a form of GFP in which fluorescent intensity can be increased 100 fold after activation with 400nm light
Image credit: Cell expressing mCherry lamin A and PA-GFP tagged lamin A. Lower portion of the nucleus was exposed to 405nm. Activated PA-GFP signal is shown in green. Image courtesy of Takeshi Shimi, Ph.D. and Robert D. Goldman, Ph.D., Northwestern University Medical School
Wild-type GFP exists in two forms, which give rise to a major and a minor absorbance peak at 397nm and 475nm, respectively. Intense illumination at 400 nm shifts the population to the 475nm form, thereby increasing the absorbance of the minor peak. This capacity for photoconversion led to the development of PA-GFP. By selecting a form of GFP with a negligible 475nm peak, photoconversion produces a much greater proportional increase in 475nm absorbance compared with standard GFP - and therefore increases contrast. PA-GFP exhibits very low green emission (max 517nm) after 488nm excitation and can be activated 100-fold by stimulation with 405nm light.
PA-GFP is useful for studying temporal and spatial dynamics of.proteins in living cells or tracking individual cells in a population of cells or in tissue. After activation, a clear increase in fluorescent intensity at the site of activation can be observed and the redistribution of activated molecules can be followed over time using time-lapse imaging. This provides an alternative, and more direct, method, of molecular tracking compared to, for example, FRAP or FLIP.
Selective activation of PA-GFP can be achieved on both (1) widefield microscopes and (2) confocal fluorescent microscopes.
Nikon's PA-GFP unit can be mounted on a standard inverted microscope equipped for fluorescence. This unit is placed on the back port of the microscope and uses a fibre-coupled laser (405 nm) to illuminate a diffraction limited spot on the specimen.
On confocal systems, depending on the type of laser used, a region of interest can be activated either by the zoom capacity of the scan mirrors or by local application of laser power. Alternatively, the laser can be controlled through an AOM or AOTF, allowing complete flexibility in defining the region of interest.
Nikon's PA-GFP unit mounted on an Eclipse Ti inverted microscope can be used both in combination with confocal and widefield fluorescent imaging.