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

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

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.

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, inflammation

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 .......

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.......

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

We thank Valentina Greco for the K14-mCherry-H2B mice. We are grateful to the Emory University Physics Department Machine Shop for generating the glass coverslip disks. This work was funded by Career Development Award #IK2 BX005370 from the US Department of Veterans Affairs BLRD Service to LS, NIH Awards RF1-AG079269 and R56-AG072473 to MJMR, and I3 Emory SOM/GT Computational and Data Analysis Award to MJMR.