installing the live imaging insert on an inverted microscope stage

Intravital Imaging Using an Inverted Microscope to Track Skin Cell Dynamics

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, 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&#160;been primarily performed using upright multiphoton microscopes due to their ability to provide high-resolution imaging at tissue depths &#62;100 &#181;m11,12. Nevertheless, these instruments&#160;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 advantages13,14. 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 &#62;50 &#181;m 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&#39;). 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&#39;). 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&#160;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.

Author Spotlight: Developing a Tool for Using Inverted Confocal Microscopes for In Vivo Intravital Imaging 

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

We developed a tool to perform intravital imaging on live mice using an inverted confocal microscope. This enables us to track skin cell dynamics throughout homeostasis and compare cell behaviors during disease onset and progression. Multiphoton microscopy is the primary approach applied to achieve intravital cell tracking.However, the disadvantages of these microscopes include limited day-to-day imaging versatility, high cost, and technical complexity. Our new tool enables the use of versatile inverted confocal microscopes for in vivo intravital imaging. By modifying the design file, the stage insert dimensions can be customized to fit any make of inverted microscope.The objective hole can be resized and repositioned to fit any animal model and tissue of choice.

Watch this Scientific Journal Video about Installing the Live Imaging Insert on an Inverted Microscope Stage at JoVE.com

Installing the Live Imaging Insert on an Inverted Microscope Stage  

To begin installing the 3D printed live imaging insert on the inverted microscope stage, carefully place the insert into the large microscope stage groove. Using screws, secure each corner of the insert. Slide the heat plate into the side opening of the insert in a plug side down orientation, so the plate lays atop the lower insert grooves with the plug port underneath the stage.Align the grooved circular opening in the insert with a 40 x silicone oil immersion objective and apply silicone oil to the center of the objective. Then using a syringe, apply a small amount of vacuum grease along the grooved circular opening. Lay the cover slip disk on top of the opening to seal it onto the insert.Finally, raise the 40 x objective until it barely touches the bottom of the glass cover slip.

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138 Views.  Emory University School of Medicine. To begin installing the 3D printed live imaging insert on the inverted microscope stage, carefully place the insert into the large microscope stage groove. Using screws, secure each corner of the insert. Slide the heat plate into the side opening of the insert in a plug side down orientation, so the plate lays atop the lower insert grooves with the plug port underneath the stage.Align the grooved circular opening in the insert with a 40 x silicone oil immersion objective and apply sili...

Video: Installing the Live Imaging Insert on an Inverted Microscope Stage  

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&#39;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.

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 ...

Mounting the Mouse on the Live Imaging Insert for Intravital Imaging

Before placing the anesthetized mouse on the live imaging insert installed on the inverted microscope stage, thread the isoflurane tubing with the attached nose cone through the corner tubing bracket on the insert. After plugging the heat plate into the controller containing an attached anal thermometer probe and switching it on, transfer the anesthetized mouse from the induction chamber onto the heat plate. Then secure the nose cone onto the mouse.Insert the anal probe into the animal and adjust the controller to maintain a stable body temperature of about 36 degrees Celsius. Cover the properly-positioned mouse with a plastic cling wrap to trap body heat and elevate the body temperature. Position the mouse such that its head aligns with the cover slip disc and immobilize its ear onto the center of the glass cover slip using a metal ear clip or tape.After completing imaging, select the Low Flow option on the electronic vaporizer to cease isoflurane delivery. Transfer the mouse to a heating pad until it becomes ambulatory. After removing the cover slip disc, wipe it clean with lens paper and lens solution.Store it properly to avoid scratches for reuse. Next, detach the anesthetic tubing from the insert bracket and unscrew the insert from the microscope stage. The acquired Z-stacks of fluorescently-labeled live mouse ear tissue showed minimal drift in the x, y and z axes over time, validating the custom 3D printed live imaging inserts.The time points from a three hour time lapse movie of the mCherry positive mouse ear epidermal cells of an adult male mouse revealed a single z plane remaining in focus over the extended time. Similar observations were made from another three-hour time lapse movie of GFP positive ear dermal fibroblasts of an adult female mouse.