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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

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

Published: June 30th, 2023

DOI:

10.3791/65529

1Department of Cell Biology, Emory University School of Medicine, 2Independent scholar, 3Center for Neurodegenerative Disease, Emory University School of Medicine, 4Department of Dermatology, Emory University School of Medicine, 5Winship Cancer Institute, Emory University School of Medicine, 6Atlanta Veterans Affairs Medical Center

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 healing8,9, inflammation

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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 .......

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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.......

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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

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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.

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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

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