NIDDK K08 DK090754 to MPH. NIGMS P41 GM103493 to RDS. This material is the result of work (by MPH) which was supported with resources and the use of facilities at the Portland Veterans Affairs Medical Center. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

All procedures described herein were approved by the Institutional Animal Care and Use Committee of Oregon Health &#38; Science University.
1. Setup Used for Demonstration
<ol>
	<li>Use an upright 2-photon microscope, a 3-axis stage controller, a headstage/pipette holder, and a 3-axis controller for the headstage/pipette holder; all four of these items are required. 
	NOTE: Many similar setups are available and will suffice as long as independent quantitative 3-axis control of the pipette and microscope stage are available, and the microscope is set up for in vivo upright imaging.</li>
</ol>
2. Materials Necessary Prior to Starting Experimental Protocols
<ol>
	<li>Use FITC-dextran, 2,000,000 Da, 5% solution in normal saline or phosphate-buffered saline for retroorbital injection to mark the renal vasculature. This molecular weight (MW) is selected because it remains in the vasculature and does not filter.</li>
	<li>Machine-pulled borosilicate glass micropipettes: Pull micropipettes to 6&#8211;10 &#181;m tip using long-taper, closed tip settings (e.g., heat = 610, velocity = 150, time = 250 ms, looped) on a micropipette puller. Bevel to 45&#176; and flame-polish lightly.</li>
	<li>Quantum-dot coating of micropipettes: Coat micropipette tips with quantum dots according to the referenced protocol.12</li>
	<li>Polysiloxane kidney support/spacer. Use polysiloxane putty to craft a kidney support/spacer. Fashion a 1 x 1 cm2 by 5 mm thick right-angle rhomboid (i.e., a truncated cube) from polysiloxane putty. Remove the middle 70% of the polysiloxane from one edge and a circle comprising about 70% of the center of the rhomboid. Allow the polysiloxane to dry for 24 h. See Figure 1.</li>
	<li>Microinjector preparation: Prefill a length of polyethylene (PE)-50 tubing and the micropipette-loaded micropipette holder with oil. If mass spectrometry is planned, perfluorodecalin is required, otherwise mineral oil may be used. Set up the microinjector with a gas-tight Hamilton-type syringe and fill with perfluorodecalin. This will be connected to the PE-50 tubing and pipette holder.</li>
	<li>Place the pipette in the pipette holder and forward fill the PE-50 tubing and pipette, then attach the proximal end of the PE-50 tubing to the oil-filled Hamilton syringe, creating a hydraulic system from pipette to syringe.</li>
</ol>
3. Lateral Pipette Access to The Kidney Below a Fluid Imaging Column via a Novel Surgical Procedure
NOTE: The assembly of the imaging support system and surgical prep is shown in Figure 1. The procedure described is performed on C57BL/6 mice weighing 20&#8211;25 g.
<ol>
	<li>Weigh the mouse.</li>
	<li>Induce anesthesia using 4% isoflurane and maintain with 1.5&#8211;2.5% isoflurane in air/oxygen mixture. Confirm that the mouse is anesthetized by absence of response to painful stimulus and reduced respiratory rate.</li>
	<li>Lubricate the eyes and position the animal lateral on a baseplate. Immobilize 4 extremities using tape.</li>
	<li>Inject normal saline, 200 &#181;L, subcutaneously and place a rectal temperature probe. Control temperature using a heating lamp during surgery and a heating pad during imaging.</li>
	<li>Remove all hair on the left side of the mouse using a depilatory cream.</li>
	<li>Locate the spleen, which is visible under the skin, and locate the left kidney on the dorsal and caudal side of spleen.</li>
	<li>Make a 0.5 cm incision on the skin and smaller incision on the peritoneum, just enough for the kidney to push through easily.</li>
	<li>Extrude the kidney with gentle pressure. Place kidney stabilizer form made with polysiloxane around the kidney and fix with cyanoacrylate adhesive. Line the kidney up with the spacer such that the lateral-most surface of the kidney extends beyond the stabilizer by about 1 mm.</li>
	<li>Fix a head plate to the stabilizer form with glue and mount the head plate to mounting bars on the base plate.</li>
	<li>Fill the well in the polysiloxane support surrounding the kidney with 1% agarose solution and place the 10 mm coverslip on top and hold until agarose is firm. Seal coverslip to the head plate with glue and create a ring around the coverslip with dental cement.</li>
	<li>Inject FITC-dextran (2,000,000 Da, 5% solution, 100&#8211;150 &#181;L) retro-orbitally and move the mouse and fixation plate to the 2-photon microscope stage quickly, maintaining anesthesia and ensuring adequate waste gas scavenging on the microscope stage.</li>
</ol>
4. Selection of a Suitable Glomerulus and Pipette Access to Bowman&#39;s Space
<ol>
	<li>Definitions:
	<ol>
		<li>Define X as left-right on the screen and left-right facing the microscope</li>
		<li>Read SX (stage X) from the stage controller</li>
		<li>Define PX as pipette X, on the pipette dial controller</li>
		<li>Define Y as up-down on the screen and forward toward the microscope and back toward the 2-photon setup</li>
		<li>Define Z as up toward the ceiling, down toward the floor, and measured on the stage with the objective Z position.</li>
		<li>Define S(O)Z as the stage (really the objective) height.</li>
	</ol>
	</li>
	<li>Find the surface of the kidney, identifiable using green fluorescent protein (GFP) filter settings in the ocular. Due to the injection of FITC-Dextran, the vasculature will be bright green.
	<ol>
		<li>Identify a suitable glomerulus. After identifying the surface of the kidney using the ocular, switch to 2-photon (non-scanning mode) and explore the imaging window. Favorable characteristics for micropuncture are the following: vertical distance below the coverslip &#62;30 &#181;m (to prevent collision between the pipette and coverslip during access) and lateral distance from the lateral kidney capsule to the glomerulus &#60;400 &#181;m (beyond this distance the deviation of the pipette may increase likelihood of a miss).</li>
	</ol>
	</li>
	<li>Record lateral and vertical distance to the puncture point, a point on the renal capsule directly to the pipette-side of the glomerulus.</li>
	<li>Raise the objective focal point into the water column, keeping the x and y stage coordinates unchanged, a distance of just about a centimeter.</li>
	<li>Drive the pipette tip into the water column and turn on 4&#39;,6-diamidino-2-phenylindol (DAPI) excitation. Move the pipette in the x and y dimensions to the point of maximal fluorescence of the tip, this will be the center of the objective. Finding the pipette in the ocular is difficult without this precise prepositioning. Because quantum dots fluoresce at the same (in this case, red) wavelength regardless of excitation wavelength, the DAPI excitation produces red fluorescence which is tightly focused on the tip, illustrated in Figure 2.</li>
	<li>Change the excitation setting to red fluorescent protein (RFP) and visualize the pipette in the ocular, then precisely center it in the ocular view.</li>
	<li>Switch to 2-photon and find the pipette under 2-photon, placing it precisely in the center of the image. This is the registration position.</li>
	<li>Save an image of the pipette.</li>
	<li>Register the stage and the micropipette controller coordinates. 
	NOTE: Use the supplemental .html file, which will execute calculations using JavaScript code, (advantageous on systems which do not have installed spreadsheet software) or a spreadsheet to calculate the offset between stage and pipette coordinates and to calculate the target coordinates for the pipette controller.</li>
	<li>Remove the pipette from the water column in the x axis, keeping z and y the same.</li>
	<li>Move the pipette Z to the target glomerulus Z (i.e., move the pipette down in the Z direction below the coverslip)</li>
	<li>Move the pipette Y to the target glomerulus Y coordinate.</li>
	<li>Move the 2-photon live view to the target glomerulus Z and then to the edge of the kidney and note the SX.</li>
	<li>Calculate the kidney edge PX using the offset from the registration SX.</li>
	<li>Move the stage toward the pipette (increase the SX) such that the edge of the kidney is far to the left of the screen, but still visible.</li>
	<li>Advance the pipette quickly to about 100 &#181;m less than the kidney edge PX calculated above.</li>
	<li>Locate the pipette tip, advancing the pipette slowly. Increase the red gain and watch the red pixel histogram (the pixel distribution shifts before the pipette is imaged in the window, due to the extreme brightness of quantum dots and off-target fluorescence).</li>
	<li>Advance to the kidney edge under live 2-photon imaging. 
	NOTE: Prior to entering the renal capsule, it is possible to redirect the pipette in the Y and Z dimensions. However, this may break the pipette tip. A more conservative measure, if the pipette is off target, is to withdraw in the X dimension up to 2 cm, redirect, and then return in the X dimension to the kidney edge. Once the pipette is within the tissue, movement in any axis other than X leads to pipette flexion which requires great experience to make use of, and frequently leads to breakage.</li>
	<li>Drive the pipette in the X axis slowly to the glom target PX, keeping an eye on the SX. (It is helpful to occasionally go back to the glomerulus to see if it has shifted at all upon insertion of the micropipette).</li>
	<li>When you are in the correct location, document position with a z-stack. 
	NOTE: With the micropipette in place, drugs, proteins, or fluorescent tracers may be injected, fluid may be aspirated for later analysis, or pressure or charge relative to another electrode may be measured.</li>
</ol>
5. Aspiration of Fluid from Bowman&#39;s Space
<ol>
	<li>Set the micropump to inject 100 nL of perfluorodecalin over 2 min to ensure patency of the pipette and reduce confounding from pipette plugging during entry. Reimage to ensure pipette position.</li>
	<li>Wait 4&#8211;6 min for additional filtration.</li>
	<li>Set the micropump to aspirate up to 300 nL at a rate of up to 50 nL/min. 
	NOTE: Changes in glomerular morphology are not observed with this rate, suggesting it does not alter the rate of delivery of fluid to the space during aspiration. As there is no oil block as in conventional micropuncture, recovery of this volume, necessary for mass spectrometry, could include some of the injected perflourodecalin and possibly tubular fluid. For assays such as ion-sensitive electrode measurements fluorescence spectroscopy, polymerase chain reaction, and other sensitive endpoints, lower volumes may be used. If mass spectrometry is not the endpoint, mineral oil and standard micropuncture techniques can be used to measure the aspirated volume prior to storage.</li>
	<li>Image once more.</li>
	<li>Withdraw the pipette and preserve the sample, adding TRIS buffer and storing at -80&#176; prior to analysis.</li>
	<li>Euthanize the mouse using an overdose of isoflurane or other approved method. 
	NOTE: Filtrate enters the space by filtration from the glomerular capillaries. The single-nephron glomerular filtration rate (SNGFR) in mice is reported between 8&#8211;14 nL/min.3 Starling forces govern SNGFR, however, and negative hydrostatic pressure in Bowman&#39;s space therefore may increase SNGFR. Standard micropuncture methods use tubular blockade with oil and neutral pressure for tubular fluid sampling, however these compounds interfere with mass spectrometry (see below); therefore, in this technique the early proximal tubule remains patent. Further, at the time of aspiration, Bowman&#39;s space contains an unknown, but positive volume of filtrate. Therefore, fluid aspiration rate may exceed SNGFR. 
	NOTE: In the experiments described here, the goal was to obtain a larger than usual sample of glomerular filtrate for mass spectrometry analysis by nanoproteomic techniques. Since use of mass spectrometry precludes use of oil blocks with mineral oil or wax (complex mixtures of organic molecules which reduce signal:noise in mass spectrometry) perfluorodecalin is used to fill the micropipette and syringe. Perflourodecalin is not known to block tubular flow, but is biologically inert and does not interfere with mass spectrometry.</li>
</ol>

<ol>
	<li>Schnermann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropuncture+analysis+of+tubuloglomerular+feedback+regulation+in+transgenic+mice.">Micropuncture analysis of tubuloglomerular feedback regulation in transgenic mice.</a> Journal of the American Society of Nephrology. 10 (12), 2614-2619 (1999).</li><li>Lorenz, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropuncture+of+the+kidney:+a+primer+on+techniques.">Micropuncture of the kidney: a primer on techniques.</a> Comprehensive Physiology. 2 (1), 621-637 (2012).</li><li>Vallon, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropuncturing+the+nephron.">Micropuncturing the nephron.</a> Pflugers Archive. European Journal of Physiology. 458 (1), 189-201 (2009).</li><li>Perrien, D. 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=Novel+methods+for+microCT-based+analyses+of+vasculature+in+the+renal+cortex+reveal+a+loss+of+perfusable+arterioles+and+glomeruli+in+eNOS-/-+mice.">Novel methods for microCT-based analyses of vasculature in the renal cortex reveal a loss of perfusable arterioles and glomeruli in eNOS-/- mice.</a> BMC Nephrology. 17, 24 (2016).</li><li>Ehling, J., 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=Quantitative+Micro-Computed+Tomography+Imaging+of+Vascular+Dysfunction+in+Progressive+Kidney+Diseases.">Quantitative Micro-Computed Tomography Imaging of Vascular Dysfunction in Progressive Kidney Diseases.</a> Journal of the American Society of Nephrology. 27 (2), 520-532 (2016).</li><li>Gimenez, 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=3D+Imaging+of+Nanoparticle+Distribution+in+Biological+Tissue+by+Laser-Induced+Breakdown+Spectroscopy.">3D Imaging of Nanoparticle Distribution in Biological Tissue by Laser-Induced Breakdown Spectroscopy.</a> Scientific Reports. 6, 29936 (2016).</li><li>Miyamoto, 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=Mass+spectrometry+imaging+reveals+elevated+glomerular+ATP/AMP+in+diabetes/obesity+and+identifies+sphingomyelin+as+a+possible+mediator.">Mass spectrometry imaging reveals elevated glomerular ATP/AMP in diabetes/obesity and identifies sphingomyelin as a possible mediator.</a> EBioMedicine. 7, 121-134 (2016).</li><li>Piehowski, P. D., Zhao, R., Moore, R. J., Clair, G., Ansong, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomic+analysis+of+mass+limited+tissue+samples+for+spatially+resolved+tissue+profiling.">Quantitative proteomic analysis of mass limited tissue samples for spatially resolved tissue profiling.</a> Methods in Molecular Biology. , (2017).</li><li>Yi, L., Piehowski, P. D., Shi, T., Smith, R. D., Qian, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+microscale+separations+towards+nanoproteomics+applications.">Advances in microscale separations towards nanoproteomics applications.</a> Journal of Chromatography A. 1523, 40-48 (2017).</li><li>Clair, G., 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=Spatially-resolved+proteomics:+Rapid+quantitative+analysis+of+laser+capture+microdissected+alveolar+tissue+samples.">Spatially-resolved proteomics: Rapid quantitative analysis of laser capture microdissected alveolar tissue samples.</a> Scientific Reports. 6, 39223 (2016).</li><li>Huang, E. L., 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=SNaPP:+Simplified+nanoproteomics+platform+for+reproducible+global+proteomic+analysis+of+nanogram+protein+quantities.">SNaPP: Simplified nanoproteomics platform for reproducible global proteomic analysis of nanogram protein quantities.</a> Endocrinology. 157 (3), 1307-1314 (2016).</li><li>Andrasfalvy, B. 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=Quantum+dot-based+multiphoton+fluorescent+pipettes+for+targeted+neuronal+electrophysiology.">Quantum dot-based multiphoton fluorescent pipettes for targeted neuronal electrophysiology.</a> Nature Methods. 11 (12), 1237-1241 (2014).</li><li>Huang, E. L., 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=SNaPP:+Simplified+nano-proteomics+platform+for+reproducible+global+proteomic+analysis+of+nanogram+protein+quantities.">SNaPP: Simplified nano-proteomics platform for reproducible global proteomic analysis of nanogram protein quantities.</a> Endocrinology. 157 (3), (2016).</li><li>Tojo, A., Endou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrarenal+handling+of+proteins+in+rats+using+fractional+micropuncture+technique.">Intrarenal handling of proteins in rats using fractional micropuncture technique.</a> American Journal of Physiology. 263 (4 Pt 2), F601-F606 (1992).</li><li>Sandoval, R. M., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+glomerular+permeability+of+fluorescent+macromolecules+using+2-photon+microscopy+in+Munich+Wistar+rats.">Quantifying glomerular permeability of fluorescent macromolecules using 2-photon microscopy in Munich Wistar rats.</a> Journal of Visualized Experiments. (74), (2013).</li><li>Sandoval, R. M., Kennedy, M. D., Low, P. S., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+and+trafficking+of+fluorescent+conjugates+of+folic+acid+in+intact+kidney+determined+using+intravital+two-photon+microscopy.">Uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy.</a> American Journal of Physiology: Cell Physiology. 287 (2), C517-C526 (2004).</li><li>Salmon, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+the+endothelial+glycocalyx+links+albuminuria+and+vascular+dysfunction.">Loss of the endothelial glycocalyx links albuminuria and vascular dysfunction.</a> Journal of the American Society of Nephrology. 23 (8), 1339-1350 (2012).</li></ol>

The authors have nothing to disclose.

We present a method to access Bowman&#39;s space of non-surface glomeruli in mice, facilitated by 2-photon microscopy. We developed this procedure to address a key limitation of glomerular micropuncture, the rarity of surface glomeruli addressable by 1-photon microscopy in mice, in order to facilitate an experimental objective, aspiration of fluid from Bowman&#39;s space for subsequent analysis. Development and practice of this technique rests on six critical steps. First, the novel surgical preparation must be carefully carried out so that the imaging water column does not run off the coverslip and the coverslip extends over the area of kidney which is the target of the pipette. Second, the glass pipette used for micropuncture must be rendered visible for 2-photon microscopy, which is accomplished using quantum dots. Third, stereotactic technique is required to precisely position a pipette in Bowman&#39;s space in three dimensions, up to 100 &#181;m below the kidney surface. Therefore, registering the coordinate systems of the pipette and the stage with precision are critical steps. Fourth, careful selection of the target glomerulus is necessary to ensure it is accessible to the pipette without impingement by the kidney support structure and imaging column. Lastly, careful consideration must be given to the analytic steps to follow the acquisition procedure, and volume and timing of acquisition of fluid samples must be matched to the analysis and to glomerular physiology.
We designed an acquisition procedure that may be extended to many analyses, including traditional micropuncture endpoints, such as flame photometry, ion-sensitive electrode measurements, or measurements of pressure, volume or charge. Additionally, we believe this technique will be amenable to novel analytic endpoints including polymerase chain reaction (perhaps following reverse transcription for miRNA) and metabolomics downstream of mass spectrometry. The special modifications employed to facilitate mass spectrometry deserve additional discussion, and they impose some limitations. First, although mass spectrometry is highly sensitive, the low protein content and volume of micropuncture samples renders analysis of protein below the dynamic range of conventional proteomic exploration, and therefore simplified nanoproteomics were necessary.8,13 Second, to optimize protein yield for early assays, we determined that 200-300 nL of aspirate was necessary, but de novo filtrate acquisition of this volume would require perhaps as long as 20 minutes of aspiration if the mouse GFR is only 8-14 nL/min3. As Tojo and Endou demonstrated that prolonged aspiration alters the albumin content of early proximal tubule fluid14, we elected to aspirate over 6 minutes; however this means that our aspiration rate exceeds the filtrate inflow rate. Users of this procedure are encouraged to consider the physiology of glomerular filtration in their experimental system in designing their workflow. Mass spectrometry, a sensitive technique, would be overwhelmed by the signal from an introduced petroleum distillate such as mineral oil, which is commonly used in micropuncture to comprise the hydraulic system for aspiration and isolate segments of the nephron. Therefore, we could not use mineral oil for this purpose, or its other common use, quantification of volume of nanoliter range samples. Instead we fill the system with perfluorodecalin which is biologically inert, does not disturb mass spectrometry, and has favorable optical characteristics. We believe the limitations imposed by perfluorodecalin are surmountable and are working on additional technical innovations which we expect will allow measurement of sample volume and blockade of the tubular segment.
Most micropuncture studies have been performed in Munich-Wistar rats, which demonstrate increased numbers of surface glomeruli, but this greatly limited physiologic study of tubular transport and other renal physiology because of the loss of the fundamental tool of molecular biology, transgenic mice2,3. Because it facilitates micropipette access to Bowman&#39;s space in mice, the novel technique therefore mitigates these critical limitations. We adopted this technique in order to access renal filtrate for proteomic studies using high-sensitivity mass spectrometry, known as nanoproteomics9. However, there are likely additional applications. For example, renal physiologic study of filtered protein has been greatly aided by use of fluorescent tracers with 2-photon microscopy15,16,17. Addition of micropuncture to 2-photon microscopy offers the possibility of performing single-nephron physiologic study with fluorescent molecules, allowing neighboring, non-injected nephrons to serve as controls. It is hoped that this clear explanation of the necessary steps will allow wide adoption in labs already equipped for 2-photon microscopy and/or micropuncture. Although it is complex, we have now performed this procedure many times and the refinements presented herein represent a stable platform for physiologic discovery.

The purpose of this procedure is to provide routine micropuncture access to Bowman&#39;s space and other glomerular structures in mice. Micropuncture studies for renal physiology have been limited to 1-photon microscopy, which can only image within a few microns of the kidney surface, and which offers limited precision in the z-dimension. Because mice have few surface glomeruli, it is not always possible to find a surface glomerulus by 1-photon microscopy, therefore most micropuncture studies have been carried out in Munich-Wistar rats, which have more numerous surface glomeruli. Therefore, the benefits of working in mouse models have been limited in micropuncture studies1,2,3. Recent advances in imaging technologies, including micro-CT4,5, nanoparticle imaging6, and imaging mass spectrometry7 have greatly enhanced the range of modalities applicable to glomerular physiology, but there remains no substitute for the unique ability to intervene and sample that micropuncture provides. Therefore extending the use of micropuncture using the techniques presented here is expected to facilitate novel renal physiology studies, in particular, evaluation of the content of renal filtrate (i.e., metabolomics) and basic physiology of transgenic mice, such as measurements of filtrate pressure and charge, previously performed only in rats.
In this technique, use of 2-photon microscopy allows visualization and micropipette access to renal structures up to about 100 &#181;m below the renal capsule. Multiple (5&#8211;10) glomeruli are therefore accessible to micropuncture in every mouse kidney thus far imaged. Although this technique shares some features with conventional renal micropuncture, it was designed de novo and extensive modifications from conventional technique are required. In this protocol we demonstrate aspiration of fluid from Bowman&#39;s space and show example results of subsequent analysis with mass spectrometry (nanoproteomics)8,9,10,11. Downstream use of mass spectrometry requires a specialized sample preparation workflow, which is also demonstrated here.

<table>
	<tbody>
		<tr>
			<td>Upright 2 photon microscope</td>
			<td>Zeiss</td>
			<td>LSM 7MP</td>
			<td></td>
		</tr>
		<tr>
			<td>3 axis microscope stage controller</td>
			<td>Sutter</td>
			<td>MP-285</td>
			<td></td>
		</tr>
		<tr>
			<td>3 axis headstage controller</td>
			<td>Sutter</td>
			<td>MP-225</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette holder</td>
			<td>Molecular Devices</td>
			<td>1-HL-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Headstage</td>
			<td>Molecular Devices</td>
			<td>CV203BU</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-dextran 2000 kDa MW</td>
			<td>Sigma-Aldrich</td>
			<td>52471-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>borosilicate glass capillary tubes</td>
			<td>Sutter</td>
			<td>B150-110-7.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum dots, 605 nm</td>
			<td>Thermofisher</td>
			<td>Q21701MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysiloxane</td>
			<td>Sugru</td>
			<td>No cat number</td>
			<td>www.sugru.com, &#34;original formula&#34;. Any color.</td>
		</tr>
		<tr>
			<td>PE-50 tubing</td>
			<td>Instech Labs</td>
			<td>BTPE-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>WPI</td>
			<td>UMP-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector controller</td>
			<td>WPI</td>
			<td>Micro4</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfluorodecalin</td>
			<td>Sigma-Aldrich</td>
			<td>306-94-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>9012-36-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip, 10 mm</td>
			<td>Harvard Apparatus</td>
			<td>64-0718</td>
			<td></td>
		</tr>
		<tr>
			<td>Headplate</td>
			<td>Custom</td>
			<td>No part number</td>
			<td>Common in neuroscience labs, many suppliers</td>
		</tr>
		<tr>
			<td>Head fixation device</td>
			<td>Custom</td>
			<td>No part number</td>
			<td>Common in neuroscience labs, many suppliers</td>
		</tr>
		<tr>
			<td>30 gauge needle</td>
			<td>Becton-Dickinson</td>
			<td>125393</td>
			<td>For retroorbital injection</td>
		</tr>
		<tr>
			<td>Tuberculin syringe</td>
			<td>Becton-Dickinson</td>
			<td>309626</td>
			<td>For retroorbital injection</td>
		</tr>
	</tbody>
</table>

micropuncture of bowman's space in mice facilitated by 2 photon microscopy

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

This procedure requires a unique surgical preparation of the kidney for 2-photon imaging and access, which is illustrated in Figure 1. This preparation shown here allows a vertical imaging column with the objective above the kidney with few density changes for best-possible optics for 2-photon microscopy simultaneously with lateral access for the pipette, driven exclusively in the horizontal (x) dimension. Partial extrusion of the kidney prevents excess tension on the renal pedicle and preserves vascular flow, and construction of a custom kidney support enables the two objectives of imaging and access. The second challenge in this procedure is precise positioning of the pipette within the kidney in 3 dimensions, which requires registration of the pipette and stage coordinate systems. The critical step for this process is illustrated in Figure 2, which shows the pipette being spotted in the water column of the 2-photon microscope under DAPI-excitation. Entering the water column and registering the coordinates of the pipette to those of the stage prior to entering the kidney is critical to enable precise stereotactic positioning of the pipette within the target Bowman&#39;s space. The pipette enters the imaging water column from the right. With DAPI excitation turned on, the red quantum dot-coated pipette fluoresces brightly in the red-orange, and it can be carefully positioned under the middle of the objective. As the excitation beam passes through the center of the objective, the pipette may be freely moved to the point of maximum fluorescence, ensuring that it will be visible in the eyepiece.
Proper pipette pulling and glomerulus selection are critical to the success of this protocol, as illustrated in Figure 3, Figure 4, Figure 5. In Figure 3A, a properly-pulled, red-fluorescent quantum dot-coated glass micropipette imaged in the fluid column during the pipette registration portion of the procedure can be seen. The tip is 6 microns in width. In Figure 3B, a poorly-pulled pipette with 12 &#181;m tip is shown. This pipette cannot penetrate the renal capsule without causing vascular trauma due to the 12 &#181;m diameter and irregular tip surface (note the bur at the top of the bevel). In Figure 3C and 3D, the importance of optimal positioning rather than imaging of the target glomerulus is shown. The beautiful, near-surface glomerulus illustrated in Figure 3C demonstrates favorable imaging (due to its surface position at 20 &#181;m below the renal capsule) but would not be suitable for access by this procedure because it is too close to the surface, and the pipette would hit the coverslip. In Figure 3D, optimally-positioned glomeruli are shown. Note the different scale used to illustrate both glomeruli (scale bars are all 50 &#181;m). These glomeruli appear less sharp because of refraction caused by depth; this image was taken at 70 &#181;m below the renal capsule. The lateral kidney edge is 250 &#181;m to the right, making both of these glomeruli accessible. During an access procedure, imaging is tightly focused on the target glomerulus as in Figure 4, and every-second image acquisition is used, allowing the investigator to precisely observe positioning of the pipette in Bowman&#39;s space.
Figure 4 illustrates a typical renal entry and the result, a pipette tip within Bowman&#39;s space. In Figure 4A, a mean intensity projection from a z-stack with orthogonal views demonstrates the pipette tip in Bowman&#39;s space. Note that there is red pipette tip spectral artifact (round ball of fluorescence) due to extremely bright fluorescence of the quantum dots arranged on the conical section of the tip. In Figure 4B, a volume projection of z-stack data demonstrates another pipette in Bowman&#39;s space. Note that the pipette dragged Bowman&#39;s capsule in the direction of travel on entry, creating apparent tenting behind the tip as described in the protocol.
In Figure 5, the results of a failed procedure are shown in which a pipette with a too-large opening broke at the renal capsule, causing bleeding. The pipette was too blunt; on attempting to pass the renal capsule, the capsule was pushed ahead of the pipette tip until breakage occurred. In this image, the renal capsule is visible, enhanced by subcaspular bleeding, in FITC-fluorescent green. FITC signal is visible within the pipette itself, indicating that blood under pressure entered the pipette lumen. The arrow points to many red blood cells visible within the pipette lumen as filling defects in the FITC-dextran.
Figure 6 depicts a representative mass spectrum obtained from Bowman&#39;s space aspirate, mouse urinary protein 17 (MUP17). Lastly, Table 1 demonstrates example results of successful aspiration procedures, listing proteins identified using nanoscale mass spectrometry on aspirate collected over 6 minutes from each of 3 mice. In each case, the pipette was imaged as it was withdrawn from Bowman&#39;s space, and no FITC fluorescence was observed within Bowman&#39;s space or the pipette lumen, indicating lack of aspirate contamination with plasma. 17 proteins, primarily of low molecular weight, were identified from a minimum of 2 unique peptides per protein. Spectral counts are low, consistent with prior estimates of protein in the glomerular filtrate, and known filtered proteins, such as vitamin D binding protein (VTDB), albumin (ALBU), and major urinary protein 17 (MUP17) are present.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58206/58206fig1.jpg" /> 
Figure 1: Partial extrusion of the kidney with custom support and immobilization for lateral access. On the left, the parts of the imaging column and kidney support are shown, with the complete assembly in center. On the right, the kidney preparation is shown before (above) and after (below) application of the support. <a href="https://www.jove.com/files/ftp_upload/58206/58206fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58206/58206fig2.jpg" /> 
Figure 2: Completed kidney prep at pipette registration step of protocol. Here, DAPI excitation is being used to position the micropipette within the water column of the 2 photon microscope. <a href="https://www.jove.com/files/ftp_upload/58206/58206fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58206/58206fig3.jpg" /> 
Figure 3: Imaging of pipettes and the kidney after injection of FITC-dextran, demonstrating suitable and unsuitable glomeruli for micropuncture. A. A well-pulled pipette with 6 &#181;m tip. B. A rough-edged, blunt tip. C. This glomerulus is well-defined, but too close to the coverslip for micropuncture. D. Suitably positioned glomeruli. Scale bars are all 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/58206/58206fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58206/58206fig4.jpg" /> 
Figure 4: Successful pipette passage leads to placement in Bowman&#39;s space-views from 2 different procedures. A. Z-stack with orthogonal projections demonstrates pipette tip in Bowman&#39;s space abutting the glomerular tuft. Scale bar is 50 &#181;m. B. Volume rendering from z-stack similarly demonstrates a pipette in Bowman&#39;s space abutting the glomerular tuft. Scale bar is 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/58206/58206fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58206/58206fig5.jpg" /> 
Figure 5: An unsuccessful procedure due to a blunt pipette, tearing the renal capsule and leading to bleeding into the pipette lumen. FITC fluorescence from extravasated plasma, and red blood cells (arrow) are visible within the pipette. Arrow points to red blood cells visible within the pipette lumen. Scale bar is 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/58206/58206fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58206/58206fig6.jpg" /> 
Figure 6: The mass spectrum for major urinary protein 17 (MUP17), obtained from nanoscale liquid chromatography/mass spectrometry analysis of Bowman&#39;s space aspirate. <a href="https://www.jove.com/files/ftp_upload/58206/58206fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protein</td>
			<td>MW (kD)</td>
			<td>Mean Spectral Count</td>
		</tr>
		<tr>
			<td>ACTA_MOUSE</td>
			<td>42</td>
			<td>2</td>
		</tr>
		<tr>
			<td>ACTB_MOUSE</td>
			<td>42</td>
			<td>1</td>
		</tr>
		<tr>
			<td>CLPX_MOUSE</td>
			<td>69</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>DHSA_MOUSE</td>
			<td>73</td>
			<td>1</td>
		</tr>
		<tr>
			<td>FOLR2_MOUSE</td>
			<td>29</td>
			<td>1</td>
		</tr>
		<tr>
			<td>GBLP_MOUSE</td>
			<td>35</td>
			<td>1</td>
		</tr>
		<tr>
			<td>ALBU_MOUSE</td>
			<td>66</td>
			<td>6.7</td>
		</tr>
		<tr>
			<td>HBA_MOUSE</td>
			<td>15</td>
			<td>2</td>
		</tr>
		<tr>
			<td>HBB1_MOUSE</td>
			<td>16</td>
			<td>1</td>
		</tr>
		<tr>
			<td>MIB1_MOUSE</td>
			<td>110</td>
			<td>1</td>
		</tr>
		<tr>
			<td>MUP17_MOUSE</td>
			<td>21</td>
			<td>1</td>
		</tr>
		<tr>
			<td>PERI_MOUSE</td>
			<td>54</td>
			<td>1</td>
		</tr>
		<tr>
			<td>RNAS4_MOUSE</td>
			<td>17</td>
			<td>2</td>
		</tr>
		<tr>
			<td>SPTB1_MOUSE</td>
			<td>2</td>
			<td>1</td>
		</tr>
		<tr>
			<td>VIME_MOUSE</td>
			<td>54</td>
			<td>1</td>
		</tr>
		<tr>
			<td>VTDB_MOUSE</td>
			<td>53</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
Table 1: List of proteins identified in Bowman&#39;s space aspirate from 3 mice. 
Supplemental Video 1: a volume rendering from a z-stack acquired after positioning a pipette in Bowman&#39;s space demonstrates the pipette tip within the space, abutting the capillary tuft. <a href="https://www.jove.com/files/ftp_upload/58206/0128a-3dview03162018-3.avi">Please click here to download this file.</a>

Watch this Scientific Journal Video about Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy at JoVE.com

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...

micropuncture-bowman-s-space-mice-facilitated-2-photon

Research

JoVE Journal

Biology

9.2K Views.  Oregon Health & Science University. We present use of 2-photon microscopy to place a micropipette within Bowman's urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

Video: Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

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

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

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

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

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

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

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

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

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

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

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

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

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

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

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

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...