Name: Mounting the Mouse on the Live Imaging Insert for Intravital Imaging
Uploaded: 2023-06-30T15:00:00
Description: Watch this Scientific Journal Video about Mounting the Mouse on the Live Imaging Insert for Intravital Imaging at JoVE.com

mounting the mouse on the live imaging insert for intravital imaging

Intravital avbildning ved hjelp av et omvendt mikroskop for å spore hudcelledynamikk

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.

Forfatter Spotlight: Utvikle et verktøy for bruk av inverterte konfokale mikroskoper for in vivo intravital avbildning 

Strømlinjeformet intravital bildebehandlingstilnærming for langsiktig overvåking av epitelvevsdynamikk på et invertert konfokalmikroskop

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 Mounting the Mouse on the Live Imaging Insert for Intravital Imaging at JoVE.com

Mounting the Mouse on the Live Imaging Insert for Intravital Imaging  

Montering av musen på Live Imaging Insert, for intravital avbildning

Før du plasserer den bedøvede musen på live-bildeinnsatsen som er installert på det inverterte mikroskopstadiet, trer du isofluranslangen med den festede nesekjeglen gjennom hjørneslangebraketten på innsatsen. Etter å ha koblet varmeplaten til kontrolleren som inneholder en vedlagt analtermometersonde og slått den på, overfør den bedøvede musen fra induksjonskammeret til varmeplaten. Fest deretter nesekeglen på musen.Sett analsonden inn i dyret og juster kontrolleren for å opprettholde en stabil kroppstemperatur på ca. 36 grader Celsius. Dekk den riktig plasserte musen med en plastplastfolie for å fange kroppsvarme og heve kroppstemperaturen. Plasser musen slik at hodet er på linje med dekselets glideplate og immobiliser øret på midten av glassdekselet ved hjelp av en øreklemme eller tape av metall.Når avbildningen er fullført, velger du alternativet Low Flow på den elektroniske fordamperen for å avslutte leveringen av isofluran. Overfør musen til en varmepute til den blir ambulerende. Etter at du har fjernet dekselplaten, tørker du den ren med linsepapir og linseoppløsning.Oppbevar den riktig for å unngå riper for gjenbruk. Deretter løsner du bedøvelsesslangen fra innsatsbraketten og skru av innsatsen fra mikroskoptrinnet. De ervervede Z-stablene av fluorescerende merket levende musørevev viste minimal drift i x-, y- og z-aksene over tid, og validerte de tilpassede 3D-trykte live-bildeinnsatsene.Tidspunktene fra en tre timers time lapse-film av de mCherry positive museøreepidermale cellene til en voksen hannmus avslørte et enkelt z-plan som forble i fokus over lengre tid. Lignende observasjoner ble gjort fra en annen tre-timers time lapse-film av GFP-positive øredermal fibroblaster av en voksen kvinnelig mus.

Research

JoVE Journal

Biology

Mounting the Mouse on the Live Imaging Insert for Intravital Imaging

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 ...