This work was supported by the Institute of Basic Science (IBS-R015-D1), the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2019M3A9E2061789), and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2019M3E5D2A01058329). We are grateful to Eunsoo Kim and Eugene Lee for their technical assistance.

All surgical procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Sungkyunkwan University and Seoul National University.
1. Preparation of solutions: artificial saliva and tastants
<ol>
	<li>Prepare artificial saliva by dissolving 2 mM NaCl, 5 mM KCl, 3 mM NaHCO3, 3 mM KHCO3, 0.25 mM CaCl2, 0.25 mM MgCl2, 0.12 mM K2HPO4, 0.12 mM KH2PO4, and 1.8 mM HCl in distilled water (&#62;1 L), and adjust the pH of the solution to 7 (see Table of Materials)12.</li>
	<li>Prepare tastants, such as sour: 10 mM citric acid; salty: 400 mM NaCl, optionally with 50 &#181;M amiloride; sweet: 40 mM acesulfame K; bitter: a mixture of 5 mM quinine, 5 mM denatonium and 20 &#181;M cycloheximide, by dissolving the tasting chemical in the artificial saliva prepared in step 1.1.</li>
</ol>
2. Preparation of the microfluidic system
NOTE: Tastants were delivered to the mouse tongue using a pressurized multichannel fluidic delivery system (refer Figure 1 and Table of Materials).
<ol>
	<li>Fill the reservoirs of the pressurized flow perfusion system with the artificial saliva and tastants.</li>
	<li>Connect the compressed air line to the regulator input and set the air pressure between 30 and 50 psi in the fluidic delivery system.</li>
	<li>Set the output pressure of the regulator to 0.4 psi and check if liquid comes out of the tube under this pressure.</li>
	<li>Connect the manifold from the reservoirs to the input port of &#181;Tongue.</li>
	<li>Connect the output port of &#181;Tongue to a syringe pump and withdraw liquid with ~300 &#181;L&#8729;min-1 to establish the steady-state condition. Observe the constant volume of a hanging droplet under the &#181;Tongue. Adjust the value of the setting parameter depending on the sample height.</li>
	<li>Disconnect the compressed air line and stop the syringe pump until protocol step 3 is completed.</li>
</ol>
3. Mouse preparation for in vivo imaging (Figure 2).
NOTE: All animal preparations were carried out during the daytime under aseptic conditions on a laboratory workbench.
<ol>
	<li>Mouse anesthesia
	<ol>
		<li>Prepare a 7-week-old or older mouse of either sex. Use a genetically modified mouse line that expresses calcium-sensing fluorescence proteins in the taste cells.</li>
		<li>The mouse is restrained for anesthesia. A mixture of 100 mg/kg ketamine and 10 mg/kg xylazine is injected intraperitoneally into the mouse13.</li>
	</ol>
	</li>
	<li>TRITC-dextran (500 kDa) in 2.5% W/V phosphate buffer saline is administrated intravenously into the mouse through a retro-orbital route to observe blood circulation during an imaging session.</li>
	<li>Attach a head fixer on the mouse skull to minimize movement artifacts.
	<ol>
		<li>The mouse head is sprayed with 70% ETOH while the mouse is placed in a supine position. Lift the head skin lightly with forceps and snip off approximately 7 mm2 with scissors.</li>
		<li>Clean the hair around the scalp, remove the periosteum under the skin, apply an instant adhesive to the skull, and attach a customized head fixer.</li>
		<li>After the instant adhesive is hardened, apply dental glue around the head fixer and illuminate with a blue light to solidify dental glue.</li>
	</ol>
	</li>
	<li>Place the mouse tongue on the bottom unit of &#181;Tongue.
	<ol>
		<li>Attach the lower lip of the mouse to the bottom unit of &#181;Tongue with an instant adhesive.</li>
		<li>Place the mouse on the board (mouse preparation board in Figure 1B) and put the bottom unit of the &#181;Tongue to the posts (&#181;Tongue hold post in Figure 1B). Make sure that the holes at the edge of the bottom unit are aligned to the post.</li>
		<li>Tighten the mouse head fixer to the head fixer holder at the board. Then, adjust the distance between the mouse head and the device. Rotate the mouse head smoothly approximately 45&#176; using the head fixer holder. This process prevents physical contact of the mouse nose with microscope objective.</li>
		<li>Draw the mouse tongue gently using plastic tweezers and attach the ventral side of the tongue to the upper side of the bottom unit of the &#181;Tongue. Then, wipe the surface of the mouse tongue with a wet cotton swab.</li>
		<li>Soak a piece of paper in the artificial saliva and place it on the exposed surface of the mouse tongue to maintain a wet condition.</li>
		<li>Place the curved washers on the posts that hold both ends of the bottom part of &#181;Tongue.</li>
	</ol>
	</li>
	<li>Place the mouse preparation on the microscope stage. Position the exposed mouse tongue under the approximate center of the microscope objective area. Be sure not to deviate from the dynamic range of the stage. Then, tighten the mouse board on the stage with screws.</li>
	<li>Place the heating pad under the mouse body and maintain the temperature at 36.5 &#176;C-37.5 &#176;C. Monitor the mouse body temperature with a temperature sensor and control the temperature of the heating pad using a feedback signal from the temperature sensor.</li>
	<li>Twist a thin piece of paper and place it at the mouth of the mouse to prevent liquid from entering the mouse trachea.</li>
	<li>Remove the wet tissue from the mouse tongue and place the prepared &#181;Tongue on the mouse tongue. Put a microfluidic channel on the tongue and adjust its position to observe the surface of the tongue through the imaging window.</li>
	<li>Secure the &#181;Tongue by gently screwing on both ends with minimal compressive pressure.</li>
</ol>
4. Imaging acquisition
<ol>
	<li>Turn on the 920 nm two-photon laser and the microscope in advance of use.</li>
	<li>Mount the water-immersion objective (16x, NA 0.80 or 25x, NA 1.1) on the microscope. Drop the distilled water on the imaging window of the &#181;Tongue and immerse the objective.</li>
	<li>In camera mode, turn on the light using mercury lamp and illuminate the surface of the tongue.</li>
	<li>By adjusting the Z-axis, search the autofluorescent signal from the filiform papillae to find the approximate focal plane. Then, using the X and Y adjustment knob, locate a taste bud.</li>
	<li>Switch to the multiphoton mode. Set the image acquisition conditions as follows: excitation wavelength: 920 nm; emission filter set: 447/60 nm, 525/50 nm, and 607/70 nm; bidirectional raster scan mode, frame size: 512 x 512.</li>
	<li>Adjust the X and Y positions to place the taste bud on the center of the image window.</li>
	<li>Search the blood vessels surrounding the taste bud at about two-third height of the taste bud. Visualize the blood circulation by TRITC-dextran (500 kDa) injection from protocol step 3.2. If the blood flow clogs, loosen the fixing screws slightly to allow blood flow.</li>
	<li>Adjust Z-axis and find the Z-plane of taste bud that contains an adequate number of taste cells.</li>
	<li>Proceed calcium imaging with 2-6 Hz for 80 s. Provide a taste solution of 20 s by switching on the reservoir of the fluidic system after imaging starts. After 20 s of taste stimulation, switch the reservoir back to artificial saliva.</li>
	<li>After sequential imaging is finished, wait for about 3-4 min in advance to the next imaging session. Keep the artificial saliva flowing to the mouse tongue to wash away the tastant remanent from the previous imaging session. Depending on the design of the experiment, repeat the session as required.</li>
	<li>When in vivo calcium imaging is complete, euthanize the mouse according to the IACUC procedure. The mouse under anesthesia is sacrificed in the CO2 chamber. 
	&#8203;NOTE: Check the depth of the anesthesia using a toe-pinch reflex. During an imaging session, artificial saliva from the reservoirs should be provided consistently. If bubbles appear at the imaging window of the &#181;Tongue, remove the bubbles by pushing them through the input or the output of the &#181;Tongue using strong liquid pressure.</li>
</ol>
5. Image analysis (Figure 3)
<ol>
	<li>Image conversion
	<ol>
		<li>Open the raw image files using Fiji14 or a similar image analysis software.</li>
		<li>Convert the image file to a RGB stack file to use the NPL Bud Analyzer code.
		<ol>
			<li>Image &#62; Color &#62; Split Channels</li>
			<li>Image &#62; Color &#62; Merge Channels and select the image from step 5.2.1.</li>
			<li>Image &#62; Color &#62; Stack to RGB</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Image registration 
	NOTE: Use the custom-written code for data analysis. Please refer to https://github.com/neurophotonic/Tastebud-analyzer.
	<ol>
		<li>Run the code named Taste_GUI.m; a GUI window named NPL Bud Analyzer will pop up. Click on the New Analysis button on the upper-right corner, then load the converted image from step 5.1. Set the frame rate above the loaded image.</li>
		<li>Draw the region of interest (ROI) over the loaded image for registration. Double click on the selected ROI and an auto-calculated registration will start.</li>
	</ol>
	</li>
	<li>Obtain the relative fluorescence intensity changes (&#916;F/F)
	<ol>
		<li>Go back to the NPL Bud Analyzer window to show the registered image from step 5.2 automatically. If the user already has a reg file, click on the Load Data button and select the _reg.tif file.</li>
		<li>Click on the CIRCLE or POLYGON button and position the ROI of taste cell over the taste bud image.</li>
		<li>This presents the raw fluorescence intensity and the calcium trace (&#916;F/F) of the selected taste cell automatically under the taste bud image.</li>
		<li>Click on Save Trace to present the calcium trace (&#916;F/F) on the right side of the GUI, while ROI is shown over the taste bud image. Repeat steps 5.3.2-5.3.4 until the ROI selection is finished. 
		NOTE: Click on the Delete Trace button if the ROI is mis-selected to eliminate the last selected ROI and calcium trace.</li>
		<li>After the ROI selection is finished, write the file name on the bottom-right corner, and click on the Finish button to export the &#916;F/F calcium trace as an .xls format, and the tase bud image with ROIs in a .bmp format.</li>
	</ol>
	</li>
	<li>Analysis of the calcium trace
	<ol>
		<li>Analyze the calcium trace obtained from step 5.3. Consider that taste cells have reacted to tastant when fluorescence intensity rises more than two standard deviations of the baseline after the tastant is delivered4, and p-values are less than 0.01, using paired or unpaired t-tests10.</li>
		<li>Consider the taste cell as a responder cell, if it responds to a certain tastant more than two times out of three trials(~60%)15.</li>
		<li>Obtain the representative calcium traces by averaging individual calcium traces acquired from step 5.4.1.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Mao, T., O'Connor, D. H., Scheuss, V., Nakai, J., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+subcellular+targeting+of+GCaMP-type+genetically-encoded+calcium+indicators.">Characterization and subcellular targeting of GCaMP-type genetically-encoded calcium indicators.</a> PLoS One. 3 (3), 1-10 (2008).</li><li>Shih, A. 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=Two-photon+microscopy+as+a+tool+to+study+blood+flow+and+neurovascular+coupling+in+the+rodent+brain.">Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 32 (7), 1277-1309 (2012).</li><li>Choi, M., Kwok, S. J. J., Yun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+fluorescence+microscopy:+Lessons+from+observing+cell+behavior+in+their+native+environment.">In vivo fluorescence microscopy: Lessons from observing cell behavior in their native environment.</a> Physiology. 30 (1), 40-49 (2015).</li><li>Caicedo, A., Samir Jafri, M., Roper, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+Ca2++imaging+reveals+neurotransmitter+receptors+for+glutamate+in+taste+receptor+cells.">In situ Ca2+ imaging reveals neurotransmitter receptors for glutamate in taste receptor cells.</a> Journal of Neuroscience. 20 (21), 7978-7985 (2000).</li><li>Tomchik, S. M., Berg, S., Kim, J. W., Chaudhari, N., Roper, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breadth+of+tuning+and+taste+coding+in+mammalian+taste+buds.">Breadth of tuning and taste coding in mammalian taste buds.</a> Journal of Neuroscience. 27 (40), 10840-10848 (2007).</li><li>Dando, R., Roper, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+communication+in+intact+taste+buds+through+ATP+signalling+from+pannexin+1+gap+junction+hemichannels.">Cell-to-cell communication in intact taste buds through ATP signalling from pannexin 1 gap junction hemichannels.</a> The Journal of Physiology. 587 (24), 5899-5906 (2009).</li><li>Chandrashekar, 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=The+cells+and+peripheral+representation+of+sodium+taste+in+mice.">The cells and peripheral representation of sodium taste in mice.</a> Nature. 464 (7286), 297-301 (2010).</li><li>Oka, Y., Butnaru, M., von Buchholtz, L., Ryba, N. J. P., Zuker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+salt+recruits+aversive+taste+pathways.">High salt recruits aversive taste pathways.</a> Nature. 494 (7438), 472-475 (2013).</li><li>Choi, M., Lee, W. M., Yun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopic+interrogation+of+peripheral+taste+sensation.">Intravital microscopic interrogation of peripheral taste sensation.</a> Scientific Reports. 5 (8661), 1-6 (2015).</li><li>Han, J., Choi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+functional+screening+of+taste+sensation+in+vivo.">Comprehensive functional screening of taste sensation in vivo.</a> bioRxiv. 16419 (371682), 1-22 (2018).</li><li>Thestrup, T., 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=Optimized+ratiometric+calcium+sensors+for+functional+in+vivo+imaging+of+neurons+and+T+lymphocytes.">Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes.</a> Nature Methods. 11 (2), 175-182 (2014).</li><li>Danilova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glossopharyngeal+nerves+to+taste+stimuli+in+C57BL+/+6J+mice.">Glossopharyngeal nerves to taste stimuli in C57BL / 6J mice.</a> BME Neuroscience. 15, 1-15 (2003).</li><li>Wu, A., Dvoryanchikov, G., Pereira, E., Chaudhari, N., Roper, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breadth+of+tuning+in+taste+afferent+neurons+varies+with+stimulus+strength.">Breadth of tuning in taste afferent neurons varies with stimulus strength.</a> Nature Communications. 6 (8171), 1-11 (2015).</li><li>Schindelin, 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=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Tan, H. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gut-brain+axis+mediates+sugar+preference.">The gut-brain axis mediates sugar preference.</a> Nature. 580 (7804), 511-516 (2020).</li><li>Roebber, J. 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The authors declare competing financial interests: J. Han and M. Choi are inventors of the patented &#181;Tongue technology described in this article, and the &#181;Tongue system is commercially available via SciTech Korea.

Described here is a detailed protocol to apply &#181;Tongue to the investigation of functional activities of taste cells in vivo. In this protocol, the functional imaging on the taste cells using genetically encoded calcium indicators is performed. In addition to the use of transgenic mice, the electrophoretic loading of calcium dyes (or voltage sensing dyes) onto the taste cells can be an alternative option.
All the taste solutions less than 1.336 of refractive index were used in thi...

The intravital fluorescence microscope is used widely to study the spatiotemporal dynamics on living tissues. Researchers are rapidly developing genetically encoded sensors that provide specific and sensitive transformations of the biological processes into fluorescence signals - which can be recorded readily using fluorescence microscopes that are widely available1,2. Although most internal organs in rodents have been investigated using the microscope, its successful application to the tongue has not yet been successful3.
Previous studies on the calcium imaging of taste cells were conducted ex vivo by thin-sectioning a tongue tissue to obtain circumvallate taste buds4,5,6 or by peeling off the taste epithelium to obtain fungiform taste buds7,8. The preparation of these samples was inevitably invasive, thus the natural microenvironments such as nerves innervation, permeability barriers, and blood circulation, were largely perturbed. The first intravital tongue imaging window was reported in 2015 by Choi et al., but reliable functional recording was not achievable because of the movement and optical artifacts caused by fluidic tastant stimuli9.
Recently, microfluidics-on-a-tongue (&#181;Tongue) was introduced10. This device integrates a microfluidic system with an imaging window on the mouse tongue. By attaining a quasi-steady-state flow of tastant stimuli throughout the imaging period, artifacts from fluidic motion could be minimized (Figure 1). The input port is fed by a series of multichannel pressure controllers, whereas the output port is connected to a syringe pump, which maintains 0.3 mL/min. Additionally, optical artifacts caused by the difference in refractive indices of tastant solutions were minimized by ratiometric analysis introducing a calcium-insensitive indicator (tdTomato) as well as the calcium indicator (GCaMP6)11. This design provided microscopic stability of taste cells in vivo even with abrupt switching between fluidic channels. Consequently, the &#181;Tongue implement a reliable functional screening of multiple tastants to the mouse taste buds in vivo.
In this protocol, the experimental procedures are explained in detail for calcium imaging of the mouse fungiform taste buds in vivo using &#181;Tongue. First, the preparation of artificial saliva and tastant solutions is described. Second, the setting up of the microfluidic system to achieve the quasi-steady-state flow is introduced. Third, the procedures used to mount the mouse tongue on the &#181;Tongue to permit image acquisition are delineated. Lastly, each step for image analysis, including correction of lateral motion artifacts and ratiometry, is specified. This protocol can be adapted readily to any research laboratory with a mouse facility and a two-photon microscope or equivalent equipment.

<table><tbody><tr><td>acesulfame K</td><td>Sigma Aldrich</td><td>04054-25G</td><td>Artificial saliva / tastant</td></tr><tr><td>calcium chloride solution</td><td>Sigma Aldrich</td><td>21115-100ML</td><td>Artificial saliva / tastant</td></tr><tr><td>citric acid</td><td>Sigma Aldrich</td><td>C0759-100G</td><td>Artificial saliva / tastant</td></tr><tr><td>cycloheximide</td><td>Sigma Aldrich</td><td>01810-5G</td><td>Artificial saliva / tastant</td></tr><tr><td>denatonium</td><td>Sigma Aldrich</td><td>D5765-5G</td><td>Artificial saliva / tastant</td></tr><tr><td>Dental glue</td><td>Denkist</td><td>P0000CJT-A2</td><td>Animal preparation</td></tr><tr><td>Image J</td><td>NIH</td><td>ImageJ</td><td>Data analysis</td></tr><tr><td>IMP</td><td>Sigma Aldrich</td><td>57510-5G</td><td>Artificial saliva / tastant</td></tr><tr><td>Instant adhesive</td><td>Loctite</td><td>Loctite 4161, Henkel</td><td>Animal preparation</td></tr><tr><td>K2HPO4</td><td>Sigma Aldrich</td><td>P3786-100G</td><td>Artificial saliva / tastant</td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P9541-500G</td><td>Artificial saliva / tastant</td></tr><tr><td>Ketamine</td><td>Yuhan</td><td>Ketamine 50</td><td>Animal preparation</td></tr><tr><td>KH2PO4</td><td>Sigma Aldrich</td><td>P0662-25G</td><td>Artificial saliva / tastant</td></tr><tr><td>KHCO3</td><td>Sigma Aldrich</td><td>237205-500G</td><td>Artificial saliva / tastant</td></tr><tr><td>MATLAB</td><td>Mathwork</td><td>MATLAB</td><td>Data analysis</td></tr><tr><td>MgCl2</td><td>Sigma Aldrich</td><td>M8266-100G</td><td>Artificial saliva / tastant</td></tr><tr><td>MPG</td><td>Sigma Aldrich</td><td>49601-100G</td><td>Artificial saliva / tastant</td></tr><tr><td>Mutiphoton microscope</td><td>Thorlab</td><td>&amp;nbsp;Bergamo II</td><td>Microscope</td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S3014-500G</td><td>Artificial saliva / tastant</td></tr><tr><td>NaHCO3</td><td>Sigma Aldrich</td><td>792519-500G</td><td>Artificial saliva / tastant</td></tr><tr><td>Objective</td><td>Nikon</td><td>N16XLWD-PF</td><td>Microscope</td></tr><tr><td>Octaflow</td><td>ALA Scientific Instruments</td><td>OCTAFLOW II</td><td>Fluidic control</td></tr><tr><td>PC</td><td>LG</td><td>Lg15N54</td><td>Fluidic control</td></tr><tr><td>PH meter</td><td>Thermoscientific</td><td>ORION STAR AZ11</td><td>Artificial saliva / tastant</td></tr><tr><td>Phosphate-buffered saline</td><td>Sigma Aldrich</td><td>806562</td><td>Artificial saliva / tastant</td></tr><tr><td>quinine</td><td>Sigma Aldrich</td><td>Q1125-5G</td><td>Artificial saliva / tastant</td></tr><tr><td>Syringe pump</td><td>Havard Apparatus</td><td>PHD ULTRA 4400</td><td>Fluidic control</td></tr><tr><td>TRITC-dextran</td><td>Sigma Aldrich</td><td>52194-1G</td><td>Animal preparation</td></tr><tr><td>Ultrafast fiber laser</td><td>Toptica</td><td>FFultra920 01042</td><td>Microscope</td></tr><tr><td>Xylazine</td><td>Bayer Korea</td><td>Rompun</td><td>Animal preparation</td></tr></tbody></table>

&#181;tongue: a microfluidics-based functional imaging platform for the tongue in vivo

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in the taste sensory organ. By integrating microfluidics into the intravital tongue imaging window, the &#181;Tongue provides reliable functional images of taste cells in vivo under controlled exposure to multiple tastants. In this paper, a detailed step-by-step procedure to utilize the &#181;Tongue system is presented. There are five subsections: preparing of tastant solutions, setting up of a microfluidic module, sample mounting, acquiring functional image data, and data analysis. Some tips and techniques to solve the practical issues that may arise when using the &#181;Tongue are also presented.

The Pirt-GCaMP6f-tdTomato mouse was used to obtain a taste bud image. The surface of the mouse tongue was covered with autofluorescent filiform papillae. Taste buds are spread sparsely over the surface of the tongue (Figure 4A). The images of the taste bud and its structure were acquired using three different filter detectors. Using the 607/70 nm filter set, the tdTomato signal from the taste cells was obtained for ratiometric analysis (Figure 4B). Using the 525...

Watch this Scientific Journal Video about ÂµTongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo at JoVE.com

 &#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

The article introduces the &#181;Tongue (microfluidics-on-a-tongue) device for functional taste cell imaging in vivo by integrating microfluidics into an intravital imaging window on the tongue.

&#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in ...

tongue-microfluidics-based-functional-imaging-platform-for-tongue

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JoVE Journal

Neuroscience

4.2K Views.  Sungkyunkwan University. The article introduces the µTongue (microfluidics-on-a-tongue) device for functional taste cell imaging in vivo by integrating microfluidics into an intravital imaging window on the tongue.

Video:  µTongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Technique to Collect Fungiform Taste Papillae from Human Tongue

Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

Medicine

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Bioengineering

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely manipulate neural firing, hormonal secretion and genetically-reprogrammed cells. However, the translation of this technology for medical applications is currently hampered by a lack of biophysical mechanisms by which targeted tissues sense and respond to LIPUS. A suitable approach to identify these mechanisms would be to use optical biosensors in combination with LIPUS to determine underlying signaling pathways. However, implementing LIPUS to a fluorescence microscope may introduce undesired mechanical artefacts due to the presence of physical interfaces that reflect, absorb and refract acoustic waves. This article presents a step-by-step procedure to incorporate LIPUS to commercially-available upright epi-fluorescence microscopes while minimizing the influence of physical interfaces along the acoustic path. A simple procedure is described to operate a single-element ultrasound transducer and to bring the focal zone of the transducer into the objective focal point. The use of LIPUS is illustrated with an example of LIPUS-induced calcium transients in cultured human glioblastoma cells measured using calcium imaging.

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a modality for non-invasive mechanical stimulation of endogenous or engineered cells with high spatial and temporal resolution. This article describes how to implement LIPUS to an epi-fluorescence microscope and how to minimize acoustic impedance mismatch along the ultrasound path to prevent unwanted mechanical artefacts.

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely ...

Engineering

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is powered by local progenitor cells and renders taste function prone to disruption by a multitude of medical treatments, which in turn severely impacts the quality of life. Thus, studying this process in the context of drug treatment is vital to understanding if and how taste progenitor function and TRC production are affected. Given the ethical concerns and limited availability of human taste tissue, mouse models, which have a taste system similar to humans, are commonly used. Compared to in vivo methods, which are time-consuming, expensive, and not amenable to high throughput studies, murine lingual organoids can enable experiments to be run rapidly with many replicates and fewer mice. Here, previously published protocols have been adapted and a standardized method for generating taste organoids from taste progenitor cells isolated from the circumvallate papilla (CVP) of adult mice is presented. Taste progenitor cells in the CVP express LGR5 and can be isolated via EGFP fluorescence-activated cell sorting (FACS) from mice carrying an Lgr5EGFP-IRES-CreERT2 allele. Sorted cells are plated onto a matrix gel-based 3D culture system and cultured for 12 days. Organoids expand for the first 6 days of the culture period via proliferation and then enter a differentiation phase, during which they generate all three taste cell types along with non-taste epithelial cells. Organoids can be harvested upon maturation at day 12 or at any time during the growth process for RNA expression and immunohistochemical analysis. Standardizing culture methods for production of lingual organoids from adult stem cells will improve reproducibility and advance lingual organoids as a powerful drug screening tool in the fight to help patients experiencing taste dysfunction.

The protocol presents a method for culturing and processing lingual organoids derived from taste stem cells isolated from the posterior taste papilla of adult mice.

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is ...

Developmental Biology

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include ...

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

µTongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

Summary

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Chapters in this video

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

Fluorescence detection methods for microfluidic droplet platforms

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level