Name: µTongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo (Video) | JoVE Journal
Uploaded: 2021-04-22T15:00:00.000+00:00
Duration: 7 min 53 s
Description: Learn intravital fluorescence microscopy of taste cells using the µTongue microfluidic system in live mice. This method enables functional calcium imaging with controlled tastant delivery while preserving neural connections and blood circulation.

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5 minutes

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/pubmed?term=((Characterization+and+subcellular+targeting+of+GCaMP-type+genetically-encoded+calcium+indicators%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Two-photon+microscopy+as+a+tool+to+study+blood+flow+and+neurovascular+coupling+in+the+rodent+brain%5BTitle%5D)+AND+%22Journal+of+Cerebral+Blood+Flow+%26+Metabolism%22%5BJournal%5D)">Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain.</a> Journal of Cerebral Blood Flow & 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/pubmed?term=((In+vivo+fluorescence+microscopy%3A+Lessons+from+observing+cell+behavior+in+their+native+environment%5BTitle%5D)+AND+%22Physiology%22%5BJournal%5D)">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/pubmed?term=((In+situ+Ca2%2B+imaging+reveals+neurotransmitter+receptors+for+glutamate+in+taste+receptor+cells%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Breadth+of+tuning+and+taste+coding+in+mammalian+taste+buds%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Cell-to-cell+communication+in+intact+taste+buds+through+ATP+signalling+from+pannexin+1+gap+junction+hemichannels%5BTitle%5D)+AND+%22The+Journal+of+Physiology%22%5BJournal%5D)">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/pubmed?term=((The+cells+and+peripheral+representation+of+sodium+taste+in+mice%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((High+salt+recruits+aversive+taste+pathways%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Intravital+microscopic+interrogation+of+peripheral+taste+sensation%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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/pubmed?term=((Comprehensive+functional+screening+of+taste+sensation+in+vivo%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">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/pubmed?term=((Optimized+ratiometric+calcium+sensors+for+functional+in+vivo+imaging+of+neurons+and+T+lymphocytes%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">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/pubmed?term=((Glossopharyngeal+nerves+to+taste+stimuli+in+C57BL+%2F+6J+mice%5BTitle%5D)+AND+%22BME+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Breadth+of+tuning+in+taste+afferent+neurons+varies+with+stimulus+strength%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Fiji%3A+An+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">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/pubmed?term=((The+gut-brain+axis+mediates+sugar+preference%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The gut-brain axis mediates sugar preference.</a> Nature. 580 (7804), 511-516 (2020).</li>
<li>Roebber, J. K., Roper, S. D., Chaudhari, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+the+anion+in+salt+%28NaCl%29+detection+by+mouse+taste+buds%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">The role of the anion in salt (NaCl) detection by mouse taste buds.</a> The Journal of Neuroscience. 39 (32), 6224-6232 (2019).</li>
<li>Kusuhara, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Taste+responses+in+mice+lacking+taste+receptor+subunit+T1R1%5BTitle%5D)+AND+%22Journal+of+Physiology%22%5BJournal%5D)">Taste responses in mice lacking taste receptor subunit T1R1.</a> Journal of Physiology. 591 (7), 1967-1985 (2013).</li>
</ol>

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

&#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...

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.

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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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.

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

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of  &lt;1 μm × &lt;1 μm × &lt;3 μm in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

Ultrasound imaging can be used to display the shape and movements of the tongue in real time during speech. The images can be used to determine the nature of speech sound errors. Visual feedback of the tongue can be used to facilitate improvements in speech sound production in clinical populations.

Ultrasound Images of the Tongue: A Tutorial for Assessment and Remediation of Speech Sound Errors

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.

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

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.

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

We have developed a generalized protocol to dissociate a large quantity of high-quality single cells from the epithelium and mesenchyme/connective tissue of embryonic and adult mouse tongues.

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice

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.

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

A pendant drop surface film balance implemented with a multi-subphase exchange, nicknamed the OCTOPUS, allows for mimicking digestive conditions by the sequential subphase exchange of the original bulk solution with simulated gastrointestinal fluids. The simulated in vitro digestion is monitored by recording in situ the interfacial tension of the digested interfacial layer.

In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids

The goal of this protocol is to provide a standardized approach for quantitatively assessing the integrity of the murine gingival epithelial barrier by topically applying fluorescence-labeled dextrans of varying molecular sizes on the gingival surface in vivo, followed by ex vivo imaging using a single-photon microscopy system.

Functional Assessment of Mouse Gingival Epithelial Barrier Using Fluorescent Tracers

Learn intravital fluorescence microscopy of taste cells using the µTongue microfluidic system in live mice. This method enables functional calcium imaging with controlled tastant delivery while preserving neural connections and blood circulation.

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

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