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.
Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.
In the past two decades the advent of live microscopy and the use of fluorescent proteins have led to major breakthroughs on every cellular process imaginable, thus advancing our understanding of cell biology 1. This field has benefited tremendously from the use of mammalian cell cultures that are extremely powerful model systems, particularly when it comes to experimental manipulations. However, they do not often provide a true representation of the biology of complex multicellular organisms 2. This issue has begun to be addressed by the development of intravital microscopy (IVM) that has opened the door to investigating key biological questions in fields such as neurobiology, immunology and tumor biology 3. So far, most of the studies based on IVM have been performed at the levels of tissues and individual cells, without providing any information about the dynamics of subcellular compartments. Recently, our laboratory and others have developed IVM techniques capable of imaging subcellular structures in live rodents 4-7, 13-15 and allowing pharmacological and genetic manipulations in vivo. This approach has been used by us to study membrane trafficking in vivo, and more specifically endocytosis and regulated exocytosis 6,7.
Our experimental model system is based on exposing, stabilizing and imaging the submandibular salivary glands (SGs) of anesthetized rodents. The choice of the SGs as a model organ for IVM is due to the fact that the glands are easily accessible by performing a minor surgery, can be externalized without compromising their physiology, and stabilized to reduce the motion artifacts due to heartbeat and respiration. In addition, SGs can be selectively manipulated genetically by injecting either viral or non-viral based vectors through the salivary duct 8,9. Finally, SGs are exocrine glands composed of polarized epithelial cells, which form the acini and the ducts, myoepithelial cells, and a complex population of stromal cells. For this reason, they are an excellent model to study exocytosis, endocytosis, gene delivery, and actin cytoskeleton, as highlighted in our recent studies 10, and offer the opportunity to study aspects of cell biology such as cell polarity, cell division, cell-cell junctions, and ion channels.
In this paper we describe in detail an imaging protocol for achieving subcellular resolution in the epithelium of the SGs of a live mouse. Specifically, we show how to image the secretory granules in the acinar cells of the SGs during regulated exocytosis. As previously shown, upon stimulation with agonists of the beta-adrenergic receptor, the secretory granules fuse with the apical plasma membrane and gradually collapse, releasing their content into the acinar canaliculi 6. Our goal is to provide the basic tools to investigators with minimal experience in surgical procedures and animal handling, so that they can successfully perform IVM at a subcellular resolution. Since the most challenging part in IVM is the preparation of the animal, here we focus on the description of the basic surgical procedures that are utilized to expose and immobilize the SGs without compromising their function. As for the procedures to label subcellular structures, several strategies, such as systemic delivery of fluorescent probes, use of transgenic animals, or a combination of both, have been described elsewhere 7,11.
Part 1: Microscope and Preparation of the Imaging Setup
Part 2: Animals and Anesthesia
Part 3: Animal Surgery and Positioning for Intravital Microscopy
Part 4: Imaging Parameters
In the GFP/mTomato mouse, the acini appear as clearly distinct structures, which express cytosolic GFP and membrane-targeted tandem-Tomato peptide (Figure 2, broken line). In individual acini, acinar cells are delineated by the tandem-Tomato peptide. GFP is also detected in the nuclei that are clearly visible inside the acinar cells (Figure 2, arrows). Cytosolic GFP is excluded from the secretory granules that appear as dark circular vesicles of approximately 1-1.5 μm in diameter (Figure 2, inset, arrowheads). To visualize exocytic events, we focus on one area of the plasma membrane that is enriched in secretory granules (Figure 3, asterisk). After the subcutaneous injection of 0.1 mg/kg isoproterenol, we observe an increase in the levels of GFP fluorescence around some of the secretory granules that are in close proximity to the plasma membrane (Figure 3, green arrows). As previously shown, these granules are fused with the plasma membrane and recruit a thick actin meshwork that retains the cytoplasmic GFP 6. In addition, after fusion with the plasma membrane, the tandem-Tomato peptide diffuses into the limiting membranes of the secretory granules (Figure 3, red arrows). After 30-40 sec the secretory granules gradually collapse and their limiting membranes are integrated into the apical plasma membrane.
Figure 1. Microscope set up, surgical procedures, and animal positioning. A. A stage insert designed to hold 35 mm Petri dishes is covered with a 40 mm coverslip. The insert is placed in the microscope stage that is pre-warmed with heated pads (orange ovals) and a heated lamp. The objective is pre-heated with an objective heater (temperature set: 38 °C). B. Surgical procedures to expose the salivary glands. Using clean scissors, an incision is made in the skin above the neck area. The scissors are inserted in the opening to separate the skin from the underlying tissue. The skin is then removed, and the connective tissue is gently peeled from one of the glands using tweezers. C. The animal is placed on its side on the pre-heated stage and the gland is gently pulled and placed in the middle of the coverslip. The incisors are hooked to the stage with silk thread (inset). A small piece of lens cleaning tissue is sandwiched between the gland and the organ holder. The organ holder is then stabilized with a clamp that is secured on the stage with masking tape (green).
Figure 2. Representative image of an imaging area of the salivary glands of a live GFP/mTomato mouse. Acini are nicely highlighted in the GFP channel (green broken line). Single cells are delineated by the td-Tomato peptide that is localized at the plasma membrane (red broken line). The GFP is localized in the cytoplasm and the nuclei (arrows), but excluded from the secretory granules (inset, arrowheads). Bar, 10 μm.
Figure 3. Representative time sequence showing the exocytosis of secretory granules upon injection of 0.1 mg/kg of isoproterenol. Secretory granules were imaged close to the plasma membrane (asterisk). After fusion with the plasma membrane, the levels of GFP around the secretory granules significantly increase (green arrows), and the td-Tomato peptide diffuses into their limiting membranes (red arrows). The secretory granules slowly collapse and their membranes are integrated into the apical plasma membrane. Bar, 5 μm.
Table 1. Materials
Table 2. Instruments
So far subcellular structures have been imaged mostly in in vitro (i.e. cell cultures) or in ex vivo (i.e. organ-cultures, tissue slices, acinar preparations) model systems that often do not recapitulate the characteristics of intact live tissues 6. In this respect, the approach presented here represents a major breakthrough since it enables imaging the dynamics of a specific membrane trafficking step (i.e. regulated exocytosis) in living mice.
This protocol represents a significant advancement with respects to other procedures designed for the in vivo imaging of SGs or other organs in the body cavity 10,11,13-15. Some of our previous studies were performed in rats that are more resilient to surgical procedures, have bigger organs, and a reduced heart rate when compared to mice. Although, the procedures in mice are harder to set up, they provide several advantages including the possibility to use a broader spectrum of disease models, transgenic and knockout animals. In addition, this approach successfully minimized the motion artifacts, providing a better control over the pressure applied onto the exposed organs thus reducing the risks of: i) reducing the blood flow, ii) damaging the tissue architecture, and iii) impairing the normal function of the organ. This is a major concern that we solved by eliminating the constraint generated by the organ holder and introducing a stabilizer 10,11.
One of the most critical steps in this protocol is to maintain the proper temperature both in the animal and in the exposed organ. Indeed, anesthesia induces severe hypothermia and maintaining the appropriate body temperature significantly reduces the chances of death before completion of the experiment. Moreover, the exposed organ rapidly exchanges heat with the external environment during both the surgical procedures and the imaging. Since the local reduction of temperature severely slow down the kinetics of the exocytic steps, it is crucial to prevent it by using the heat lamp, the objective heater, and by keeping the gland moist with either the coupling gel or warm saline. Full enclosure of the microscope with an environmental chamber is also good solution for controlling the temperature. Another critical step is to achieve the appropriate balance between stabilization and maintenance of the blood flow. Indeed, in a preparation that is not properly stabilized, it is very difficult to follow the kinetics of the secretory granules whose sizes are in the micron range. On the other hand, the reduction of the blood supply results in the formation of large vacuoles or a significant inhibition of regulated exocytosis. To achieve this balance it is important to constantly monitor the blood flow and check for signs of cellular damage while applying the pressure to stabilize the organ.
The two main limitations of this protocol are the speed of acquisition and the depth of imaging. Although galvanometer-based confocal microscopes have enough temporal resolution to visualize the gradual collapse of the secretory granules, they do not allow following pre-fusion and fusion events that occur on the order of milliseconds. This aspect can be improved with the use of spinning disk microscopes or laser scanning microscopes equipped with resonant scanners. In terms of depth, confocal microscopy limits the acquisition to the first 30-50 μm below the surface of the salivary glands. This enables imaging 1 layer of acinar structures, and does not allow imaging the salivary ducts. This limitation can be overcome by using two-photon microscopy that ensures imaging up to a depth of 100-150 μm, although at the expense of spatial resolution.
Although for this study we have selected a transgenic mouse that enables simultaneous imaging of the secretory granules and the apical plasma membrane, the procedures described here can be extended to study several other intracellular processes. This can be achieved by using fluorescently tagged molecules that label specific cellular structures and can be administered either systemically or directly onto the SGs. Alternatively, new transgenic mouse models expressing fluorescently tagged-molecules have been developed, such as the glucose transport Glut4, the F-actin marker LifeAct, the nuclear marker histone 2B mouse, and the autophagy marker LC3-GFP. Finally, the basic strategy we have developed to immobilize and image the SGs at high resolution can be potentially extended to other organs (e.g. pancreas, liver, kidney, and mammary glands) by introducing few modifications in the size or the shape of the stabilizer.
This research was supported by the Intramural Research Program of the NIH, National Institute of Dental and Craniofacial Research.
Reagent | |||
Isoflourane (Forane) | Baxter | 101936-40 | Handle under chemical hood |
Ketamine (ketaved) | Fort Dodge Animal Health | 57457-034-10 | Stock solution 100 mg/ml |
Xylazine (Anased) | Akorn, Decatur | 61311-481-10 | Stock solution 100 mg/ml |
Neomycin/Polymyxin B | Bausch and Lomb | 24208-785-55 | Use to lubricate the eye of the mouse |
Carbomer-940 | Ashalnd, Inc. | 4607-1 | |
D-Sorbitol | Sigma-Aldrich | S1876 | |
Triethanolamine | Sigma-Aldrich | 90279 | Add drop wise to prevent the solution to solidify |
Isoproternol | Sigma-Aldrich | 16504 | Prepare fresh solutions in saline when needed. Stocks can be stored at -20 °C |
Instrument | |||
Isoflurane V 1.9 (Vaporizer) | Braintree Scientific | 190AF | |
Portable Downdraft table equipped with HEPA filter | Hazard Technology | PDDT | |
Heat lamp, Model HL1 | Braintree Scientific | HL-1 US | |
MicroTherma 2T Thermometer | Braintree Scientific | TW2 | |
Operating Scissors (11.5 cm straight | World Precision Instruments | 5003708-12 | |
#7 curved tip tweezers | World Precision Instruments | 14187 | |
Microscissors | World Precision Instruments | 503365 | |
Black Braided Silk suture #4.0 | George & Tiemann & Co | 160-1219-4 | |
Gauze sponges 2″ x 2″ | Tyco Healthcare | 9022 | |
Lens cleaning tissue | Olympus | CL-TISSUE (M97) AX6476 |