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Method Article

Intravital Two-Photon Imaging of Touch Sensory Axon Morphology in Mouse Skin

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DOI:

10.3791/69589

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December 30th, 2025

 ,  ,  , 
1Department of Pharmacology, Vanderbilt University

* These authors contributed equally

In This Article

Summary

This study presents an in vivo two-photon imaging approach for visualizing the structures of low-threshold mechanoreceptor (LTMR) axon terminals in the forepaw skin of mice. Using repeated, high-resolution imaging of individual axons, this method provides a new platform for studying the structure and function of the cutaneous sensory circuit during development and in adults.

Abstract

Low-threshold mechanoreceptors (LTMRs) are somatosensory neurons that detect innocuous light touch stimuli such as vibration, hair deflection, and pressure. They form subtype-specific axonal terminals in the periphery and project axons centrally to the spinal cord to transmit tactile information. Current understanding of LTMR development and organization comes from fixed tissues that cannot reveal the dynamic and temporal processes of axonal wiring and remodeling. Here, a two-photon imaging method is presented for visualizing LTMR axon morphology in the mouse forepaw during development and in adults. Two-photon microscopy can achieve high-resolution imaging within intact skin, enabling repeated imaging of the same axon terminals across postnatal time points. Two-photon imaging can be combined with new mouse genetic tools to reveal axon development with subtype-specificity. Chronic imaging of LTMRs enables studies of circuit assembly and repair following injury. These approaches provide an in vivo system that will provide new insights into the cellular mechanisms that regulate somatosensory axon development and regeneration.

Introduction

Touch is the earliest sense to develop and plays a central role in how organisms experience and respond to the physical world. Touch is detected by low-threshold mechanoreceptors (LTMRs), which are subtypes of dorsal root ganglion (DRG) neurons that detect innocuous mechanical stimuli, including indentation, vibration, and hair deflection1,2. LTMRs are pseudounipolar and extend peripheral axons into the peripheral tissues to form subtype-specific terminals. Their central axons relay tactile information to the central nervous system by forming synaptic connections with spinal cord neurons3. However, how touch circuits are established and maintained is poorly understood.

Earlier foundational studies in cats during the 1960s and 1970s first identified these functionally distinct mechanoreceptors and their response properties in hairy skin4,5. LTMR subtypes are classified by their unique morphology, conduction velocity, and response properties2. The major LTMR subtypes are Aβ rapidly adapting (RA) and slowly adapting (SA) LTMRs, Aδ-LTMRs, and C-LTMRs2,6,7. Aβ RA-LTMRs, Aδ-LTMRs, and C-LTMRs form longitudinal lanceolate complexes around hair follicles2. Aδ-LTMRs form polarized lanceolate endings around hair follicles on the caudal side, a feature essential for their direction-selective responses to hair deflection7. Axonal endings in the lanceolate complexes are closely aligned with hair shafts and are enveloped by finger-like processes from terminal Schwann cells, which provide trophic support and influence axonal morphogenesis8,9,10. In glabrous skin, Aβ RA-LTMRs axons terminate in Meissner corpuscles, which are ovoid-shaped structures located within dermal papillae that are tuned to motion across the skin2,11.

The organization of LTMR axonal terminals is regulated by both intrinsic programs and skin-dependent cues during development12,13. When damaged, LTMR dysfunction is associated with pathological conditions such as mechanical allodynia12,14. Yet, the mechanisms for LTMR development and maintenance in the intact skin remain unclear. Much of what is known about their development stems from fixed tissues, which cannot capture the dynamic processes that may take place in vivo6,15,16,17,18. For example, Aβ RA-LTMRs and Aδ-LTMRs exhibit active innervations around hair follicles and axonal pruning during early postnatal development19. How hair follicle innervation and maintenance are achieved in vivo remains unknown.

Two-photon laser-scanning microscopy, an advanced imaging technique that allows visualization of fluorescently labeled structures within living tissues, has enabled high-resolution in vivo imaging of dynamic physiological processes20,21. During two-photon imaging, brief pulses of near-infrared light excite fluorophores only at the focal plane, thereby confining the signal to a small volume and minimizing out-of-focus fluorescence21,22. This confinement significantly reduces phototoxicity and allows three-dimensional, spatially resolved imaging of structures20,23,24. Furthermore, the longer excitation wavelengths used in two-photon microscopy scatter less within tissue, allowing for deeper penetration and lower noise in complex environments20,25,26. For example, two-photon imaging revealed structural synaptic plasticity and neuronal activities in spinal cord dorsal horn neurons27,28,29. In addition, chronic in vivo two-photon imaging of hair follicles revealed insights about their homeostasis in the mouse ear skin23. Hence, two-photon microscopy is well-suited for studying LTMR structures in the skin in vivo, especially when repeated, long-term imaging is required to track developmental and chronic changes.

When paired with genetically modified mouse lines expressing fluorescent reporters, two-photon microscopy can reveal neuronal morphology in the skin3. In the somatosensory system, yellow fluorescent protein (YFP) expressed under the control of the thy1 promoter (Thy1-YFP)30 selectively labels Aβ-LTMR lanceolate complexes in hairy skin and Meissner corpuscles in glabrous skin30,31. In Thy1-YFP mice, labeled axons are positive for myelin basic protein and neurofilament, which are markers for myelinated Aβ axons30,31. Additionally, TrkBCreER, in which tamoxifen-inducible Cre recombinase (CreER) is driven by the expression of the TrkB (tropomyosin receptor kinase B) gene, selectively labels Aδ-LTMRs during development7. In the skin, two-photon microscopy is most effective for imaging LTMRs in anesthetized mice, as movement by the mouse would impact the high-resolution imaging acquisition. It takes time to visualize fluorescent proteins at the axonal terminals after CreER-induced expression, and the minimum waiting period needs to be determined for each transgenic line. In general, this methodology can be applied to visualizing LTMRs across mice of all developmental stages and in adults.

This protocol explains the procedures for using two-photon microscopy to investigate LTMR axon morphology in the hairy and glabrous skin of developing and adult mice at high resolution. The protocol includes several tips for optimal high-resolution and chronic imaging. This integrative approach enables investigation of the cellular mechanisms driving the assembly and plasticity of touch sensory axons in vivo.

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Protocol

All experimental procedures were performed in compliance with institutional animal care regulations and were approved by the Institutional Animal Care and Use Committee (IACUC) at Vanderbilt University. For the safety of the experimenters, all the procedures were performed with lab coats, sterile gloves, and masks. All tools were sterilized thoroughly prior to use, and all experimental surfaces were cleaned with 70% ethanol before and after each experiment. The mice used in this study were adolescents and adult animals of both sexes and were derived from strains of Thy1-YFP, TrkBCreER, and Ai140. The reagents and the equipment used are listed in the Table of Materials.

1. Preparation of instruments

  1. Turn on the laser. Allow ample time for the laser to warm up. In this work, the Chameleon Discovery NX laser was used, which has a pulse duration of 100 femtoseconds (fs) and a repetition rate of 80 ± 0.5 megahertz (MHz).
  2. Turn on the two-photon microscope and open the control and acquisition software on the computer.
  3. Turn on the heating pad and set it to 37 °C to prevent hypothermia during anesthesia.

2. Preparation of anesthesia and the forepaw for imaging

  1. To induce anesthesia, place the mouse in the induction chamber and set the system to 3.5% isoflurane until the mouse loses its righting reflex and shows smooth, regular respirations (following institutionally approved protocols). Room air is used as a carrier and should be set to 1.0 liter per minute (L/min).
  2. Place the mouse on the heating pad on the imaging platform and reduce the isoflurane to 1.5% and oxygen to 0.5 L/min. Isoflurane should be delivered through the nose cone. For mice younger than postnatal day 20, isoflurane is set to 2-4% to maintain anesthesia. Monitor anesthetic depth every 10 min by assessing loss of the pedal and palpebral reflexes, respiratory rate and depth, and overall muscle tone.
  3. To minimize movement artifacts during imaging, including breathing-related motion, the mouse should be placed on a separate stage from the paw. For this protocol, the mouse body rests on a platform directly adjacent to, but not touching, the imaging platform where the paw is mounted.
  4. Apply ophthalmic ointment to the eyes to prevent dryness during imaging.
  5. Gently apply depilatory cream to the paw using a cotton swab to remove the hair. After letting it sit on the skin for a minute, remove the cream, and then clean the mouse paw with 70% ethanol and water.
  6. Gently stretch the skin to stabilize the skin surface. Using a sterile 30 G needle and black ink, gently pierce the skin and tattoo with two adjacent dots on either side of the intended image region. Ensure the dots are spaced about 2 millimeters (mm) apart. After the tattooing is completed, properly dispose of the needle in a sharps container.
  7. On a glass microscope slide, place four dots of vacuum grease on the corners and place a small piece of clay (e.g., DAS Air-Hardening Modeling Clay) approximately 1 mm thick in the middle of the slide. Place the slide under the objective lens and center the mouse's paw directly on the piece of clay (Figure 1A,B). Gently roll the mouse's paw on the slide to flatten it and press it into the clay. Carefully place a 22 mm x 60 mm coverslip on top of the paw to secure the slide. Adjust the angle of the mouse skin by putting light pressure on each of the corners of the coverslip until the surface of the coverslip and skin are parallel to the imaging plane. Using gentle force during imaging helps prevent potential skin injury.
  8. For imaging the glabrous skin, place the slide under the objective lens and center the mouse's paw on the slide as detailed in step 2.7. Use tweezers to rotate the paw so the glabrous skin faces up. Use the flat end of the tweezers to gently press the paw into the clay. Once the paw is flat, place the coverslip on top and press down as described in step 2.7.

3. Two-photon microscope imaging of LTMR morphology

NOTE: The following parameters can be adjusted based on the specific experiment. For this experiment, a 20x water-immersion objective lens with a numerical aperture of 1 and a working distance of 2.00 mm was used. Steps 3.1-3.6 describe the specific parameters used for the Thy1-YFP mice's right forepaw, which can serve as a reference guide.

  1. Place a drop of distilled water on the coverslip, directly over the mouse paw.
  2. Lower the objective lens until a water column is formed.
  3. Once the objective lens is centered over the mouse paw, close the microscope box and use the LED screen to focus on the sample via the X, Y, and Z controllers. The LED screen displays a live image illuminated by the microscope's built-in LED.
    NOTE: LED mode is used to confirm that the objective lens is centered on the field of interest before switching to two-photon mode. The LED image shows surface features such as blood vessels and hair follicles, serving as a visual guide for alignment and targeting. The tattoos on the mouse paw appear as black spots on the LED screen. Using these landmarks, find and focus on the area between the black spots. This ensures that the same area of the mouse paw is imaged repeatedly.
  4. Once the sample is found, turn off the LED screen. To perform two-photon imaging from the tissue, switch the microscope from the mirror setting to the dichroic setting by flipping a lever on the upper-right side of the microscope.
  5. Close the blackout curtains and switch on the detector to visualize YFP.
  6. Using Prairie View, open the shutter, and switch the software to imaging mode. For imaging Thy1-YFP, turn on the tunable laser set at 960 nanometers (nm) to a gain of 700-800. When imaging GFP, set the tunable laser to 920 nm.
    1. For postnatal day 10 or younger mice, set the initial laser power to 50, increasing until the desired signal intensity is achieved. This lower initial laser power minimizes light penetration and the risk of photodamage. After the laser wavelength, gain, and power are set, begin scanning.
      ​NOTE: For capturing a high-resolution 1.00x field-of-view image or an image of Meissner corpuscles in glabrous fingertips, it is recommended to use an image size of 1024 x 1024 with an imaging field of view of 601.9 µm x 601.9 µm, and a pixel size of 0.588 µm x 0.588 µm. For imaging lanceolate endings around hair follicles, it is recommended to have an image size of 1024 x 1024 at 8.00x zoom, an imaging field of view of 75.2 µm x 75.2 µm, and a pixel size of 0.147 µm x 0.147 µm. It is important to note that the imaging field of view and pixel size change with the zoom setting.
  7. Use the X, Y, and Z controllers to find the imaging field, adjust the zoom as needed, and start image acquisition. An optimal image is typically obtained at a starting position about 10-20 µm under the skin surface.
    NOTE: A high-resolution image of the hairy skin typically has a step size of 1 µm or 2 µm with approximately 40-120 slices. Meanwhile, a high-resolution image of the glabrous skin typically has a step size of 2 µm to 4 µm with approximately 80-100 slices. This results in a typical z-range of 100-250 µm, and the overall maximum imaging depth is approximately 300 µm.
  8. The imaging session will be limited to 25 min to minimize the duration of isoflurane exposure and reduce the risk of anesthesia-related adverse effects to the animal. The extra isoflurane is absorbed by an anesthesia charcoal filter canister, which can be disposed of in regular trash.

4. Postoperative care

  1. Remove the mouse paw from the glass slide and return the mouse to a recovery cage.
  2. Keep the mouse cage warm on a heating pad until the mouse fully recovers from the anesthesia.
  3. Place the mouse back into its home cage, observing the mouse for an additional 30 min to ensure the mouse integrates with its cage mates.

5. In vivo chronic imaging of LTMR morphology

  1. Place the mouse under the two-photon microscope by following steps 2.4-3.3.
  2. Using the LED screen, locate the ink dots from the previous imaging session. Orient the screen between the dots to ensure that the desired area is captured. During scanning mode on the microscope, check for landmarks, such as large nerves or hair follicle patterns, to ensure locating the same area.
  3. Once the imaging area is found, follow steps 3.4-3.6 to continue imaging.
  4. Check the quality and intensity of images, as well as the morphology of major axons, obtained by two-photon excitation of the skin area. Ensure that the image fields are comparable to those taken during the previous imaging session.

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Results

In this work, two-photon microscopy was used to visualize Aβ RA-LTMR and Aδ-LTMR axonal structures in the skin during development and in adults. Mature lanceolate endings, occurring in mice about postnatal day 20 and older, are fully developed19,32. The overall organization of axon terminals in the hairy skin of adult mice was imaged, as shown in the maximum intensity projection (Figure 2A). The depth and shape of these axon terminal...

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Discussion

This study demonstrates the use of two-photon microscopy to examine LTMR morphology in the intact skin of neonatal and adult mice. This longitudinal imaging approach overcomes the limitations of tissue fixation and enables real-time observation of axonal structures in intact tissues. The Aβ RA-LTMR and Aδ-LTMR axonal morphologies visualized through two-photon imaging are consistent with observations from previous PFA-fixed tissues2,6,

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Disclosures

The authors report no relevant disclosures.

Acknowledgements

We would like to thank Dr. Hannah Elam, Dr. Snighda Mukerjee, Rachelle Larivee, and Dr. Tegy J. Vadakkan for all of their support and assistance throughout this research. We thank Ryan Michael Nuera for help with setting up the isoflurane system. This work is supported by the Howard Hughes Medical Institute Hanna Gray Fellowship (Grant# GT17518).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
20x Objective Lens – Super NA 1.0Olympus1-U2B965Two-Photon Microscope Setup
22x60 Cover SlipVWR16004-330Two-Photon Microscope Setup
Air-Hardening Modeling ClayDASTwo-Photon Microscope Setup
Anesthesia Air Pump (HF), 110VRWD Life Science Inc.R510-30Isoflurane System
Anesthesia Concentric MaskRWD Life Science Inc.R510-MIsoflurane System
Anesthesia Induction Chamber – Mouse and RatRWD Life Science Inc.V105Isoflurane System
Bruker Ultima Investigator Plus Laser Scanning MicroscopeBruker Nano, Inc. Two-Photon Microscope Setup
Butterfly NeedlesFisher Scientific1484041Two-Photon Microscope Setup
Chameleon Discovery NX with TPC: DualCoherent1385777Two-Photon Microscope Setup
Cone Mask with Tubing for Neonatal MiceRWD Life Science Inc.68680Isoflurane System
Dual emission filters (et525/70m and et595/50m) and t565lpxr  dichroic beam splitterNikonINVP-01Two-Photon Microscope Setup
Epi-Fluorescence Filter Set ET-GFP ET470/40x and ET252/50m; dichroic T495LPXR Nikon518065Two-Photon Microscope Setup
EthanolDecon Laboratories Inc.Two-Photon Microscope Setup
Far Infrared Warming Pad with Controller (AC power)Kent ScientificRT-0515Two-Photon Microscope Setup
GaAsp Detectors (2) Hamamatsu Photonics HED-UP-2 Two-Photon Microscope Setup
Gas Filter CanistersRWD Life Science Inc.R510-31-6Isoflurane System
High Vacuum GreaseSigma AldrichZ273554Imaging System
Isoflurane, USP (250 mL)Piramal Critical Care6679-017-25Isoflurane System
Laser Cover Box Teco TechnologyImaging System
Mice: B6.129S6(Cg)-Ntrk2tm3.1(cre/ERT2)Ddg/JJAX27214
Mice: B6.Cg-Igs7tm140.1(tetO-EGFP,CAG-tTA2)Hze/JJAX30220
Mice: B6.Cg-Tg(Thy1-YFP)16Jrs/JCharles River3709
Nair Hair Remover CreamAmazon28001Two-Photon Microscope Setup
Raw Data FilesZenodo10.5281/zenodo.17370834
Rigid Stand with Large Rectangular Insert HolderThorlabsMP15MTwo-Photon Microscope Setup
Small Animal Anesthesia Operation PlatformRWD Life Science Inc.R510-PGLIsoflurane System
Soothe Lubricant Eye OintmentBausch + LombTwo-Photon Microscope Setup
Superfrost Plus Microscope SlidesFisher Scientific12-550-15Two-Photon Microscope Setup

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Somatosensory AxonsLTMR AxonsIn Vivo ImagingChronic ImagingHair Follicle InnervationAxon DevelopmentMeissner Corpuscles
Intravital Two-Photon Imaging of Touch Sensory Axon Morphology in Mouse Skin
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