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Neuroscience

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

Published: April 22nd, 2021

DOI:

10.3791/62361

1Department of Biomedical Engineering, Sungkyunkwan University, 2Center for Neuroscience Imaging Research, Institute for Basic Science, 3School of Biological Sciences, Seoul National 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.

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 µTongue 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 µTongue 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 µTongue are also presented.

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 imag....

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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

  1. 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 (.......

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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.......

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Described here is a detailed protocol to apply µTongue 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.......

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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.

....

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

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