Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins
September 1st, 2013
Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.
The overall goal of this procedure is to visualize subcellular structures in murine salivary glands. This is accomplished by first surgically exposing the salivary glands without damaging them. In the second step, the anesthetized animal is immobilized on the microscope stage.
Next, the salivary glands are carefully positioned so as not to compromise their function in the final step. The mouse is stabilized with a custom holder to minimize any motion artifacts due to the animal's heartbeat or respiration. Ultimately, the dynamics of the subcellular structures can be imaged by intra vital microscopy.
Prior to anesthesia, weigh the mice to determine the proper dose of anesthetic. Next, induce pre anesthesia by isof fluorine inhalation. Once the animals are unconscious, confirm sedation every 15 minutes by pop pinch and keep the mice under heat lamp for the duration of the surgery.
Monitor the body temperature of the animals with a rectal probe thermometer. Now shave the neck of the mouse with a pair of electric clippers, and then use an ethanol soaked gauze sponge to wipe down the ventral side of the neck, drying the excess alcohol with the kim wipe. Next, use a pair of dull curved tweezers to lift up a small piece of skin at the lower side end of the massiter muscles and make a small incision.
Insert the scissors into the incision, pointing the tips upwards towards the underside of the skin and opening the blades several times until the skin has separated from the underlying tissue. Then starting near the base of the salivary glands where the nerve duct and blood vessels connect the gland to the rest of the body. Use the scissors to cut a three millimeter wide, 10 millimeter long strip of loosened skin.
After cleaning the wound area, the resultant opening will be oval in shape and measure roughly eight by 12 millimeters. Both salivary glands will be visible. Use a syringe to apply optical coupling gel into the middle of the cut, and then use fresh pieces of sterile gauze to wipe the gel towards the outside of the opening until all the blood and loose hair is removed.
Now, use a pair of number seven tweezers to find the tip of the right salivary gland, grabbing the connective tissue a few millimeters above the top of the gland. Then tear the connective tissue around the gland without injuring the parenchyma. Once the gland is exposed, gently separate the connective tissue from the gland, washing away any bleeding with saline.
Make sure that all the connective tissues are separated and the gland is fully exposed. Prior to bringing the animal to the microscope, place a thick piece of gauze and a heat pack onto the microscope stage, covering the top of the heat pack with a thin piece of gauze as well. Place a cover slip over the center of the stage.
Then position the mouse on its right side on top of the gauze, securing the animal with a piece of masking tape on the ventral side of the front right paw and a silk suture hooked around the incisors. Create some tension between the thread and the paw and attach them onto the stage. With more masking tape, the salivary gland should rest naturally.
In the middle of the cover slip. Use plastic wedges coupled to the microscope stage to further stabilize the mouse's head and thorax. Next place a small piece of lenss cleaning tissue over the gland.
Extend the gland slightly and place a customized holder over the gland. Then tightly, couple the stabilizer to the stage with a metal clamp and masking tape. Cover the animal with gauze and a heat pad and monitor the exposed glands temperature with a flexible thermal coupled probe equipped thermometer Immediately after stabilizing the animal, use epi fluorescence illumination to focus on the surface of the gland.
Assess the blood flow in the capillaries and the overall morphology of the tissue. To excite the GFP gene, use a 488 nanometer laser. Set the peak power to 0.5 milliwatts to avoid photobleaching for the tandem tomato protein.
Use a 561 nanometer laser, setting the peak power to one milliwatt. Now assess the stability of the tissue by pre scanning the selected area. If the preparation is not stable, adjust the animal accordingly to find the proper focal plane.
In the pre mode. Gradually move the objective towards the cover slip until the aser structures, secretory granules and plasma membrane become clearly visible. Then to image the dynamics of the secretory granules, use a scan speed of 0.5 to two seconds per frame and 256 by 256 pixels with a 0.2 micrometer per pixel spatial scale.
In order to optimally visualize the granules and the plasma membrane, set the optical thickness between 0.9 to 1.2 micrometers. Next, record an image of the field of view to be used as a reference point. Finally, stimulate exocytosis by subcutaneously injecting 0.1 milligram per kilogram of freshly prepared isoproterenol and saline.
One minute after the injection. Fusion of the secretory granules with the plasma membrane will be detected by an increase in the GFP fluorescence around the granules and the diffusion of the tandem tomato peptide into their limiting membranes. In the GFPM tomato mouse, the asinine appear as clearly distinct structures that express cytosolic GFP and membrane targeted tandem tomato peptide as indicated by the broken lines.
In individual asinine aser cells are delineated by the tandem tomato peptide as indicated by the arrows. GFP is also detected in the nuclei that are clearly visible inside the aser cells. Cytosolic GFP indicated by the arrowhead is excluded from the secretory granules that appear as dark circular vesicles of approximately one to 1.5 micrometers in diameter.
To visualize exophytic events, a representative area of the plasma membrane that is enriched in secretory granules as indicated by the asterisk is shown after the injection of the isoproterenol. An increase in the levels of GFP fluorescence denoted by the green arrows around some of the secretory granules that are in close proximity to the plasma membrane can be observed. In addition, after fusion with the plasma membrane, the tandem tomato peptide diffuses into the limiting membranes of the secretory granules as highlighted by the red arrows.
After 30 to 40 seconds, the secretary granules gradually collapse and their limiting membranes are integrated into the apical plasma membrane. The approach presented here represents a major breakthrough in cell biology as it enables imaging of the dynamics of a specific membrane trafficking process in living mice.
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Overview
This article presents a detailed method for visualizing subcellular structures in murine salivary glands using intravital microscopy. The procedure involves surgical exposure of the glands, immobilization of the animal, and careful positioning to minimize motion artifacts.
Key Study Components
Area of Science
- Neuroscience
- Biology
- Imaging Techniques
Background
- Intravital microscopy allows for real-time imaging of biological processes.
- It is particularly useful for studying dynamic cellular structures.
- Salivary glands serve as a model for observing subcellular dynamics.
- Understanding these dynamics can provide insights into various physiological processes.
Purpose of Study
- To visualize the dynamics of subcellular structures in live mice.
- To develop a reliable method for imaging secretory granules.
- To enhance the understanding of cellular functions in salivary glands.
Methods Used
- Surgical exposure of salivary glands without damage.
- Immobilization of the anesthetized animal on the microscope stage.
- Careful positioning of the salivary glands to maintain function.
- Use of a custom holder to stabilize the mouse and reduce motion artifacts.
Main Results
- Successful imaging of subcellular structures in live salivary glands.
- Demonstration of the dynamics of secretory granules.
- Validation of the method for future studies in live animal models.
- Insights into the physiological roles of these structures.
Conclusions
- The method provides a robust approach for studying subcellular dynamics.
- Intravital microscopy can be applied to various biological research areas.
- This technique enhances our understanding of cellular processes in vivo.
Frequently Asked Questions
Executive Industry Relevance
Intravital microscopy enables real-time visualization of subcellular dynamics in live animal models, providing critical mechanistic insights for target validation and pathway analysis. By imaging secretory granule exocytosis and membrane trafficking in salivary glands, this method supports preclinical de-risking of GPCR-mediated signaling pathways relevant to metabolic and secretory disease targets. The approach enhances predictive confidence in early discovery by linking molecular perturbations to functional subcellular outputs in a physiologically relevant context.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables direct observation of beta-adrenergic receptor-stimulated secretory granule fusion, supporting functional validation of GPCR targets in live tissue.
- Operational Value: Provides a reproducible platform to interrogate kinase, phosphatase, or cytoskeletal regulators of exocytosis using fluorescent biosensors.
- Therapeutic Value: Facilitates mechanistic de-risking of compounds targeting cAMP/PKA pathways by quantifying subcellular translocation kinetics in real time.
Screening & Assay Development
- Assay Readiness: Stabilized salivary gland preparation allows consistent baseline imaging of GFP and tandem-Tomato signals for compound library screening.
- Quantitative Output: Enables measurement of granule fusion frequency, membrane diffusion rates, and collapse kinetics as functional readouts for pathway modulation.
- Scalability: The surgical stabilization and imaging workflow can be adapted to multi-animal studies with standardized thermal and motion control.
Translational & Preclinical Research
- Disease Relevance: Models regulated secretion in exocrine tissues, applicable to pancreatitis, Sjogren’s syndrome, or cystic fibrosis transmembrane regulator (CFTR) dysfunction studies.
- Biomarker Continuity: Fluorescent reporters for granule membranes and cytosol enable correlation of imaging endpoints with biochemical assays of secretion.
- Preclinical De-risking: Supports go/no-go decisions by linking target engagement to functional subcellular outcomes before organ-level phenotyping.
Pipeline & Workflow Integration
This method fits within the discovery continuum from target validation through lead optimization, where real-time subcellular imaging informs mechanism of action and pathway selectivity.
- Discovery Biology: Supports hypothesis testing of GPCR effector pathways by visualizing downstream effector recruitment and membrane dynamics in intact tissue.
- Screening: Enables assay-ready preparations for evaluating compound effects on exocytosis kinetics with spatial and temporal resolution.
- Analytics: Generates quantitative data on fluorescence intensity shifts, diffusion rates, and vesicle collapse timing to compare experimental conditions.
- Translational Research: Connects molecular targeting to functional secretion in a disease-relevant exocrine model, supporting extrapolation to human pathophysiology.
- Enterprise Reuse: The stabilization and imaging platform can be reused across multiple fluorescent reporters and disease models with minimal revalidation.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity in signal transduction by providing direct visualization of effector translocation and membrane integration events.
- Operational Value: Standardized immobilization and imaging protocol improves reproducibility across laboratories and study timelines.
- Strategic Value: Enables earlier identification of false-positive hits by confirming functional subcellular activity beyond binding or bulk biochemical assays.
- Portfolio Impact: Increases confidence in target selection by validating pathway engagement in a physiologically intact, dynamic cellular environment.
Implementation Considerations
- Requires expertise in murine surgery, anesthesia, and intravital microscopy setup.
- Dependent on confocal microscope with 488 nm and 561 nm laser lines, thermal stage, and motion stabilization hardware.
- Necessitates standardized animal preparation protocols to ensure gland accessibility and functional stability across experiments.
- Adaptation to other tissues may require modified stabilization approaches due to differences in organ mobility and accessibility.
- Photobleaching limits necessitate careful laser power calibration and frame rate selection for longitudinal imaging.
