 4 and 5 and continue directly to step 6.</li>
</ol>
4. Extracellular matrix (ECM) digestion
NOTE: Hyaluronidase digestion of the ECM must be performed to facilitate the proper penetration of the antibodies into deep regions of the tissues28.
<ol>
	<li>Immerse individual organs in 15 mL of hyaluronidase reaction buffer for 2 h at 37 &#176;C in a 50 mL conical tube in flat position protected from light. 
	NOTE: To prepare hyaluronidase reaction buffer, dissolve 10 mM of CAPSO; 150 mM of Sodium Chloride (NaCl), and 0.05% of NaN3 (see Table of Materials) in double-distilled water and adjust pH to 10.</li>
	<li>Prepare Enzyme Solution by mixing 75 &#181;L of 20 mg/mL of hyaluronidase stock into 425 &#181;L of hyaluronidase reaction buffer. To prepare 20 mg/mL of hyaluronidase stock, dissolve hyaluronidase in 50 mM of Carbonate buffer, 150 mM of NaCl, 0.01% of BSA, and 0.05% of NaN3 (see Table of Materials). Adjust pH to 10 and aliquot in volumes of 77 &#181;L at -30 &#176;C.</li>
	<li>Discard hyaluronidase reaction buffer using a pipette and immerse the organ in the Enzyme Solution (500 &#181;L in a 15 mL conical tube) in a standing position protected from light for 24 h at 37 &#176;C with gentle shaking (5 x g).</li>
	<li>Wash the sample in 15 mL of hyaluronidase wash buffer in a 50 mL conical tube in a horizontal position protected from light for 2 h (three times) at 37 &#176;C with gentle shaking (5 x g).
	<ol>
		<li>To prepare hyaluronidase wash buffer, dissolve 50 mM of Carbonate buffer, 150 mM of NaCl, 0.1% (v/v) of Triton X-100, 5% (v/v) of Methanol, and 0.05% NaN3. Adjust pH to 10. To prepare 10x Carbonate buffer-NaCl stock, dissolve 2.96 g of Sodium Carbonate, 1.86 g of Sodium Hydrogen Carbonate, and 8.77 g of NaCl in 100 mL of double-distilled water with 0.05% NaN3 (see Table of Materials) and adjust pH to 10.</li>
	</ol>
	</li>
</ol>
5. Immunostaining
<ol>
	<li>Label vasculature using anti-&#945;-SMA (alpha-small muscle actin, see Table of Materials) antibodies following the steps below.
	<ol>
		<li>Generate primary plus conjugated Fab fragment secondary antibody complex. Start this reaction 1.5 h prior to the staining procedure.
		<ol>
			<li>Calculate the required amount of primary and secondary antibodies (mix at a 1:0.5 ratio by weight). 
			NOTE: For the primary antibody anti-&#945;-SMA, 3.5 &#181;g is needed to label the entire heart or a fragment of skeletal muscle of similar dimensions. For 2.5 mg/mL product, 3.5/2.5 = 1.4 &#181;L of antibody solution is needed. For secondary antibody AlexaFluor 647 anti-mouse Fab fragment, 1.75 &#181;g is needed to label the entire heart or a fragment of skeletal muscle of similar dimensions. For 1.7 mg/mL product, 1.75/1.7 = 1 &#181;L of antibody solution is needed.</li>
			<li>Mix primary and secondary antibodies in an amber 2 mL tube and incubate for 1.5 h at 37 &#176;C.</li>
		</ol>
		</li>
		<li>For buffer exchange, mix 7.5 mL of 2x HV1 3D immunostaining buffer (included in the kit, see Table of Materials) with 7.5 mL of double-distilled water and immerse the tissue sample for 1.5 h at 32 &#176;C with gentle shaking (5 x g) in a 15 mL conical tube in a horizontal position. Start this reaction simultaneously as the generation of antibody complex (1.5 h prior to the immunostaining procedure).</li>
	</ol>
	</li>
	<li>Perform 3D immunostaining following the steps below.
	<ol>
		<li>In a 15 mL conical tube, prepare the antibody staining solution following Supplementary File 1.</li>
		<li>Collect the tissue sample from the buffer exchange media (step 5.1.2) and immerse it in the antibody staining solution. Incubate tissues individually for 1 week at 32 &#176;C with gentle shaking (5 x g) of the tubes in a standing position protected from light. Seal the tube with paraffin film to avoid evaporation.</li>
		<li>Move to 4 &#176;C and incubate ON in a standing position.</li>
		<li>Cool 1x HV1 3D immunostaining wash buffer (included in the kit, see Table of Materials) to 4 &#176;C and wash the sample with 15 mL buffer (two times) for 30 min each at 4 &#176;C with gentle shaking (5 x g). Keep 15 mL conical tubes in a horizontal position until step 5.2.7.</li>
		<li>Dilute formalin to 1% in 1x HV1 3D immunostaining wash buffer and immerse the sample in 8 mL of the solution for 24 h at 4 &#176;C with gentle shaking (5 x g).</li>
		<li>Incubate in fresh 1% formalin solution for 1 h at 37 &#176;C with gentle shaking (5 x g).</li>
		<li>Wash in 15 mL of PBS for 2 h at 25 &#176;C with gentle shaking (5 x g).</li>
	</ol>
	</li>
	<li>Boost tdTomato signal using anti-Red Fluorescent Protein (RFP) antibodies following the steps below.
	<ol>
		<li>Follow the same incubation times and temperatures as in step 5.2.2. Calculate the amount of primary and secondary antibodies (see Table of Materials) and mix them at a 1:0.5 ratio by weight. 
		NOTE: For primary antibody anti-RFP, 5 &#181;g is needed to label the entire heart or a fragment of skeletal muscle of similar dimensions. For 1.25 mg/mL product, 5/1.25 = 4 &#181;L of the solution is required. For secondary antibody Alexa Fluor 647 anti-rabbit Fab fragment, 2.5 &#181;g is needed to label the entire heart or a fragment of skeletal muscle of similar dimensions. For 1.5 mg/mL product, 2.5/1.5 = 1.7 &#181;L of the solution is needed.</li>
		<li>Prepare the antibody staining solution as mentioned in Supplementary File 1.</li>
	</ol>
	</li>
	<li>Boost GFP signals using anti-GFP nanobodies following the steps below.
	<ol>
		<li>Follow the same incubation times and temperatures as mentioned in step 5.2.2. 
		NOTE: Anti-GFP nanobodies (see Table of Materials) are conjugated with Alexa Fluor 647, so the antibody complex generation is unnecessary.</li>
		<li>Prepare the antibody staining solution as mentioned in Supplementary File 1.</li>
	</ol>
	</li>
</ol>
6. RI matching
<ol>
	<li>Immerse transparent organs in 5 mL of 50% water-diluted CUBIC-R+ solution (see Table of Materials) at RT (ON) with gentle shaking (5 x g) in a 50 mL conical tube. Keep tubes in a standing position during the entire RI matching step. 
	NOTE: Recycled CUBIC R+ solutions from previous experiments could be reused (up to four times) in this step. To prepare CUBIC-R+ solution, dissolve 45% of 2,3-Dimethyl-1-phenyl-5-pyrazolone (Antipyrine), 30% of Nicotinamide or N-Methylnicotinamide, and 0.5% of N-butyldiethanolamine in double-distilled water or use the commercial CUBIC-R+ buffer (see Table of Materials). 
	CAUTION: CUBIC-R+ causes skin irritation, serious eye irritation, and damage to organs. Wear protective gloves, and ensure eye and face protection. Do not breathe dust, fumes, gas, mist, or vapors. Dispose of to an approved waste disposal plant.</li>
	<li>Replace 50% CUBIC-R+ with 5 mL of 100% CUBIC-R+ and incubate with gentle shaking (5 x g) at RT for 2 days. Then, transfer the tissues onto a stack of lint-free wipes and carefully turn the tissues to remove CUBIC-R+ solution from the organ surfaces for 5 min.</li>
</ol>
7. Mounting
<ol>
	<li>After drying the tissues, transfer them to 5 mL of Mounting Solution (RI = 1.520) (see Table of Materials) in a six-well cell culture plate and incubate them (ON) at RT. Frequently turn the tissues to eliminate air bubbles, especially on the surfaces of the organs. 
	NOTE: Organs can be stored for more than 6 months in Mounting Solution or CUBIC-R+.</li>
	<li>Adhere the tissues to the microscope sample holder by using cyanoacrylate-based gel glue.</li>
	<li>Immerse the samples in the microscope quartz cuvette filled with 120 mL of Mounting Solution and image them transversally to their longitudinal axis. Adhere the heart with the apex and the aorta horizontally aligned. 
	&#8203;NOTE: Cleared and mounted organs can be sectioned with a vibratome and imaged at high magnification by confocal microscopy. Embed the organs in 2% agarose and cut sections of 100-500 &#181;m. Organs can also be manually sectioned with a sharp blade to produce thick-tissue sections (&#62;1000 &#181;m). After sectioning, place the slices into glass-bottom 35 mm Petri dishes, mount using the same mounting solution, seal with nail polish, and image with a confocal microscope.</li>
</ol>
8. Image acquisition
<ol>
	<li>Image the mounted samples with a light-sheet microscope (see Table of Materials). Set magnification and step size between individual slices to 3 &#181;m, and use right and left light sheet lasers with 5 &#181;m thicknesses and 100% width. Set the exposure time constant at 50-100 ms, and adjust the laser power from 10% to 80% depending on the fluorescence signal intensity.
	<ol>
		<li>For the co-detection of tdTomato-expressing parasites and DiR-stained dormant amastigotes (Figure 2C), use red (Ex/Em 561/620-660 nm) and infra-red (Ex/Em 785/845-55 nm) channels, respectively. For the co-detection of T cells and tdTomato-expressing parasites in CD8 reporter mouse (Figure 2D), use green (Ex/Em 488/525-50 nm) and red channel, respectively.</li>
		<li>For the coinfections assays (Figure 2E), as well as for the co-detection of parasites expressing GFP in nuclei reporter mice (Figure 2F), use the green and red channels, respectively. For the detection of tdTomato-expressing parasites, nuclear dye, and heart vasculature (Figure 3B,C), use the red, green, and far-red (Ex/Em 640/680-730 nm) channels, respectively.</li>
		<li>Detect anti-RFP antibodies with the far-red channel and anti-GFP nanobodies using the green channel (Figure 3D).</li>
	</ol>
	</li>
	<li>Convert the acquired TIFF image stacks and 3D reconstruct the organs using Imaris v9.7.2 software (see Table of Materials).</li>
</ol>
9. Surface reconstruction and quantification with Imaris software
<ol>
	<li>Select surfaces detection algorithm tool and initiate the wizard in the image analysis software.</li>
	<li>Perform an initial analysis in a 3D region of interest (randomly selected) and then apply it to the entire 3D organ reconstruction. After choosing a region of interest (ROI), select the channel to be analyzed, uncheck the smooth button, and select background subtraction. 
	NOTE: Background subtraction calculates a unique local background value for every voxel and subtracts this from the original pixel value. The diameter of the largest sphere which fits into the object must be up to 200 &#181;m for T. cruzi-infected cells, 10 &#181;m for T cells, and 5 &#181;m for individual parasites.</li>
	<li>Use the histogram to adjust the classification and the filter drop-down list to select the measurements for classification. 
	NOTE: Use the histogram to adjust the classification: ellipticity (oblate) and sphericity measurements are recommended.</li>
	<li>Finalize the wizard, press statistics button, and retrieve the total number of parasite-infected cells or T cells as the number of disconnected components per time point.</li>
</ol>

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C., 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+three-dimensional+single-cell-resolution+whole-brain+atlas+using+CUBIC-X+expansion+microscopy+and+tissue+clearing.">A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing.</a> Nature Neuroscience. 21, 625-637 (2018).</li><li>Zhao, 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=Cellular+and+molecular+probing+of+intact+human+organs.">Cellular and molecular probing of intact human organs.</a> Cell. 180 (4), 796-812 (2020).</li><li>Erturk, A., 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=Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO.">Three-dimensional imaging of solvent-cleared organs using 3DISCO.</a> Nature Protocols. 7, 1983-1995 (2012).</li><li>Renier, N., 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=iDISCO:+a+simple,+rapid+method+to+immunolabel+large+tissue+samples+for+volume+imaging.">iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.</a> Cell. 159 (4), 896-910 (2014).</li><li>Chung, K., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLARITY+for+mapping+the+nervous+system.">CLARITY for mapping the nervous system.</a> Nature Methods. 10, 508-513 (2013).</li><li>Susaki, E. A., 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=Versatile+whole-organ/body+staining+and+imaging+based+on+electrolyte-gel+properties+of+biological+tissues.">Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues.</a> Nature Communications. 11, 1982 (2020).</li><li>Kubota, S. I., 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=Whole-body+profiling+of+cancer+metastasis+with+single-cell+resolution.">Whole-body profiling of cancer metastasis with single-cell resolution.</a> Cell Reports. 20 (1), 236-250 (2017).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), e3564 (2012).</li><li>Bustamante, J. M., 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+modified+drug+regimen+clears+active+and+dormant+trypanosomes+in+mouse+models+of+Chagas+disease.">A modified drug regimen clears active and dormant trypanosomes in mouse models of Chagas disease.</a> Science Translational Medicine. 12 (567), (2020).</li><li>Wang, H., Khoradmehr, A., Tamadon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FACT+or+PACT:+A+comparison+between+free-acrylamide+and+acrylamide-based+passive+sodium+dodecyl+sulfate+tissue+clearing+for+whole+tissue+imaging.">FACT or PACT: A comparison between free-acrylamide and acrylamide-based passive sodium dodecyl sulfate tissue clearing for whole tissue imaging.</a> Cell Journal. 21 (2), 103-114 (2019).</li><li>Hofmann, J., Gadjalova, I., Mishra, R., Ruland, J., Keppler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+tissue+clearing+and+multi-organ+volumetric+imaging+enable+quantitative+visualization+of+sparse+immune+cell+populations+during+inflammation.">Efficient tissue clearing and multi-organ volumetric imaging enable quantitative visualization of sparse immune cell populations during inflammation.</a> Frontiers in Immunology. 11, 599495 (2020).</li><li>Messal, H. A., 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=Antigen+retrieval+and+clearing+for+whole-organ+immunofluorescence+by+FLASH.">Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH.</a> Nature Protocols. 16, 239-262 (2021).</li><li>Kolesova, H., Capek, M., Radochova, B., Janacek, J., Sedmera, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+different+tissue+clearing+methods+and+3D+imaging+techniques+for+visualization+of+GFP-expressing+mouse+embryos+and+embryonic+hearts.">Comparison of different tissue clearing methods and 3D imaging techniques for visualization of GFP-expressing mouse embryos and embryonic hearts.</a> Histochemistry and Cell Biology. 146 (2), 141-152 (2016).</li><li>Chen, Y., 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+versatile+tiling+light+sheet+microscope+for+imaging+of+cleared+tissues.">A versatile tiling light sheet microscope for imaging of cleared tissues.</a> Cell Reports. 33, 108349 (2020).</li></ol>

The authors declare that they have no competing interests.

The absence of extensive, whole-organ imaging of parasites and the immune response limits the understanding of the complexity of the host-parasite interactions and impedes the evaluation of therapies for Chagas disease. The present study adopted the CUBIC pipeline to clarify and stain intact organs and tissues of T. cruzi-infected mice.
Multiple tissue clearing protocols were tested in this study (PACT32, ECi33, FLASH34...

Chagas disease, caused by the protozoan parasite T. cruzi, is among the world&#39;s most neglected tropical diseases, causing approximately 13,000 annual deaths. The infection often progresses from an acute to a chronic stage producing cardiac pathology in 30% of the patients, including arrhythmias, heart failure, and sudden death1,2. Despite the strong host immune response elicited against the parasite during the acute phase, low numbers of parasites chronically persist throughout the host&#39;s life in tissues such as the heart and skeletal muscle. Several factors, including the delayed onset of adaptive immune responses and the presence of non-replicating forms of the parasite, may contribute to the capacity of T. cruzi to avoid a complete elimination by the immune system3,4,5,6. Furthermore, non-replicating dormant forms of the parasite display a low susceptibility to trypanocidal drugs and may in part be responsible for the treatment failure observed in many cases7,8.
The development of new imaging techniques provides an opportunity to gain insight into the spatial distribution of the parasites in the infected tissues and their relationship with the immune cells involved in their control. These characteristics are crucial for a better understanding of the processes of parasite control by the immune system and tracking the rare dormant parasites present in chronic tissues.
Light-sheet fluorescence microscopy (LSFM) is one of the most comprehensive and unbiased methods for 3D imaging of large tissues or organs without thin-sectioning. Light-sheet microscopes utilize a thin sheet of light to only excite the fluorophores in the focal plane, reduce photobleaching and phototoxicity of samples, and record images of thousands of tissue layers using ultra-fast cameras. The high level of tissue transparency necessary for the proper penetration of the laser light in tissues is obtained by homogenizing the refractive index (RI) following tissue delipidation and decolorization, which reduces the scattering of light and renders high-quality images9,10,11.
Tissue clearing approaches have been developed for the imaging of whole mice12,13,14, organoids15,16,17, organs expressing reporter fluorescent markers18,19,20,21,22,23, and recently a limited number of human tissues24. The current methods for tissue clearing are classified into three families: (1) organic solvent-based methods such as DISCO protocols25,26, (2) hydrogel-based methods, such as CLARITY27, and aqueous methods, such as CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis)18,19,28,29. CUBIC protocols maintain organ shape and tissue integrity, preserving the fluorescence of endogenously expressed reporter proteins. The most recent update of this technique, CUBIC-HistoVision (CUBIC-HV), also permits the detection of epitopes using fluorescently-tagged antibodies and DNA labeling28.
In the present protocol, the CUBIC pipeline for detecting T. cruzi expressing fluorescent proteins in clarified intact mouse tissues was used. Optically transparent tissues were LSFM imaged, 3D reconstructed, and the precise total number of T. cruzi infected cells, dormant amastigotes, and T cells per organ were automatically quantified. Also, this protocol was successfully adopted to obtain uniform labeling of cleared organs with antibodies and nuclear stains. These approaches are essential for understanding the expansion and control of T. cruzi in infected hosts and are useful for fully evaluating chemo- and immuno-therapeutics for Chagas disease.

<table><tbody><tr><td>1-methylimidazole</td><td>Millipore Sigma</td><td>616-47-7</td><td></td></tr><tr><td>2,3-Dimethyl-1-phenyl-5-pyrazolone (Antipyrine</td><td>TCI</td><td>D1876</td><td></td></tr><tr><td>6-wells cell culture plates</td><td>ThermoFisher Scientific</td><td>140675</td><td></td></tr><tr><td>AlexaFluor 647 anti-mouse Fab fragment</td><td>Jackson Immuno Research Laboratories</td><td>315-607-003</td><td></td></tr><tr><td>AlexaFluor 647 anti-rabbit Fab fragment</td><td>Jackson Immuno Research Laboratories</td><td>111-607-003</td><td></td></tr><tr><td>anti-GFP nanobody Alexa Fluor 647</td><td>Chromotek</td><td>gb2AF647-50</td><td></td></tr><tr><td>anti-RFP</td><td>Rockland</td><td>600-401-379</td><td></td></tr><tr><td>anti-&amp;alpha;-SMA</td><td>Sigma</td><td>A5228</td><td></td></tr><tr><td>B6.C+A2:A44g-Gt(ROSA)26Sor&lt;sup&gt;tm14(CAG-tdTomato)Hze&lt;/sup&gt;/J mouse</td><td>The Jackson Laboratory</td><td>Strain #007914</td><td>Common Name: Ai14 , Ai14D or Ai14(RCL-tdT)-D</td></tr><tr><td>B6.Cg-Gt(ROSA)26Sor&lt;sup&gt; tm14(CAG-tdTomato)Hze&lt;/sup&gt;/J mouse</td><td>The Jackson Laboratory</td><td>Strain #007914</td><td>Common Name: Ai14 , Ai14D or Ai14(RCL-tdT)-D</td></tr><tr><td>BOBO-1 Iodide</td><td>ThermoFisher Scientific</td><td>B3582</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>#A7906</td><td></td></tr><tr><td>C57BL/6J-Tg(Cd8a*-cre)B8Asin/J mouse</td><td>The Jackson Laboratory</td><td>Strain #032080</td><td>Common Name: Cd8a-Cre (E8III-Cre)</td></tr><tr><td>CAPSO</td><td>Sigma</td><td>#C2278</td><td></td></tr><tr><td>Cleaning wipes Kimwipes</td><td>&amp;nbsp;Kimberly-Clark</td><td>T8788</td><td></td></tr><tr><td>Confocal Laser Scanning Microscope</td><td>Zeiss</td><td>LSM 790</td><td></td></tr><tr><td>CUBIC-HV 1 3D immunostaining kit</td><td>TCI</td><td>C3699</td><td></td></tr><tr><td>CUBIC-HV 1 3D nuclear staining kit</td><td>TCI</td><td>C3698</td><td></td></tr><tr><td>CUBIC-L</td><td>TCI</td><td>T3740</td><td></td></tr><tr><td>CUBIC-P</td><td>TCI</td><td>T3782</td><td></td></tr><tr><td>CUBIC-R+</td><td>TCI</td><td>T3741</td><td></td></tr><tr><td>Cyanoacrylate-based gel superglue</td><td>Scotch</td><td>571605</td><td></td></tr><tr><td>DiR (DiIC18(7); 1,1&amp;rsquo;-dioctadecyl-3,3,3&amp;rsquo;,3&amp;rsquo;-tetramethylindotricarbocyanine iodide) Company: Biotium</td><td>Biotium</td><td>#60017</td><td></td></tr><tr><td>Ethylene diamine tetra acetic acid (EDTA)</td><td>Millipore Sigma</td><td>60-00-4</td><td></td></tr><tr><td>Falcon Centrifuge tubes 15 mL</td><td>Corning</td><td>CLS430791</td><td></td></tr><tr><td>Falcon Centrifuge tubes 50&amp;nbsp; mL</td><td>Corning</td><td>CLS430290</td><td></td></tr><tr><td>Formalin</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Heparin</td><td>ThermoFisher Scientific</td><td>J16920.BBR</td><td></td></tr><tr><td>Hyaluronidase</td><td>Sigma</td><td>#H3884 or #H4272</td><td></td></tr><tr><td>Imaris File Converter x64</td><td>BitPlane</td><td>v9.2.0</td><td></td></tr><tr><td>Imaris software</td><td>BitPlane</td><td>v9.3</td><td></td></tr><tr><td>ImSpector software</td><td>LaVision BioTec, Miltenyi Biotec</td><td>v6.7</td><td></td></tr><tr><td>Intravenous injection needle 23-G</td><td>Sartori, Minisart Syringe filter</td><td>16534</td><td></td></tr><tr><td>Kimwipes</td><td></td><td></td><td>lint free wipes</td></tr><tr><td>Light-sheet fluorescent microscope</td><td>Miltenyi Biotec</td><td>ULtramicroscope II imaging system</td><td></td></tr><tr><td>Methanol</td><td>ThermoFisher Scientific</td><td>041838.K2</td><td></td></tr><tr><td>Micropipette tips, 10 &amp;micro;L, 200 &amp;micro;L and 1,000 &amp;micro;L</td><td>Axygen</td><td>T-300, T-200-Y and T-1000-B</td><td></td></tr><tr><td>Motorized pipet dispenser</td><td>Fisher Scientific, Fisherbrand</td><td>03-692-172</td><td></td></tr><tr><td>Mounting Solution</td><td>TCI</td><td>M3294</td><td></td></tr><tr><td>N-butyldiethanolamine</td><td>TCI</td><td>B0725</td><td></td></tr><tr><td>Nicotinamide</td><td>TCI</td><td>N0078</td><td></td></tr><tr><td>N-Methylnicotinamide</td><td>TCI</td><td>M0374</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Thermo Fisher Scientific</td><td>14190-094</td><td></td></tr><tr><td>RedDot 2 Far-Red Nuclear Stain</td><td>Biotium</td><td>#40061</td><td></td></tr><tr><td>Sacrifice Perfusion System</td><td>Leica</td><td>10030-380</td><td></td></tr><tr><td>Scissors</td><td>Fine Science Tools</td><td>91460-11</td><td></td></tr><tr><td>Serological pipettes</td><td>Costar Sterile</td><td>4488</td><td></td></tr><tr><td>Shaking incubator</td><td>TAITEC</td><td>BR-43FM MR</td><td></td></tr><tr><td>Sodium azide (NaN&lt;sub&gt;3&lt;/sub&gt;)</td><td>ThermoFisher Scientific</td><td>447815000</td><td></td></tr><tr><td>Sodium carbonate (Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;)</td><td>ThermoFisher Scientific</td><td>L13098.36</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>ThermoFisher Scientific</td><td>447302500</td><td></td></tr><tr><td>Sodium hydrogen carbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>ThermoFisher Scientific</td><td>014707.A9</td><td></td></tr><tr><td>SYTOX-G Green Nucleic Acid Stain</td><td>ThermoFisher Scientific</td><td>S7020</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr></tbody></table>

quantitative 3d imaging of trypanosoma cruzi-infected cells, dormant amastigotes, and t cells in intact clarified organs

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi (T. cruzi), is an obligate intracellular parasite with a diverse biology that infects several mammalian species, including humans, causing cardiac and digestive pathologies. Reliable detection of T. cruzi in vivo infections has long been needed to understand Chagas disease&#39;s complex biology and accurately evaluate the outcome of treatment regimens. The current protocol demonstrates an integrated pipeline for automated quantification of T. cruzi-infected cells in 3D-reconstructed, cleared organs. Light-sheet fluorescent microscopy allows for accurately visualizing and quantifying of actively proliferating and dormant T. cruzi parasites and immune effector cells in whole organs or tissues. Also, the CUBIC-HistoVision pipeline to obtain uniform labeling of cleared organs with antibodies and nuclear stains was successfully adopted. Tissue clearing coupled with 3D immunostaining provides an unbiased approach to comprehensively evaluate drug treatment protocols, improve the understanding of the cellular organization of T. cruzi-infected tissues, and is expected to advance discoveries related to anti-T. cruzi immune responses, tissue damage, and repair in Chagas disease.

CUBIC fixed tissues were washed with PBS to remove fixatives and then incubated with CUBIC-L cocktails, a basic buffered solution of amino alcohols that extract pigments and lipids from the tissue resulting in decolorization of tissue while maintaining tissue architecture. Grid lines in the paper can be seen through the tissues indicating an appropriate clearing of the organs (Figure 2A). After delipidation, tissues were washed and immersed in CUBIC-R+ and mounting solution ...

Watch this Scientific Journal Video about Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs at JoVE.com

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

The present protocol describes light-sheet fluorescent microscopy and automated software-assisted methods to visualize and precisely quantify proliferating and dormant Trypanosoma cruzi parasites and T cells in intact, cleared organs and tissues. These techniques provide a reliable way to evaluate treatment outcomes and offer new insights into parasite-host interactions.

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi ...

quantitative-3d-imaging-trypanosoma-cruzi-infected-cells-dormant

Research

JoVE Journal

Immunology and Infection

3.0K Views.  University of Georgia. The present protocol describes light-sheet fluorescent microscopy and automated software-assisted methods to visualize and precisely quantify proliferating and dormant Trypanosoma cruzi parasites and T cells in intact, cleared organs and tissues. These techniques provide a reliable way to evaluate treatment outcomes and offer new insights into parasite-host interactions.

Video: Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Developmental Biology

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 &#181;m) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Using this protocol, we were able to image 160 &#181;m thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron.

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

Neuroscience

Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates

American trypanosomiasis is transmitted to humans by triatomine bugs through the ingestion of contaminated food, by blood transfusions or accidently in hospitals and research laboratories. In addition, the Trypanosoma cruzi infection is transmitted congenitally from a&#160;chagasic mother to her offspring, but the male partner&#39;s contribution to in utero contamination is unknown. The findings of nests and clumps of amastigotes and of trypomastigotes in the theca cells of the ovary, in the goniablasts and in the lumen of seminiferous tubules suggest that T. cruzi infections are sexually transmitted. The research protocol herein presents the results of a family study population showing parasite nuclear DNA in the diploid blood mononuclear cells and in the haploid gametes of human subjects. Thus, three independent biological samples collected one year apart confirmed that T. cruzi infections were sexually transmitted to progeny. Interestingly, the specific T. cruzi antibody was absent in the majority of family progeny that bore immune tolerance to the parasite antigen. Immune tolerance was demonstrated in chicken refractory to T. cruzi after the first week of embryonic growth, and chicks hatched from the flagellate-inoculated eggs were unable to produce the specific antibody.&#160;Moreover, the instillation of the human semen ejaculates intraperitoneally or into the vagina of naive mice yielded T. cruzi amastigotes in the epididymis, seminiferous tubule, vas deferens and uterine tube with an absence of inflammatory reactions in the immune privileged organs of reproduction. The breeding of T. cruzi-infected male and female mice with naive mates resulted in acquisition of the infections, which were later transmitted to the progeny. Therefore, a robust education, information and communication program that involves the population and social organizations is deemed necessary to prevent Chagas disease.

The Trypanosoma cruzi agent of Chagas disease produces long-lasting asymptomatic infections that abruptly develop into clinically recognized pathology. The following research protocol describes a short-run family-based epidemiological study to unravel the T. cruzi infection transmitted sexually from parent to progeny.

American trypanosomiasis is transmitted to humans by triatomine bugs through the ingestion of contaminated food, by blood transfusions or accidently in ...

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central ...

Whole&#45;Organ Pathogen Tracking via Fluorescent Reporter Quantification

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

Author Spotlight: Unraveling Bacterial Responses to Antibiotics and Immune System in Tissues

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

In Vivo Bioluminescent Model of Chagas Disease for Drug Evaluation

Demystifying In Vivo Bioluminescence Imaging of a Chagas Disease Mouse Model for Drug Efficacy Studies

To control and decrease the public health impact of human protozoan diseases such as Chagas disease, leishmaniasis, and human African trypanosomiasis, expediting the development of new drugs and vaccines is necessary. However, this process is filled with difficulties such as highly complex parasite biology and disease pathogenesis and, as typical for neglected tropical diseases, comparatively limited funding for research and development. Thus, in vitro and in vivo study models that can sufficiently reproduce infection and disease key features while providing rational use of resources are essential for progressing research for these conditions. One example is the in vivo bioluminescence imaging (BLI) mouse model for Chagas disease, which provides highly sensitive detection of long wavelength light generated by Trypanosoma cruzi parasites expressing luciferase. Despite this technique becoming the standard approach for drug efficacy in vivo studies, research groups might still struggle to implement it due to a lack of proper practical training on equipment handling and application of quality control procedures, even when suitable BLI equipment is readily available. Considering this scenario, this protocol aims to guide from planning experiments to data acquisition and analysis, with details that facilitate the implementation of protocols in research groups with little or no experience with BLI, either for Chagas disease or for other infectious disease mouse models.

Author Spotlight: Real-Time Monitoring of Parasite Burden and Host Response

The protocol provided here describes the detailed steps to perform drug efficacy studies in a bioluminescent model of Trypanosoma cruzi infection, with a focus on data acquisition, analysis, and interpretation. Troubleshooting and quality control procedures to minimize technical issues are also provided.

To control and decrease the public health impact of human protozoan diseases such as Chagas disease, leishmaniasis, and human African trypanosomiasis, ...

3D Visualization of Immune Cell Populations in HIV-Infected Tissues via Clearing, Immunostaining, Confocal, and Light Sheet Fluorescence Microscopy

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 million individuals infected worldwide and no widely accessible cure. Despite intensive efforts, a detailed understanding of virus and host cell interactions in tissues during infection and in response to therapy remains incomplete. To address these limitations, water-based tissue clearing techniques CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) and CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/in situ-hybridization-compatible Tissue hYdrogel) are applied to visualize complex virus host-cell interactions in HIV-infected tissues from animal models and humans using confocal and light sheet fluorescence microscopy. Optical sectioning of intact tissues and image analysis allows rapid reconstruction of spatial information contained within whole tissues and quantification of immune cell populations during infection. These methods are applicable to most tissue sources and diverse biological questions, including infectious disease and cancer.

Tissue clearing, combined with immunofluorescence microscopy, allows spatial visualization and quantification of immune-cell populations and virus proteins within intact tissues. Optical sectioning of cleared tissues with confocal and light sheet fluorescence microscopy can generate 3D models of complex tissue environments and reveal spatial heterogeneity exhibited during HIV infection.

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 ...

Biology

Ultrastructural Expansion Microscopy in In Vitro Life Cycle of Trypanosoma cruzi

Ultrastructural Expansion Microscopy in Three In Vitro Life Cycle Stages of Trypanosoma cruzi

We describe here the application of ultrastructure expansion microscopy (U-ExM) in Trypanosoma cruzi, a technique that allows increasing the spatial resolution of a cell or tissue for microscopic imaging. This is performed by physically expanding a sample with off-the-shelf chemicals and common lab equipment.
Chagas disease is a widespread and pressing public health concern caused by T. cruzi. The disease is prevalent in Latin America and has become a significant problem in non-endemic regions due to increased migration. The transmission of T. cruzi occurs through hematophagous insect vectors belonging to the Reduviidae and Hemiptera families. Following infection, T. cruzi amastigotes multiply within the mammalian host and differentiate into trypomastigotes, the non-replicative bloodstream form. In the insect vector, trypomastigotes transform into epimastigotes and proliferate through binary fission.The differentiation between the life cycle stages requires an extensive rearrangement of the cytoskeleton and can be recreated in the lab completely using different cell culture techniques. 
We describe here a detailed protocol for the application of U-ExM in three in vitro life cycle stages of Trypanosoma cruzi, focusing on optimization of the immunolocalization of cytoskeletal proteins. We also optimized the use of N-Hydroxysuccinimide ester (NHS), a pan-proteome label that has enabled us to mark different parasite structures.

This study shows a detailed protocol to perform ultrastructure expansion microscopy in three in vitro life cycle stages of Trypanosoma cruzi, the pathogen responsible for Chagas disease. We include the optimized technique for cytoskeletal proteins and pan-proteome labeling.

We describe here the application of ultrastructure expansion microscopy (U-ExM) in Trypanosoma cruzi, a technique that allows increasing the spatial ...

A Double-Labeling Immunofluorescence Technique to Visualize Host and Pathogen Proteins in an Infected Cell

Source: Gachet-Castro, C. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62219">Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions.</a>&#160;J. Vis. Exp.&#160;(2021)
This video showcases double-labeling immunostaining with two different primary antibodies raised in the same species, binding specifically to parasite and host proteins. Furthermore, labeled secondary antibodies targeting these primary antibodies allow the distinct visualization of host and parasite proteins, facilitating the study of host-pathogen interaction.

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

Summary

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Chapters in this video

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

High-Resolution Three-Dimensional Whole-Organ Tomography of Microbial Infections

Demystifying In Vivo Bioluminescence Imaging of a Chagas Disease Mouse Model for Drug Efficacy Studies

3D Visualization of Immune Cell Populations in HIV-Infected Tissues via Clearing, Immunostaining, Confocal, and Light Sheet Fluorescence Microscopy

Ultrastructural Expansion Microscopy in Three In Vitro Life Cycle Stages of Trypanosoma cruzi

A Double-Labeling Immunofluorescence Technique to Visualize Host and Pathogen Proteins in an Infected Cell