title: Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window.
abstract: Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90%1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell's arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution2are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window2,4,5however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.
authors: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine.
authors: Carolina
authors: C
authors: Rodriguez-Tirado
authors: Medical Research Council Centre for Reproductive Health, Queen's Medical Research Institute, University of Edinburgh.
authors: Takanori
authors: T
authors: Kitamura
authors: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine; Department of Obstetrics/Gynecology and Woman's Health, Albert Einstein College of Medicine.
authors: Yu
authors: Y
authors: Kato
authors: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine; Department of Obstetrics/Gynecology and Woman's Health, Albert Einstein College of Medicine; Medical Research Council Centre for Reproductive Health, Queen's Medical Research Institute, University of Edinburgh.
authors: Jeffery W
authors: JW
authors: Pollard
authors: Department of Anatomy & Structural Biology, Albert Einstein College of Medicine; Gruss-Lipper Biophotonics Center Integrated Imaging Program, Albert Einstein College of Medicine.
authors: John S
authors: JS
authors: Condeelis
authors: Department of Anatomy & Structural Biology, Albert Einstein College of Medicine; Gruss-Lipper Biophotonics Center Integrated Imaging Program, Albert Einstein College of Medicine; david.entenberg@einstein.yu.edu.
authors: David
authors: D
authors: Entenberg
mesh_terms: D000069416:Intravital Microscopy; D008168:Lung; D055806:Microvessels; D014618:Vacuum