T. S. is supported by grants from the DFG German Research Foundation (332853055), Else Kr&#246;ner-Fresenius-Stiftung (2015_A197), and the Medical Faculty of the RWTH Aachen (RWTH Returner Program). V. G. P. is supported by research fellowships from Deutsche Gesellschaft fur Nephrologie, the Alexander von Humboldt Foundation, and the National Health and Medical Research Council of Australia. D. H. E is supported by Fondation LeDucq. R. K. is supported by grants from the DFG (KR-4073/3-1, SCHN1188/5-1, SFB/TRR57, SFB/TRR219), the State of Northrhinewestfalia (MIWF-NRW) and the Interdisciplinary Centre for Clinical Research at RWTH Aachen University (O3-11).

NOTE: All experimental procedures described here were approved by the Institutional Animal Care and Use Committee (IACUC) of Oregon Health and Science University, Portland, Oregon, USA, and relevant local authorities in Aachen, Germany.
1. Retrograde Abdominal Aortic Perfusion and Fixation of Mouse Kidneys
<ol>
	<li>Prepare solutions the same day or evening before and store in a fridge overnight. Warm solutions to room temperature (RT) before using.</li>
	<li>Make a fresh batch of 3% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS). About 50-100 mL PFA is needed per mouse.
	<ol>
		<li>To make 1 L of 3% PFA: Weigh 30 g of PFA and add 800 mL of distilled water in the fume hood. Stir and heat to 50-60 &#176;C. Do not heat above 70 &#176;C. 
		CAUTION: PFA is toxic. Prepare PFA in the fume hood.</li>
		<li>Slowly add several drops of 2N NaOH. Wait a few min until PFA goes into the solution or add a few more drops. Some small chunks will not dissolve.</li>
		<li>Remove the solution from heat. Add 50 mL of 20x PBS. Chill on ice to RT.</li>
		<li>Adjust the pH to 7.3-7.4 with HCl. Add distilled water to 1 L.</li>
		<li>Remove any undissolved particles by filtering 1x PBS and 3% PFA with 0.22 &#181;m filter.</li>
	</ol>
	</li>
	<li>Add 1,000 units of Heparin to 1 L of 1x PBS. Transfer 1x PBS containing heparin and 3% PFA in 1x PBS into separate 50 mL plastic syringes. 
	NOTE: If available, pressure-controlled pump set at 80-100 mmHg or hydrostatic pressure (drip method, the height of the perfusion solutions: 160-200 cm above animal) can be used for kidney perfusion.</li>
	<li>Connect the PBS, PFA and a blunted 21 G butterfly needle to a three-way stopcock. Make sure there are no air bubbles in the whole system.</li>
	<li>Label a 15 mL conical tube and dispense 10 mL of PFA into it.</li>
	<li>Deeply anesthetize a male or female C57BL/6 mouse 12-24 weeks of age using 120 mg/kg bodyweight ketamine and 16 mg/kg bodyweight xylazine. The animal must be checked for complete absence of responsiveness by pinching the reflexes before proceeding to surgery (e.g., toe pinch reflex).</li>
	<li>Once the animal has reached a surgical plane of anesthesia, place it on its back under the dissecting microscope. Surgically open the abdomen with a midline abdominal incision using an operating scissors and expose the abdominal aorta.</li>
	<li>Clamp the abdominal aorta right above the branching to the iliac artery with a curved hemostat. Then clamp the abdominal aorta right below the renal arteries with a micro serrefine. Make a small incision (1 mm) on the abdominal aorta between the two clamps with vannas scissors. Insert the butterfly needle into the incision slowly and be careful not to rip the abdominal aorta open.</li>
	<li>Ligate the right renal artery with an 5-0 silk suture and remove the right kidney for other analysis if only one kidney is needed for perfusion and fixation.</li>
	<li>Remove the micro serrefine, transect the portal vein with vannas scissors, and immediately perfuse with 50 mL of PBS containing heparin, then switch and perfuse with 50 mL of 3% PFA. 
	NOTE: High perfusion pressure through abdominal aorta is required to open renal tubules for better antibody diffusion through tissue. Perfusion through heart may not open renal tubules.</li>
	<li>Collect the perfused kidney carefully and avoid puncturing or squeezing the tissue.</li>
	<li>Remove the capsule and cut the kidney into 1 mm thick coronal slices. Use a slicer matrix (Table of Materials) to standardize slice thickness. 
	NOTE: Alternatively, consider using a vibratome.</li>
	<li>Immerse the kidney slices with the prepared PFA in the labeled 15 mL conical tube.</li>
	<li>Dispense 10 mL of PFA to another labeled 15 mL conical tube. Flush the three-way stopcock and butterfly needle with PBS before moving to the next mouse.</li>
	<li>Carry out post-fixation overnight at RT protected from light.</li>
</ol>
2. Tissue Preparation and Immunostaining
<ol>
	<li>After post-fixation, wash the kidney slice twice with 1x wash buffer (Table of Materials) for 1 h on a horizontal rocker at RT.</li>
	<li>Perform antigen retrieval. Heat up 300 mL of 1x antigen unmasking solution (Table of Materials) in a 500 mL beaker to 92-98 &#176;C. Enclose the slice in embedding cassette permeable to the heated buffer with stirring for 1 h at 92-98 &#176;C. Remove the beaker from heat and leave it to cool to RT. 
	NOTE: Some vendors test their antibodies for immunohistochemistry application and will include a suggested antigen retrieval method in the datasheet. Therefore, some epitopes may require a more basic buffer (e.g., pH 9).</li>
	<li>Transfer the slice into 10 mL of 1x wash buffer with 0.1% Triton X-100 and rock overnight at RT. Wash the slices 2x with 10 mL of fresh 1x wash buffer for 1 h the next day.</li>
	<li>Dilute the primary antibody in 500 &#181;L of normal antibody diluent (Table of Materials). Begin with a concentration of &#173;&#173;&#173;1:50-1:100. Gently rock the kidney slice in diluted primary antibody for 4 d at 37 &#176;C. 
	NOTE: Since each antibody has unique properties, temperature during antibody incubation and dilutions of antibody need to be optimized for individual probes. For secondary antibody-only controls, incubate kidney tissue in diluent without primary antibody. Instead of commercial antibody diluent, 1x PBS with 0.1% Triton X-100 and 0.01% sodium azide can be used.</li>
	<li>Wash the kidney slice in 10 mL of 1x wash buffer overnight at RT with one change of wash buffer after 8 h.</li>
	<li>Dilute the secondary antibodies (e.g., 1:100 for Alexa Fluor-conjugated secondary antibodies) in 500 &#181;L of normal antibody diluent. Incubate the kidney slices in diluted secondary antibody for 4 days at 37 &#176;C. From this step onwards, protect the kidney slices from light.</li>
	<li>Wash the kidney slices in 10 mL of 1x wash buffer overnight at RT with one change of wash buffer after 8 h.</li>
</ol>
3. Tissue Clearing
<ol>
	<li>Transfer kidney slice to 5 mL of high grade 100% ethanol (Table of Materials) for 2 h at RT with gentle rocking (with one change to fresh ethanol after 1 h). This step is for tissue dehydration. 
	NOTE: High grade ethanol is required to achieve a high tissue translucency in the next step. Methanol or tetrahydrofuran are alternative dehydration reagents with high delipidation potential.</li>
	<li>Immerse kidney slice in 2 mL of ECi (Table of Materials) with gentle rocking at RT (with one change to fresh ECi after 2 h) overnight. 
	NOTE: The freezing/melting point of ECi is 6-8 &#176;C. Therefore, do not store samples in the fridge. Conduct immersion in a properly ventilated fume hood and avoid direct contact with clothes and skin (ECi is a non-toxic Food and Drug Administration-approved compound but has a strong odor). Use regular Eppendorf tubes or glass vessels (no polystyrene vessels).</li>
	<li>Tissue translucency can be achieved after ECi immersion and when the kidney slices are ready for imaging.</li>
</ol>
4. ConfocalImaging and Image Analysis
NOTE: For imaging, other microscopy techniques can be used as long as the refractive index matching solution is compatible with the objective lens. This protocol uses an inverted confocal microscope.
<ol>
	<li>Add 600-1,000 &#956;L of ECi into the glass bottom dish (Table of Materials). 
	NOTE: Avoid use of regular cell culture dishes, because ECi is an organic solvent that will dissolve the plastic dishes. Similarly, ECi might attack plastic parts/insulation rings on objective lenses. Refer to the appropriate reports for an overview of compatible imaging dishes30 and self-made 3-D printed chambers28.</li>
	<li>Transfer the translucent kidney slice into the dish. Place a round coverslip on the kidney slice to apply light pressure towards the glass bottom. Seal the dish with paraffin film (Table of Materials) to avoid leakage of ECi. 
	NOTE: Whole organs or several millimeter-thick tissue slices may require a border (dental cement or silicone elastomer; see Table of Materials) around the tissue to make an ECi-pool for the sample.</li>
	<li>Place the dish onto the microscope imaging platform.</li>
	<li>Take several z-stacks and perform stitching. Start with a z-step size of 5 &#956;m. 
	NOTE: Consider using long working distance (&#62;5 mm) and high numerical aperture (&#62;0.9) objectives for imaging of very thick tissue slices or organs. After imaging, transfer the tissue back to ethanol and store in wash buffer or PBS with 0.02% sodium azide.</li>
	<li>Analyze the image using 3-D rendering with software (Table of Materials).</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+of+acrylamide+sodium+dodecyl+sulphate+(SDS)-based+tissue+clearing+(FASTClear):+a+novel+protocol+of+tissue+clearing+for+3-D+visualization+of+human+brain+tissues.">Free of acrylamide sodium dodecyl sulphate (SDS)-based tissue clearing (FASTClear): a novel protocol of tissue clearing for 3-D visualization of human brain tissues.</a> Neuropathology and Applied Neurobiology. 43 (4), 346-351 (2017).</li><li>Xu, 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=Fast+free-of-acrylamide+clearing+tissue+(FACT)-an+optimized+new+protocol+for+rapid,+high-resolution+imaging+of+3-D+brain+tissue.">Fast free-of-acrylamide clearing tissue (FACT)-an optimized new protocol for rapid, high-resolution imaging of 3-D brain tissue.</a> Scientific Reports. 7 (1), 9895 (2017).</li><li>Tainaka, 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=Whole-body+imaging+with+single-cell+resolution+by+tissue+decolorization.">Whole-body imaging with single-cell resolution by tissue decolorization.</a> Cell. 159 (4), 911-924 (2014).</li><li>Matryba, P., Bozycki, L., Pawlowska, M., Kaczmarek, L., Stefaniuk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+perfusion-based+CUBIC+protocol+for+the+efficient+whole-body+clearing+and+imaging+of+rat+organs.">Optimized perfusion-based CUBIC protocol for the efficient whole-body clearing and imaging of rat organs.</a> Journal of Biophotonics. 11 (5), e201700248 (2018).</li></ol>

The authors have nothing to disclose.

Optical clearing techniques have received wide attention for 3-D visualization and quantification of microanatomy in various organs. Here, solvent-based clearing method (ECi) was combined with immunolabeling for 3-D imaging of whole tubules in kidney slices. This method is simple, inexpensive, and quick. However, other research questions may be best answered with other clearing protocols5. It is also important to keep in mind that solvent-based methods cause tissue-shrinkage at variable degrees, m...

The growing interest in studying entire organs or large multicellular structures have led to the development of optical clearing methods that involve imaging of transparent tissue in three dimensions. Until recently, the best methods to estimate cell number, length, or volume of whole structures have been stereology or exhaustive serial sectioning, which is based on the systemic sampling of tissue for subsequent analysis in two dimensions1,2,3. However, these methods are time-consuming and need a high level of training and expertise4. Optical clearing methods overcome these problems by equilibrating the refractive index throughout a sample to make tissue translucent for 3-D imaging5,6,7.
Several optical clearing methods have been developed which fall into two main categories: solvent-based and aqueous-based methods. Aqueous-based methods can be further divided into simple immersion8,9, hyperhydration10,11, and hydrogel embedding12,13. Solvent-based methods dehydrate the tissue, remove lipids and normalize the refractive index to a value around 1.55. Limitations of most solvent-based methods are quenching of endogenous fluorescence of commonly used reporter proteins such as GFP, solvent toxicity, capacity to dissolve glues used in some imaging chambers or objective lenses, and shrinkage of tissue during dehydration14,15,16,17,18,19,20,21. However, solvent-based methods are simple, time-efficient, and can work in a number of different tissue types.
Aqueous-based methods rely on the immersion of the tissue in aqueous solutions that have refractive indices in the range of 1.38-1.528,11,12,22,23,24. These methods were developed to preserve endogenous fluorescent reporter protein emission and prevent dehydration-induced shrinkage, but limitations of most aqueous-based clearing methods include a longer duration of the protocol, tissue expansion, and protein modification (i.e. partial denaturation of proteins by urea in hyperhydrating protocols such as ScaleA2)7,11,23,25. ScaleS addressed tissue expansion by combining urea with sorbitol, which counterbalances by dehydration the urea-induced tissue expansion, and preserved the tissue ultrastructure as evaluated by electron microscopy10. Tissue shrinkage or expansion affects the absolute sizes of structures, distances between objects, or cell density per volume; thus, the measurement of size changes upon clearing of the tissue may help interpret the obtained results7,26.
In general, a protocol for optical clearing consists of multiple steps, including pretreatment, permeabilization, immunolabeling (if required), refractive index matching, and imaging with advanced light microscopy (e.g., two-photon, confocal, or light-sheet fluorescence microscopy). Most of the clearing approaches have been developed to visualize neuronal tissue, and emerging studies have validated their application in other organs5. This comprehensive tool has been previously demonstrated to allow reliable and efficient analysis of kidney structures, including glomeruli27,28, immune infiltrates28, vasculature28, and tubule segments29, and it is an ideal approach to better the understanding of glomerular function and tubule remodeling in health and disease.
Summarized here is a solvent-based method that combines immunostaining of kidney tubules; optical clearing with cheap, non-toxic, and ready-to-use chemical ethyl cinnamate (ECi); and confocal microscopy imaging that allows complete tubule visualization and quantification. This method is simple, combines antigen-retrieval of kidney slices with staining of commercial antibodies, and does not require specialized equipment, which makes it accessible to most laboratories.

<table>
	<tbody>
		<tr>
			<td>0.22 &#181;m filter</td>
			<td>Fisher Scientific</td>
			<td>09-761-112</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>Fisher Scientific</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>21 gauge butterfly needle</td>
			<td>Braun</td>
			<td>Venofix</td>
			<td></td>
		</tr>
		<tr>
			<td>3-way stopcock</td>
			<td>Fisher Scientific</td>
			<td>K420163-4503</td>
			<td></td>
		</tr>
		<tr>
			<td>3D analyis software</td>
			<td>Bitplane AG</td>
			<td>IMARIS</td>
			<td></td>
		</tr>
		<tr>
			<td>3D analyis software</td>
			<td>Cellprofiler</td>
			<td></td>
			<td>free open-source software</td>
		</tr>
		<tr>
			<td>5-0 silk suture</td>
			<td>Fine Science Tools</td>
			<td>18020-50</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml plastic syringes</td>
			<td>Fisher Scientific</td>
			<td>14-817-57</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-BrdU monoclonal antibody</td>
			<td>Roche</td>
			<td>11296736001</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody diluent</td>
			<td>Dako</td>
			<td>S0809</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31-647</td>
			<td>BioLegend</td>
			<td>102516</td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate-based antigen retrieval solution</td>
			<td>Vector Laboratories</td>
			<td>H-3300</td>
			<td></td>
		</tr>
		<tr>
			<td>curved hemostat</td>
			<td>Fisher Scientific</td>
			<td>13-812-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako Wash Buffer</td>
			<td>Agilent</td>
			<td>S3006</td>
			<td></td>
		</tr>
		<tr>
			<td>dissecting microscope</td>
			<td>Motic</td>
			<td>DSK-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding cassettes</td>
			<td>Carl Roth</td>
			<td>E478.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>100983</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl cinnamate</td>
			<td>Sigma-Aldrich</td>
			<td>112372</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexible film/Parafilm M</td>
			<td>Sigma-Aldrich</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-AQP2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-9882</td>
			<td></td>
		</tr>
		<tr>
			<td>Guinea pig anti-NKCC2</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>DOI: 10.1681/ASN.2012040404</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Carl Roth</td>
			<td>P074.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sagent Pharmaceuticals</td>
			<td>401-02</td>
			<td></td>
		</tr>
		<tr>
			<td>hemostat</td>
			<td>Agnthos</td>
			<td>312-471-140</td>
			<td></td>
		</tr>
		<tr>
			<td>horizontal rocker</td>
			<td>Labnet</td>
			<td>S2035-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging dish</td>
			<td>Ibidi</td>
			<td>81218</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>MWI Animal Health</td>
			<td>501090</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro serrefine</td>
			<td>Fine Science Tools</td>
			<td>18052-03</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher Scientific</td>
			<td>S318-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating scissors</td>
			<td>Merit</td>
			<td>97-272</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Thermo Fischer Scientific</td>
			<td>O4042-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-phoshoThr53-NCC</td>
			<td>PhosphoSolutions</td>
			<td>p1311-53</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone elastomer</td>
			<td>World Precision Instruments Kwik-Sil</td>
			<td>KWIK-SIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue slicer</td>
			<td>Zivic Instruments</td>
			<td>HSRA001-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Acros Organics</td>
			<td>AC215682500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Lancer</td>
			<td>Series 1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>MWI Animal Health</td>
			<td>AnaSed Inj SA (Xylazine)</td>
			<td></td>
		</tr>
	</tbody>
</table>

optical clearing and imaging of immunolabeled kidney tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

Kidneys are complex organs comprised of 43 different cell types31. Most of these cells form large multicellular structures such as glomeruli and tubules, and their function is highly dependent on interactions with each other. Classical 2-D histological techniques partially capture these large structures and may miss focal changes within intact structures31. Thus, 3-D analysis using optical clearing techniques helps to understand how they function in health and disease.
...

Watch this Scientific Journal Video about Optical Clearing and Imaging of Immunolabeled Kidney Tissue at JoVE.com

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

optical-clearing-and-imaging-of-immunolabeled-kidney-tissue

Research

JoVE Journal

Medicine

10.3K Views.  University Hospital RWTH Aachen. The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Video: Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, the fundamental physical diffraction limit prevents interrogation of nanoscale anatomy and subtle pathological changes when using conventional optical imaging approaches. Here, we describe a simple and inexpensive protocol, called expansion pathology (ExPath), for nanoscale optical imaging of common types of clinical primary tissue specimens, including both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. This method circumvents the optical diffraction limit by chemically transforming the tissue samples into tissue-hydrogel hybrid and physically expanding them isotropically across multiple scales in pure water. Due to expansion, previously unresolvable molecules are separated and thus can be observed using a conventional optical microscope.

Nanoscale imaging of clinical tissue samples can improve understanding of disease pathogenesis. Expansion pathology (ExPath) is a version of expansion microscopy (ExM), modified for compatibility with standard clinical tissue samples, to explore the nanoscale configuration of biomolecules using conventional diffraction limited microscopes.

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...