installing the live imaging insert on an inverted microscope stage

Imágenes intravitales con un microscopio invertido para rastrear la dinámica de las células de la piel

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.

Autor destacado: Desarrollo de una herramienta para el uso de microscopios confocales invertidos para la obtención de imágenes intravitales in vivo 

Enfoque simplificado de imágenes intravitales para la monitorización a largo plazo de la dinámica del tejido epitelial en un microscopio confocal invertido

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  

Instalación del inserto de imágenes en vivo en una platina de microscopio invertido

Para comenzar a instalar el inserto de imágenes en vivo impreso en 3D en la platina del microscopio invertido, coloque con cuidado el inserto en la ranura grande de la platina del microscopio. Con tornillos, asegure cada esquina del inserto. Deslice la placa calefactora en la abertura lateral del inserto con el lado del tapón hacia abajo, de modo que la placa quede encima de las ranuras inferiores del inserto con el puerto del enchufe debajo de la platina.Alinee la abertura circular ranurada en el inserto con un objetivo de inmersión en aceite de silicona de 40 x y aplique aceite de silicona en el centro del objetivo. Luego, con una jeringa, aplique una pequeña cantidad de grasa al vacío a lo largo de la abertura circular ranurada. Coloque el disco deslizante de la cubierta en la parte superior de la abertura para sellarlo en el inserto.Por último, levanta el objetivo 40 x hasta que apenas toque el fondo de la cubierta de cristal.

installing-the-live-imaging-insert-on-an-inverted-microscope-stage

Research

JoVE Journal

Biology

Installing the Live Imaging Insert on an Inverted Microscope Stage

138 vistas. Para comenzar a instalar el inserto de imágenes en vivo impreso en 3D en la platina del microscopio invertido, coloque con cuidado el inserto en la ranura grande de la platina del microscopio. Con tornillos, asegure cada esquina del inserto. Deslice la placa calefactora en la abertura lateral del inserto con el lado del tapón hacia abajo, de modo que la placa quede encima de las ranuras inferiores del inserto con el puerto del enchufe debajo de la platina.Alinee la abertura circular ...

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

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

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 ...