Dynamic live cell experiments such as FRAP (Fluorescence Recovery After Photo-bleaching) require the ability to target concentrated laser illumination onto a user-defi ned ROI (Region Of Interest), while minimizing photo-bleaching associated with sequential image capture. The UltraVIEW® PhotoKinesis™ Accessory enables these techniques. In addition, it can be used to perform vesicle tracking experiments, when used with photoswitchable or photoactivatable proteins.
Photoactivatable proteins such as PA-GFP (1), Dronpa (2) and PS-CFP2 (3) represent one of the most promising approaches to in vivo investigations of protein lifetimes, transport and turnover rates. In this study PSCFP2 was used, it is a photoactivatable dualcolor protein capable of irreversible photo-conversion from a cyan to green fl uorescent form in response to 405 nm light irradiation. High pH stability before photo-activation allows targeting PS-CFP2 to acidic organelles such as endosomes and lysosomes. PS-CFP2 was used as a photoswitchable tag to study traffickingof SNX1. SNX1 has been implicated in the regulation of intracellular sorting of internalized cell surface receptors. The UltraVIEW PhotoKinesis accessory can be used to investigate the photo-switching capabilities of PSCFP2 and intracellular trafficking of SNX1.
MethodThe Sorting Nexin SNX1 was successfully sub-cloned into PSCFP2-C1 and PS-CFP2-N1. Hela cells were seeded at 15×104 cells in glass bottomed chambers (MatTek) for 24 hours. The cells were then transiently transfected with 3 µg of PS-CFP2-C1, PSCFP2- N1 and 1 µg GFP-SNX1 using Genejuice® transfection reagent (Novagen®). Live cell imaging was performed 24-48 h later in Dulbecco's modified Eagle medium (DMEM).
Fluorescence imaging of living cells was performed with the UltraVIEW ERS confocal live cell imager. We applied 405 nm laser power, 6 mW, for PS-CFP2 photoswitching of the whole cell using the PhotoKinesis Accessory. Total photoswitching time for living cells was between 1-2 seconds, with 2×2 binning mode, 1 second exposure times and using the ×100 objective 1.45 NA oil immersion lens, for PS-CFP imaging through ECFP (Ex 405 nm, Em 420-475 nm) and EGFP (Ex 488 nm, Em 500-550 nm) standard filters.
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