Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Summary

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.

Abstract

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.

Introduction

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....

Protocol

Part 1: Microscope and Preparation of the Imaging Setup

1. Any inverted confocal microscope should be suitable for IVM. In this study an Olympus IX81 inverted microscope, equipped with a Fluoview-1000 laser scanning confocal unit was used. To achieve the best possible resolution, the use of high NA objectives, such as the water-immersion UPLSAPO 60x/1.20 NA, and the oil immersion Plan-Apo 60x/1.42 NA is recommended. Oil immersion objectives have higher collection efficiencies and although they require the use of a coverslip between the tissue and the objective, they provide a better imaging quality when used to image areas within 15-20 μm from the surfac....

Results

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 (

Discussion

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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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