FLIM based pH-imaging
pH imaging can be carried out using fluorescence techniques. Fluorescein, for instance exhibits a pH dependent fluorescence emission intensity. However, intensity based imaging is not suitable for quantitative pH measurements. Dyes like carboxy SNARF-1 exhibit a significant pH-dependent emission shift. This pH dependence allows quantification of pH from the ratio of the fluorescence intensities from the dye at two emission wavelengths. pH not only affects quantum efficiency and emission wavelength of the pH sensitive dyes, but also their fluorescence lifetime. FLIM based pH sensing turns out to be a more reliable method for pH quantification than the ratio imaging approaches.
Sanders, R., A. Draaijer, H.C. Gerritsen, P.M. Houpt, andY.K. Levine. 1995. Quantitative Ph Imaging in Cells Using Confocal Fluorescence Lifetime Imaging Microscopy. Anal. Biochem. 227(2):302-308.
TPE-FLIM based pH imaging in biofilm
pH-imaging with pH sensitive colloids
Recently, we synthesized pH sensitive colloids for accurate pH imaging in biological systems. These particles provide a well defined matrix for the pH sensitive dye (carboxy-fluorescein. Therefore, unwanted interactions with for instance proteins can be avoided. Therefore, a higher accuracy in the pH determination is expected.