Fig. 5 shows that ¯uorescence may also be used to measure
the pH in samples as small as a protein crystal (ca 1 nl). It is
known that the protonation state of ¯uorescein affects the
absorption and ¯uorescence properties of this molecule, which
can therefore be used as a pH indicator in solution (Yguerabide
et al., 1994). Since emission spectra from ¯uorescein are
usually dominated by the di-anionic form of the molecule, the
¯uorescence intensity decreases as the pH is lowered.