were recorded at a frequency of 1 Hz with an exposure time of
20 ms. Although the sample is slightly opaque at this
temperature and diffuses light signi®cantly, residual stray
radiation from the excitation laser does not interfere with the
¯uorescence line, which appears of remarkable signal to noise
quality. The peak emission, at 505 nm, is signi®cantly blue
shifted relative to the value commonly observed at room
temperature (ca 510 nm). Fig. 2(b) shows the stability of the
¯uorescence emission of the same solution, as recorded by the
spectrometer over 5000 s. Counting is reported at two wavelengths
of the emission spectrum (491 and 513 nm) located
near in¯exion points of the latter. A slight linear decay of the
¯uorescence intensity is observed over time, which is estimated
to be ca 3% per hour and probably results from photobleaching.
Instabilities of relatively high frequency (3.3%
r.m.s. deviation) are also observed, which result from residual
absorption by turbulent wet air circulating around the cold
nitrogen stream. The ratio between the two countings, which is
a sensitive marker of the peak emission wavelength, is
remarkably stable (0.42% r.m.s. deviation).
Fig. 3 shows two one-shot emission spectra from a crystal of
PYP, collected at 100 and 220 K. PYP is thought to mediate
the build up of a negative phototactic response to blue light
(Genick et al., 1998). Rod-shaped crystals of about 50 50
250 mm were grown in space group P65. Spectra were obtained
in the same conditions as those of Fig. 1. Despite the small size
of the crystal, its large optical density in the range 400±470 nm
prevented the collection of useful absorption spectra.
However, proper orientation of the crystal allowed ¯uorescence
spectra of excellent quality to be obtained, although
these mainly probed the sample surface. Similar emission
spectra could be obtained using excitation at 355 nm, a
wavelength well remote from the absorption maximum of
PYP (not shown). This allowed the bulk of the crystal to be
probed, but rapidly resulted in serious photo-bleaching of the