Abstract
BackgroundThe development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues. However, the use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy.ResultsHere we describe a method for the simultaneous imaging, by multiphoton microscopy, of Green Fluorescent Protein, Cyan Fluorescent Protein and collagen in vivo in living tumors. This novel method enables: 1) the simultaneous visualization of overall cell shape and sub-cellular structures such as the plasma membrane or proteins of interest in cells inside living animals, 2) direct comparison of the behavior of single cells from different cell lines in the same microenvironment in vivo.ConclusionUsing this multi-fluor, multiphoton technique, we demonstrate that motility and metastatic differences between carcinoma cells of differing metastatic potential can be imaged in the same animal simultaneously at sub-cellular resolution.
Highlights
The development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues
We chose to simultaneously image Green Fluorescent Protein (GFP) and Cyan Fluorescent Protein (CFP) because their expression is well tolerated by most cell types and they are excited by standard Ti-sapphire lasers
While YFP has been used as part of a FRET pair in vivo [17], GFP was chosen over YFP because YFP is not effectively excited by wavelengths below 900 nm [18], YFP and GFP excitation closely mirror each other and are difficult to separate, and a large number of already available cell lines and transgenic animals use GFP
Summary
The development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues. The use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy. In recent years multi-photon laser scanning microscopy has demonstrated that it has both the resolution and tissue penetration to significantly improve the analysis of tumor cell behavior in vivo [4,5,6]. Green Fluorescent Protein (GFP) has been widely used to label cells; the use of a single fluorophore, though successful, has been limiting. Tumor development and cell migration from intravasation to metastatic growth has been studied in vivo in orthotopic models and transgenic mice [7] using GFP, but limited by the fact that only one cell type can be examined without introducing a dye from an external source. GFP and RFP have been used to study separate cell populations (page number not for citation purposes)
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