100) and 632.8 nm (20 mW) (helium-neon laser, NEC ref.
GLG 5410). To avoid temperature elevation and potential
photo-bleaching, ¯uorescence measurements are generally
performed in a pulsed way. Using a mechanical shutter
synchronized with the spectrometer CCD readout, spectra
with exposure times of typically 10±30 ms are collected at a
frequency of 1±10 Hz. To prevent scattered excitation light
perturbing the ¯uorescence spectrum, a long-pass ®lter is
inserted when necessary in front of the detection objective,
cutting out light at the excitation wavelength. Depending on
sample optical density, which in some cases can be very high
(>2), either right-angle or front-face geometry is used
(Lakowicz, 1999). The latter geometry allows dif®culties
associated with strong inner-®ltering to be partially overcome
at the cost of mostly probing the crystal surface. By using two
spectrometer cards in tandem, the two in-line mirror objectives
can record ¯uorescence simultaneously. In this case, if
one spectrum is obtained with the right-angle geometry and
the other with the front-face geometry, a comparison of both
spectra allows an estimation of spectral distortion resulting
from inner ®ltering.