Microfluorometry of cytosolic pH
Measurements of the global cytosolic pH are made by an ratiometric approach that uses the pH sensitive dye BCECF (Invitrogen).
For measurements of the global pH, BCECF-loaded tissues are illuminated alternatively with 442 nm and 502 nm light and emissions are measured at 530+10 nm (Deltascan, PTI).
Alternatively, for measurements of the pH in individual cells, BCECF-loaded cells are alternatively illuminated with 454 nm and 488 nm light and emissions are measured at >530 nm (510 META, Carl Zeiss). For both approaches, the ratio of the emission intensity correlates with the cytosolic pH. The benefit of a ratiometric approach is that it is relatively independent of changes in cell volume.
Papers from the lab
Wangemann et al (1993)
Wangemann & Shiga (1994)
Wangemann et al (1995)
Wangemann et al (2007) (pdf)