Streamlined Intravital Imaging Approach for Long-Term Monitoring of Epithelial Tissue Dynamics on an Inverted Confocal Microscope

Summary

The protocol presents a new tool to simplify intravital imaging using inverted confocal microscopy.

Abstract

Understanding normal and aberrant in vivo cell behaviors is necessary to develop clinical interventions to thwart disease initiation and progression. It is therefore critical to optimize imaging approaches that facilitate the observation of cell dynamics in situ, where tissue structure and composition remain unperturbed. The epidermis is the body's outermost barrier, as well as the source of the most prevalent human cancers, namely cutaneous skin carcinomas. The accessibility of skin tissue presents a unique opportunity to monitor epithelial and dermal cell behaviors in intact animals using noninvasive intravital microscopy. Nevertheless, this sophisticated imaging approach has primarily been achieved using upright multiphoton microscopes, which represent a significant barrier for entry for most investigators. This study presents a custom-designed, 3D-printed microscope stage insert suitable for use with inverted confocal microscopes, streamlining the long-term intravital imaging of ear skin in live transgenic mice. We believe this versatile invention, which may be customized to fit the inverted microscope brand and model of choice and adapted to image additional organ systems, will prove invaluable to the greater scientific research community by significantly enhancing the accessibility of intravital microscopy. This technological advancement is critical for bolstering our understanding of live cell dynamics in normal and disease contexts.

Introduction

Intravital microscopy is a powerful tool that allows the monitoring of cell behaviors in their unperturbed in vivo environments. This unique method has provided key insights into the inner workings of complex mammalian organ systems, including the lung¹, brain², liver³, mammary gland⁴, intestine⁵, and skin⁶. Furthermore, this approach has revealed cell behavioral alterations during tumor development⁷, wound healing^8,9, inflammation¹⁰, and other diverse pathologies in situ. In this study, we focus on enhancing the accessibility of intravital microscopy to image live epithelial and stromal dynamics in intact mouse skin. Understanding cell behaviors in mammalian skin is of high clinical importance due to the remarkable regenerative and tumorigenic capacity of this tissue.

Intravital imaging in mice has been primarily performed using upright multiphoton microscopes due to their ability to provide high-resolution imaging at tissue depths >100 µm^11,12. Nevertheless, these instruments lack the workhorse versatility and more general accessibility of inverted confocal microscopes, which are more user-friendly and cost-effective, provide the ability to image cultured cells, do not require complete darkness during image acquisition, and are generally safer, among other notable advantages^13,14. In this study, we present a new tool that significantly enhances intravital imaging accessibility by adapting this approach for inverted confocal microscopes.

Here, we present a 3D-printed custom stage insert design that incorporates several key features to facilitate stable, long-term intravital imaging of mouse ear skin on an inverted confocal microscope (Figure 1, Figure 2, Figure 3, Figure 4, and Figure 5). These specialized features include an offset objective hole that allows the full body of an adult mouse to lay entirely flat during imaging. This minimizes the vibrational interference of mouse body movements on imaging and eliminates the need to administer ketamine and xylazine to dampen breathing, a practice often coupled with intravital imaging⁶. In addition, corner brackets on the insert correctly position an isoflurane nose cone to align with the face of the mouse, a metal ear clip immobilizes the mouse ear to a custom-built coverslip disk, and an optional detachable closed-loop biofeedback heat plate lies flush within the insert to support the mouse body temperature during long imaging sessions. The custom coverslip disk, which provides a flat surface essential for the mouse head and ear to lay flat, was generated in a machine shop by removing the walls of a generic coverslip-containing cell culture dish. The use of a 40x silicone oil immersion lens (1.25 numerical aperture [N.A.], 0.3 mm working distance) in conjunction with the coverslip disk and custom stage insert provides high resolution images >50 µm deep into the ear dermis.

To test the functionality of this new inverted microscope stage insert, we captured z-stacks spanning all epidermal epithelial layers over a 3 h time course in the ear of a live transgenic K14-H2B-mCherry¹⁵ adult mouse (epithelial nuclei in this mouse line contain a red fluorescent label) (Figure 6A-A'). We also captured z-stacks spanning several fibroblast layers within the skin dermis over a 3 h time course in the ear of a live transgenic Pdgfra-rtTA¹⁶; pTRE-H2B-GFP¹⁷ adult mouse (fibroblast nuclei in this mouse line contain a green fluorescent label following doxycycline induction) (Figure 6B-D'). Our high-resolution data demonstrate consistent stability by lack of drift in the x-, y-, and z-planes, thus proving the effectiveness of this new intravital imaging tool for use on inverted microscopes. Importantly, the dimensions of this 3D-printed stage insert can be adjusted, as described in Supplementary File 1, Supplementary File 2, and Supplementary File 3, to fit any inverted microscope, and the positioning of the objective opening can be moved to alternative locations within the insert to better suit imaging a particular tissue and/or animal model of interest. This invention can thus empower individual laboratories, or investigators with core facility confocal access, to adapt this tool for their unique intravital imaging needs, thereby streamlining evaluation of diverse in vivo cell biology.

Protocol

This research was performed in compliance with Emory University and Atlanta Veterans Affairs Medical Center animal care and use guidelines and has been approved by the Institutional Animal Care and Use Committee (IACUC).

1. Installing the live imaging insert on the inverted microscope stage

1. Construct the insert using .stl files (Supplementary File 1, Supplementary File 2, and Supplementary File 3) specifying the 3D dimensions and design (see Figure 1 and Figure 2A), a 3D printer, and polylactic acid (PLA).
2. Carefully place the insert (Figure 2B,C) into the large microscope stage groove (Figure 2C and Figure 3A). Use screws to secure the insert via four screw holes located in each corner of the insert (Figure 2B-C).
   NOTE: The insert is bidirectional and can be rotated 180° depending on the orientation of the microscope stage, and anesthesia apparatus.
3. Slide the heat plate into the insert plug side-down (Figure 2D) so the unit lays atop the lower insert grooves with the plug port passing underneath the stage (Figure 3B).
4. Align the grooved circular opening in the insert with a 40x silicone oil immersion objective (Figure 3C) and apply silicone oil to the center of the objective.
   1. If imaging over longer time periods (>1 h), apply a generous dollop of oil that will persist at the coverslip/objective interface throughout imaging. Do not allow oil to spill over the edges of the lens.
5. Use a syringe to apply a small quantity of vacuum grease along the grooved circular opening and lay the coverslip disk atop to seal onto the insert (Figure 3D).
   NOTE: The coverslip disk is created by removing the walls of a 35 mm x 10 mm glass-bottom cell culture dish (performed by a machine shop).
6. Raise the 40x objective until the oil kisses the bottom of the glass coverslip.

2. Isoflurane configuration and mouse prep

1. Position the low-flow electronic vaporizer components (anesthesia chamber, tubing, vaporizer, charcoal canister) to allow the nose cone and attached tubing to reach the insert (Figure 3A).
   CAUTION: Isoflurane is an inhalation anesthetic and should be handled with care to avoid spills and minimize human exposure.
2. Measure the weight of the isoflurane bottle prior to use and log. Attach the cap from the electronic vaporizer to the isoflurane bottle.
3. Connect the power cord to the electronic vaporizer. Close the blue nose cone clips and open the white induction chamber clips to allow airflow through the chamber into the charcoal canister. Remove the red ambient air cap from the left side of the machine to allow air flow.
4. Allow the system to warm up for 5 min at 200 mL/min (low flow) and 2% isoflurane.
   NOTE: Although the nose cone setting is selected, the blue nose cone line should remain closed, and the white induction chamber line should remain open.
5. Once the system is properly equilibrated, purge the lines to remove remaining isoflurane from the chamber.
6. Place the mouse in the induction chamber and select High Flow. Complete all mouse preparation (i.e., hair removal, topical drug delivery, eye ointment application, etc.) prior to laying the animal's body atop the insert. Confirm the mouse is fully anesthetized using the toe pinch reflex method.
   1. With the leg extended, use a fingernail to firmly pinch the toe without causing physical damage. If the mouse exhibits a positive reaction to the stimulus (i.e., leg retraction, foot twitch, etc.), continue administering anesthetic within the chamber until no reaction is observed. Once appropriately anesthetized, the mouse breathing rate should slow to ~55-65 breaths per minute¹⁸.
7. When the mouse is fully anesthetized, select High Flow again to stop isoflurane delivery. Purge the chamber before opening and adjust the clips (blue: open; white: closed) to deliver isoflurane through the nose cone. Select Low Flow while the nose cone is attached to the mouse to continue isoflurane delivery.
8. Thread isoflurane tubing with the attached nose cone through the corner tubing bracket on the insert (Figure 3A and Figure 5).

3. Mouse placement on the insert for intravital imaging

1. Plug the heat plate into the controller (containing an attached anal probe), power on, and allow the plate to reach 36 °C (Figure 4A).
2. Remove the anesthetized mouse from the induction chamber and lay across the heat plate (Figure 4B and Figure 5A). Perform the transfer from chamber to insert rapidly to minimize the time the mouse is active without inhalant anesthesia.
3. Secure the nose cone onto the mouse (Figure 4B,C, and Figure 5). If necessary, use tape to further secure the angle and positioning of the nose cone.
4. Insert the anal probe and adjust the controller temperature until the mouse body temperature is stable at ~36 °C (Figure 4A,B). After the mouse is properly positioned, use plastic cling wrap to trap heat, if needed, to further support the appropriate body temperature (Figure 4C).
5. Position the mouse so the head aligns with the coverslip disk and immobilize the ear onto the center of the glass coverslip using a metal ear clip (Figure 5A,B) or tape (Figure 5C).
   NOTE: The pressure of the metal ear clip on the ear can be adjusted by loosening the screw that secures the clip to the insert. A metal spring can be added for increased clip tightening flexibility.
   1. To change the ear clip location, unscrew the bolt with a 2.5 mm Allen wrench and transfer to the secondary site (Figure 2B,C). When reassembling the ear clip, place the clip against the insert, followed by the washer on top. Use a bolt to securely fasten the clip with slight force to pivot the clip.
6. Adjust the objective z-positioning until the cells are within the focal place (Figure 6A,D). Set the z-stack and time-lapse parameters according to the experimental goals and commence image acquisition.
   NOTE: If the correct setup is achieved, the mouse ear can be imaged for at least 3 consecutive h (Figure 6A',D').
   1. Once the z-stack boundaries are set, adjust the laser power and gain to ensure no z-plane is oversaturated to minimize photobleaching. Determine the time-lapse parameters using the total thickness of the z-stack, step numbers (Nyquist sampling recommended), and time intervals.
   2. Determine the imaging parameters (time intervals, total imaging time, etc.) using multiple factors, including animal viability under anesthesia, laser-induced photobleaching/phototoxicity, and the goal of live imaging (i.e., cell division dynamics, cell-cell interactions, etc.).

4. Termination of imaging

1. Turn on the water-circulating heat pad and set to Continuous Cycle and 35 °C for at least 30 min prior to the termination of image acquisition.
2. Upon imaging completion, select Low Flow on the electronic vaporizer to stop isoflurane delivery and move the mouse to a heat pad until ambulatory.
3. Once awake, move the mouse back to the transfer container while continuing to maintain on a heat pad until fully ambulatory. Consistently monitor the mouse while on the heat pad until it is responsive.
4. On the electronic vaporizer, click Select Menu > Anesthetic Control > Empty. Remove the isoflurane bottle from the system and place the manufacturer's cap back onto the bottle.
5. Measure the weight of the isoflurane bottle and charcoal canister and enter weights into log. Once the charcoal canister weighs 50 g over baseline measurement, dispose of the canister and replace it with a new unit. Return the isoflurane to the lockbox.
6. Remove the coverslip disk and wipe clean with lens paper and lens solution. Store properly to avoid scratches for reuse.
7. Remove the anesthesia tubing from the insert bracket and unscrew the insert from the microscope stage.

Results

Proper assembly of the live imaging insert on an inverted confocal microscope and appropriate orientation of a transgenic mouse atop the insert is validated by acquiring z-stacks of fluorescently-labeled, live ear tissue over a time course ≥1 h with minimal evidence of drift in the x-, y-, and z-axes. Images should be captured at consistent intervals (interval time will depend on the biological question, strength of fluorescence signal, etc.) so that cell dynamics and image drift can be tracked over time. Thro...

Discussion

In this study, we present a new tool that facilitates stable, long-term intravital imaging of intact mouse skin epithelia on inverted confocal microscopes. This invention is made of PLA, which is the most common and inexpensive 3D-printable material; all in-house 3D-printing costs for this insert amount to <$5. The two separate insert pieces (Figure 1, Supplementary File 1, and Supplementary File 2) can be easily assembled using set screws (see

Materials

Name	Company	Catalog Number	Comments
3D Printer	Qudi Tech	i-Fast	3D prints using PLA material
40x 1.25NA silicone objective lens	Nikon
AxR Laser Scanning Confocal Microscope	Nikon
Cotton Tipped Swab	VWR	76337-046	Cream/ointment application
Doxycycline hyclate	Sigma-Aldrich	D9891	Induces GFP labeling of fibroblast nuclei in Pdgfra-rtTA; pTRE-H2B-GFP mice
Flathead Screwdriver (2.5 mm)			Affiix insert to microscope stage
Flathead Screws x 4 (#6-32)	Nikon		Screw insert into microscope stage
Glass Bottom Culture Dish	chemglass Life Sciences	CLS-1811-002	Modified by removing walls of dish for use as coverslip disk compatible with live insert; 35 mm wide disk contains 20 mm wide glass coverslip; dish walls were removed by machine shop
Heat Plate controller	Physitemp	TCAT-2LV	Animal Temperature Controller - Low Voltage; anal prob attachment for mouse body temperature monitoring
Hex Wrench (1.5 mm)			For M3 setscrew adjustments
Hex Wrench (2.5 mm)			Adjust tension on metal ear clip
Intravital Imaging Insert
Isoflurane	Med-Vet International	HPA030782-100uL	Mouse anesthesia
Labeling Tape (or Scotch Tape)	VWR	10127-458	Alternative to metal ear clip to immobilize ear to coverslip
Metal fastener			used as ear clip
Mouse: C57BL/6-Pdgfraem1(rtTA)Xsun/J	The Jackson Laboratory	RRID: IMSR_JAX:034459	Fibrroblast-specific promoter driving doxycycline-inducible rtTA expression
Mouse: K14-H2BPAmCherry	Courtesy of Dr. Valentina Greco at Yale University		Labels epidermal epithelial cell nuclei with mCherry; referred to in text as "K14-H2B-mCherry"
Mouse: pTRE-H2B-GFP: STOCK Tg(tetO-HIST1H2BJ/GFP)47Efu/J	The Jackson Laboratory	RRID: IMSR_JAX:005104	Labels fibroblast nuclei with GFP when combined with Pdgfra-rtTA and induced with doxycycline
Multipurpose Sealing Wrap	Glad		Enhance mouse warmth
Optixcare	VWR	MSPP-078932779	Eye lubricant
Set screws x 3 (M3; 6 mm)	Thorlabs	SS3M6	Attachment for heatplate module
Silicone Immersion Oil			Applied to 40x silicone objective
Small Animal Heating Plate	Physitemp	HP-4M	Provides heat to animal
Somnoflow Low-Flow Electronic Vaporizer	Kent Scientific	SF-01	Mouse anesthesia
Vacuum Grease	Flinn Scientific	AP1095	Seals coverslip disk to insert
Veet			hair removal
Water circulating heat pad	Stryker Medical	TP700	for mouse revival post-imaging