TITLE: Protocol for longitudinal imaging of the taste bud in vivo with two-photon laser scanning microscopy
AUTHORS: Brittany N Walters, Zachary D Whiddon, Aaron W McGee, Robin F Krimm

[DESCRIPTION]
Here we combined chronic in vivo two-photon laser scanning microscopy with genetic sparse labeling of gustatory neurons to repeatedly observe taste cells and arbors (nerve fibers) in taste buds across time. This method expands the investigative possibilities offered by previously developed methods, and we demonstrate a number of these, such as: differentiation of cells and observation of cell turnover, arbor plasticity, and a few stains/dyes that can be used in vivo to observe nuclei and organelles in the taste bud. A workflow for reconstructing the composite z-stacks with grayscale data of both cells and arbors using ImageJ, Neurolucida 360, and Neurolucida Explorer software are also provided. Together, the method and software options for analysis presented here demonstrate a novel approach for repeatedly observing taste cells and arbors in vivo in the taste bud.         

[BEFORE_START]
The goal of this protocol is to outline one method for using in vivo two-photon laser scanning microscopy to repeatedly image taste buds in the mouse tongue. The first step toward this goal is to construct the imaging platform that the mouse will be placed on for imaging as well as to construct the accessory components (nosecone, metal plate, and cover glass holder) that are required for delivering anesthesia and stabilizing the tongue. (The cover glass holder design was adapted from the holder design presented by Choi et al. 2015.) Subsequent steps in this protocol outline the equipment required to perform in vivo two-photon laser scanning microscopy, the process for stabilizing the mouse with anesthesia, and finally, the steps to acquiring papillae maps of the tongue epithelium for repeated imaging of taste buds. 

[GUIDELINES]
A comprehensive list of reagents/materials/equipment/mice with corresponding order numbers is available under the 'Materials' section.
Aim to withdraw and position the mouse tongue in the same orientation beneath the tongue holder at each imaging session.  

[STEPS]
SECTION: Preparation of Materials
1. Construct the breadboard, nosecone, metal plate, and cover glass holder from the products listed under the 'Materials' section.
SECTION: Preparation of Materials
1.7. Construct the custom-built imaging platform from the products listed under the 'Materials' section. 
SECTION: Preparation of Materials
1.9. Secure the Mini-Post Holders to the breadboard using the 4-40 M3 Washers and Cap Screws (Figure 1, panel A, Part B) which are included in the Hardware Kit for Mini-Series. In addition, secure the metal plate on top of the two Mini-Post Holders. 
SECTION: Preparation of Materials
1.10. Insert two Mini-Series Optical Posts (Figure 1, panel A, Part C), through the Mini-Post Holders at locations b8 and f2 of the breadboard.  
SECTION: Preparation of Materials
1.11. Slide two Mini-Post Swivel Post Clamps, 360° Continuously Adjustable (Figure 1, panel A, Part D), onto the two Mini-Series Optical Posts.  
SECTION: Preparation of Materials
1.12. Insert one Mini Series Post, 6mm, 1.5", into the Mini-Post Swivel Post Clamp (Figure 1, panel A, Part E).  
SECTION: Preparation of Materials
1.13. Attach two Cage Plate Stops (Figure 1, panel A, Part F), onto the Mini Series Post. Use the screws provided with the Cage Plate Stops to secure them to the Mini Series Post. 
SECTION: Preparation of Materials
1.14. Insert one Mini-Series Optical Post L=3" (Figure 1, panel A, Part G) with the nosecone welded to it, into the Mini-Post Swivel Post Clamp.  
SECTION: Preparation of Materials
1.15. A mixture of isoflurane and oxygen from the anesthesia system is delivered via silicone tubing to the nosecone. Slide the silicone tubing over the top and bottom prongs of the nosecone. 
SECTION: Turning on Equipment
76. 
3. 
SECTION: Preparing mouse for imaging
3.1. Place the mouse in the anesthesia induction chamber. Adjust the dial on the V-1 Tabletop Laboratory Animal Anesthesia System to supply a constant flow of oxygen/isoflurane mixture (5.0% isoflurane) to the mouse. 
SECTION: Preparing mouse for imaging
3.2. Place the mouse in the supine position on the custom-built imaging platform.  Adjust the dial on the V-1 Tabletop Laboratory Animal Anesthesia System to supply a constant flow of oxygen/isoflurane mixture (0.8-1.5% isoflurane) for the duration of the imaging session. 
SECTION: Preparing mouse for imaging
3.3. Assess anesthetic depth by checking the pedal withdrawal reflex. Position the nose inside the nosecone. Align the chin of the mouse with the rounded cut-out portion of the metal piece. 
SECTION: Preparing mouse for imaging
3.4. Insert the lubricated rectal temperature probe. Apply veterinary ophthalmic ointment to the eyes using cotton tipped applicators.   
SECTION: Preparing mouse for imaging
3.5. Withdraw the tongue with plastic blunt-ended forceps and wet it with artificial saliva. Position the tip of the tongue on the custom-constructed stainless steel metal plate.  
SECTION: Preparing mouse for imaging
3.6. Lower the cover glass holder until it meets the tip of the tongue. If desired, a fixed spacer can be placed between the cover glass holder and the metal plate to limit the possibility of tissue damage. 
4.  
SECTION: Acquiring papillae maps
4.1. Position the custom-built imaging platform under the MOM. Insert the 10X 0.3 numerical aperture (NA) W-ACHROPLAN (Zeiss) objective into the MOM. Adjust the z-axis rotary knob on the stage controller to lower the objective until it is roughly 1 mm above the cover glass holder.   
SECTION: Acquiring papillae maps
4.2. The ScanImage Software will populate multiple dialogue boxes when the program is initialized. In the Main Controls dialogue box, fill in the details of the experiment under basename such as: mouse number, taste bud number, date of imaging experiment.  
SECTION: Acquiring papillae maps
4.3. Create a new folder on the desktop to store the acquired images. In the Main Controls dialogue box, click “File” -> “Set Save Path”. A file explorer dialogue box will open, and from the panel on the left, select “Desktop”, then select the new folder.   
SECTION: Acquiring papillae maps
4.4. Turn off the lights and close the black drape in front of the MOM to begin imaging. Shift on the switches for both PMT channels.    
SECTION: Acquiring papillae maps
4.5. Click “FOCUS” in the Main Controls dialogue box to begin imaging. Press the fine focus button twice on the stage controller for smooth scanning.    
SECTION: Acquiring papillae maps
4.6. Turn the y-axis rotary knob of the stage controller to the left to move the 10X objective upward until the epithelium of the tongue comes into view. Turn the x-axis rotary knob to the right to scroll to the anterior portion of the tongue.   
SECTION: Acquiring papillae maps
4.7. When the right half of the tongue has been identified for imaging, use the z-axis rotary knob to scroll through the z-slices.    
SECTION: Acquiring papillae maps
4.8. In the Movement dialogue box, under stack, click “end” to register the initial z-slice of the z-stack which should be taken at the level of the basal lamina. In the same box, under stack, click “start” to register the final z-slice of the z-stack which should be taken at the level of the epithelium.   
SECTION: Acquiring papillae maps
4.9. In the Main Controls dialogue box, click “ABORT” -> “GRAB” to acquire the image. Repeat this process using steps 4.5-4.9 to create a papillae map of the left half of the tongue.       
SECTION: Viewing papillae maps using ImageJ software
6. Drag and drop the file of the papillae maps from the file folder to the ImageJ toolbar. Adjust the brightness and contrast of the image by selecting “Image” -> “Adjust” -> “Brightness/Contrast” in the toolbar.   
4.10. Adjoin the maps at the tongue midline to prevent duplicate imaging of taste buds.     
SECTION: Viewing papillae maps using ImageJ software
6.1. A B&C panel will appear with options for adjusting the Minimum, Maximum, Brightness & Contrast. Scroll through the z-slices in the image stack and adjust these options until the individual taste buds can be identified.      
SECTION: Viewing papillae maps using ImageJ software
6.2. Adjoin the maps at the tongue midline to prevent duplicate imaging of taste buds.     
SECTION: Imaging taste buds using the papillae map
5. 
SECTION: Imaging taste buds using the papillae map
5.1. Remove the 10X objective and replace it with a 40X 1.0 NA water immersion objective (Zeiss). If you are using a water immersion objective, then add droplets of distilled water to the top of the coverslip. Use the z-axis rotary knob on the stage controller to lower the objective until it is roughly 1 mm above the cover glass holder.    
SECTION: Imaging taste buds using the papillae map
5.2. Turn off the lights and close the black drape in front of the MOM. Shift on the switches for both PMT channels.  
SECTION: Imaging taste buds using the papillae map
5.3. Click “FOCUS” in the Main Controls dialogue box to begin imaging. Press the fine focus button twice on the stage controller for smooth scanning.    
SECTION: Imaging taste buds using the papillae map
5.4. Move the 40X objective upward in the water column by turning the y-axis rotary knob of the stage controller to the left. Using the x-axis rotary knob, locate one of the taste buds, and use the z-axis rotary knob to scroll through the z-slices of the taste bud.    
SECTION: Imaging taste buds using the papillae map
5.5. In the Movement dialogue box, under stack, click “end” to register the initial z-slice of the z-stack which should be taken at the base of the taste bud. In the same box, under stack, click “start” to register the final z-slice of the z-stack which should be taken when the taste pore of the taste bud comes into view. In the Main Controls dialogue box, click “ABORT” -> “GRAB” to begin imaging.   
SECTION: Imaging taste buds using the papillae map
5.6. It is critical to number the taste buds on the papillae map immediately after it has been imaged. To do so, click the “A” icon text tool in the toolbar. Drag the textbox to the location of one taste bud, enter the text, and press Ctrl+b to add the text.    
SECTION: Imaging taste buds using the papillae map
5.7. Once each taste bud has been assigned a number, select “File” -> “Save As” -> “Tiff”. Select the appropriate folder and click “Save”. Repeat this process for all remaining taste buds imaged. Be sure to adjust the basename after each image is acquired so that it accurately reflects the number assigned to the taste bud to be imaged. 
SECTION: Imaging taste buds using the papillae map
5.8. When finished imaging, shift off the switches for both PMT channels. Open the black drape in front of the MOM, and turn on the lights.   
SECTION: Imaging taste buds using the papillae map
5.9. Use the z-axis rotary knob on the stage controller to raise the 40X objective. Clean the 10X and 40X objectives using a small amount of ethanol sprayed on the Premium Grade Optical Tissue.   
SECTION: Removing mouse
7. 
SECTION: Removing mouse
7.1. Remove the custom-built imaging platform from beneath the MOM. Raise the cover glass holder. Move the nosecone away from the nose and remove the rectal temperature probe.   
SECTION: Removing mouse
7.2. Remove the mouse from the custom-built imaging platform and place a droplet of artificial saliva onto its tongue. Return the mouse to its home cage for recovery. Wait for the mouse to regain mobility. 
SECTION: Turning off equipment
8. 
SECTION: Turning off equipment
8.1. Turn the isoflurane regulator to 0% and close the O₂ tank. Turn the switch on the low-voltage animal temperature controller to OFF.  
SECTION: Turning off equipment
8.2. Turn off the Chameleon Ti:Sapphire mode-locked, tunable laser by turning the key from ON to STANDBY. Turn off the Fidelity-2 1070-nm laser by turning the key from ON to STANDBY. Turn off the Optical Beam Shutter Controller by turning the key to OFF.   
SECTION: Turning off equipment
8.3. Press the OFF button on the Compact Power and Energy Meter Console. Flip the switch to OFF on the Motorized Micromanipulator Stage Controller. Flip the switch on the Photomultiplier tube to OFF. Turn the switch on the Low Noise Current PreAmplifier to OFF.       
SECTION: Deinterleaving, Merging, and Colorizing z-stacks using ImageJ software
9.     
SECTION: Deinterleaving, Merging, and Colorizing z-stacks using ImageJ software
9.1. To view the individual taste bud images, open Fiji ImageJ software (https://imagej.net/software/fiji/downloads). Select “File” -> “Open” to view the image.   
SECTION: Deinterleaving, Merging, and Colorizing z-stacks using ImageJ software
9.2. The composite stacks that are generated contain grayscale data for both cells and arbors in different channels. Arbor data is stored in odd slices and cell data is stored in even slices. This grayscale composite image can be deinterleaved by  selecting “Image” -> “Stacks” -> “Tools” -> “Deinterleave”.    
SECTION: Deinterleaving, Merging, and Colorizing z-stacks using ImageJ software
9.3. To pseudo-color both channels, select “Image” -> “Color” -> “Merge Channels”. A dialogue box named Merge Channels will populate. To pseudo-color the cells green and arbors red, select “filename.tif#1” in the dropdown box next to C2 (green) and “filename.tif#2” in the dropdown box next to C1 (red). If it is not already checked, then place a check in the box, “Create Composite”. Click “OK”.  
SECTION: Deinterleaving, Merging, and Colorizing z-stacks using ImageJ software
9.4. Adjust the brightness and contrast of the image. To save the image, select “File” -> “Save As” -> “Tiff”. Repeat steps 9.1-9.3 to create pseudo-colored composites for all remaining taste buds. 
SECTION: Importing Colorized Composite z-stack files into Neurolucida 360
10. To view the individual taste bud images, open the Neurolucida 360 software. Select “File” -> “Open” -> “Image Stack”. A dialogue box called Image Scaling should appear. The following parameters should be set: The distance between images (size of voxel in Z) should be set to 1 μm. Distance Type should be set to Physical Distance. Correction Factor for Physical Distance should be set to Water. Place a check in the box next to Override X and Y scaling. X and Y Scaling Applied to Images should be set for X and Y to 0.156 μm/voxel.  
SECTION: Importing Colorized Composite z-stack files into Neurolucida 360
10.1. The cells and arbors should import into the program pseudo-colored in the colors previously selected in ImageJ.  
SECTION: Importing Colorized Composite z-stack files into Neurolucida 360
10.2. Refer to Ohman and Krimm 2021 for further instructions regarding how Neurolucida 360 can be used to trace arbors.  
SECTION: Troubleshooting issues with repeatedly imaging taste buds
11. If you are having difficulties locating the individual papillae repeatedly, then troubleshooting
suggestions are listed below:  
SECTION: Troubleshooting issues with repeatedly imaging taste buds
11.1. Cannot identify taste buds. On each map, identify a unique feature that can help you identify two taste buds. This could be their remarkable proximity to each other relative to other taste buds on the tongue or the particular angle that these two (or three) taste buds sit at on the tongue. If you identify this feature on the map with the 10x objective, then you should be more easily able to navigate to it with the 40x objective.  
SECTION: Troubleshooting issues with repeatedly imaging taste buds
11.2. Unsure if the correct taste bud has been identified. Because the arbor beneath the taste bud is stable, the number / shape of arbors can be used to positively identify a taste bud.  
SECTION: Turning on equipment
2.6. Turn ON the switch to the low-voltage animal temperature controller. Shift ON the power button to the Low Noise Current PreAmplifier.   
SECTION: Turning on equipment
2.7. Open the valve of the O₂ tank.  
SECTION: Turning on equipment
2.5. Turn on the Motorized Micromanipulator Stage Controller by turning the switch to ON. Turn on the Photomultiplier tube (PMT) by turning the switch to ON. Leave the individual switches for both
channels OFF.   
SECTION: Turning on equipment
2.4. Turn on the Optical Beam Shutter Controller by turning the key to ON and pressing the “Enable” button in the lower right-hand panel. Turn on the Compact Power and Energy Meter Console by pressing the ON button.  
SECTION: Turning on equipment
2.3. Turn on the Chameleon Ti:Sapphire mode-locked, tunable laser (Coherent) by turning the key from STANDBY to ON. Press the “Open Shutter” button on the laser. Tune the laser to 920 nm. Press “Menu Select” to accept the change in wavelength. Turn on the Fidelity-2 1070-nm laser by turning the key from STANDBY to ON.  
SECTION: Turning on equipment
2.2. Insert the 612/69 nm single-band bandpass filter (filter signals to better observe tdTomato), and 510/84 nm single-band bandpass filter (filter signals to better observe Green Fluorescent Protein) into the MOM. 
SECTION: Turning on equipment
2.1. Open the Matlab ScanImage software associated with the moveable objective microscope (MOM).  
2. 
SECTION: Preparation of Materials
1.1. Cut the 12" x 12" x 3/8" Mini-Series Aluminum Breadboard so that its dimensions are 12" x 6" x 3/8". Bolt the breadboard to the Heavy Duty Lab Jack. 
SECTION: Preparation of Materials
1.2. The length × width × height dimensions of the custom-constructed metal plate are 6.3 cm × 1 cm × 1 mm. The length × width dimensions of the inner cut out portion of the metal plate are 1 cm × 0.4 cm, and of the rounded portion of the metal plate are 0.8 cm x 0.3 cm.    
SECTION: Preparation of Materials
1.4. Construct the cover glass holder from a thin piece of metal. Use a marker to outline the shape of the cover glass holder on the metal, including the inner rim where the coverslip will be applied. (Dimensions: the entire length of the cover glass holder is 3.2 cm and the height is 0.9 cm. The length × width of the outer circular portion of the cover glass holder is 0.9 cm × 0.9 cm. The length × width of the inner circular portion is: 0.5 cm × 0.5 cm).  
SECTION: Preparation of Materials
1.5. From the Dremel set, first load the EZ Lock Mandrel No 402 onto the rotary tool and proceed by loading the accessory stainless steel wire brush. With these accessories on the rotary tool, manually drill along the outline of the cover glass holder. Change the accessory of the rotary tool to Mandrel No. EZ407SA which will fit the sanding band needed to sand the rough edges of the cover glass holder. (Please refer to the Dremel user manual for a complete guide of operating and safety instructions.) 
SECTION: Preparation of Materials
1.6. Once the cover glass holder has been formed, apply superglue to the inner rim of the cover glass holder and press one, 24x60 mm, #1 platinum cover glass into the glue. Allow the coverslip to dry and adhere to the cover glass holder before using a diamond tip scribe pen to remove the pieces of coverslip that extend from the sides. 
SECTION: Preparation of Materials
1.8. Position the four Mini-Post Holders with Swivel Base, 3/4" (Figure 1, panel A, Part A), onto the breadboard at the four locations indicated: b8 & c10, f8 & g9, f4 & g3, and f2 & g1. 
SECTION: Preparation of Materials
1.3. The length × width × height dimensions of the custom-constructed nosecone are 1 cm × 1 mm × 1 cm. Two prongs should also be welded to the top and bottom of the nosecone for delivery of isoflurane and oxygen via silicone tubing. The length × width dimensions of the prongs to which the tubing is attached are 0.65 cm × 0.4 cm.