High Resolution Microscopy in Live-Cell Imaging

Abstract

We have used PALM (photoactivation localization microscopy) imaging to study the formation and disassembly of focal adhesions of live HeLa cells in a high resolution pulse chase experiment using a monomeric variant of the photoactivatable protein IrisFP [1]. mIrisFP is a photoactivatable fluorescent protein that combines irreversible photoconversion from a green- to a red-emitting form with reversible photoswitching between a fluorescent and a nonfluorescent state in both forms [2]. A subpopulation of mIrisFP molecules is photoconverted to the red form by irradiating a specific region of the cell with a pulse of violet light. Migration of the tagged proteins out of the conversion region can be studied by subsequently localizing the proteins in other regions of the cell by PALM imaging, exploiting the photoswitching capability of the red species. Online image analysis was performed by using our recently software [3]. [1] Adam et al., Proc. Natl. Acad. Sci. USA. 105 (2008) 18343. [2] Fuchs et al., Nat. Methods 7 (2010) 627. [3] Hedde et al., Nat. Methods 6 (2009) 689.