Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Carolina Rodriguez-Tirado; Takanori Kitamura; Yu Kato; Jeffery W. Pollard; John S. Condeelis; David Entenberg

doi:10.3791/54603

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Podsumowanie
Streszczenie
Wprowadzenie
Protokół
Wyniki
Dyskusje
Ujawnienia
Podziękowania
Materiały
Odniesienia
Przedruki i uprawnienia

Podsumowanie

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Streszczenie

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 resolution ² are 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 system ³. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window ^2,4,5 however, 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.

Wprowadzenie

High resolution intravital optical imaging has proven to be crucial to understanding many biological processes, allowing single-cell and sub-cellular parameters to be measured and quantified. In cancer research, intravital imaging of tumor and stromal cells has led to the discovery of many microenvironmental interactions ^6-11 that are only present in the intact animal.

Discoveries about microenvironments associated with intravasation and dissemination of tumor cells in breast cancer using single cell resolution optical imaging in vivo has even led to novel markers for prognosis and response to treatment in breast cancer ....