Primary Photophysical Processes in Chromoproteins - Fluorescent Proteins: From Understanding to Design - page 43

The large spectral width required to support very short laser pulses gives rise to
some problems. The refractive index of most optical materials is wavelength
dependent (called the material's dispersion) with the consequence that the velocity
of light in the medium is wavelength dependent. Specifically in the case of normal
dispersion, blue light travels slower than red light, giving rise to positive group
velocity dispersion (GVD) [ 33 , 34 ]. Consequently, on transmission through any
optical element, the spectrally broad pulse becomes temporally broader. Thus
pulses emitted from the laser are typically temporally much broader than their
bandwidth would suggest due to dispersion of intracavity elements and have to be
compressed. Pulse compression is achieved by routing the plane-polarised pulse
through a pair of prisms set at Brewster's angle (to minimise reflection losses) and
retroreflecting the pulse back through the same prism pair. The dispersion of the
spectrally broad beam and the different path lengths each wavelength follows in the
(dispersive) glass of the prism contribute both positive and negative GVD. By
controlling the separation and position of the prisms, it is possible to compensate
the positive GVD of the cavity with negative GVD in the prism pair to yield the
shortest pulse. Indeed negative GVD can be introduced to 'pre-compensate' posi-
tive GVD in optical elements, which the pulse must go through before reaching the
sample.
For the up-conversion measurement of the fluorescence decay time, the com-
pressed pulse is first routed to a thin non-linear crystal to generate the second
harmonic [ 36 , 37 ]. The ca. 800 nm fundamental and 400 nm second harmonic
beams are separated at a dichroic mirror. The 400 nm beam is reflected by a series
of mirrors and focused into the sample by a spherical mirror. Since the 400 nm
beam is broadened by transmission through any optics it must be recompressed,
since the shortest pulses are required at the sample . In our experiment, the bulky
prism compressor is replaced by a compact dispersive mirror pair [ 38 ]. The 400 nm
excitation pulse generates fluorescence from the sample, which is collected by a
reflective Cassegranian microscope objective, which efficiently collects the emitted
fluorescence without introducing a dispersive element (a lens) which would
degrade time resolution (in a wavelength dependent manner). The objective focuses
the light collected onto a non-linear crystal for sum-frequency generation (SFG).
The residual 800 nm pulse is routed through a variable optical delay and passes
through a half wave plate that sets the polarisation to the magic angle with respect to
the excitation polarisation, to eliminate the contribution of orientational relaxation
to the measured fluorescence kinetics. The beam then passes through a pair of
dispersive mirrors to recompress the pulse before being focused into the non-linear
SFG crystal.
In the SFG crystal, the train of fluorescence decays at wavelength

l F (appearing
at the laser repetition rate) is spatially overlapped with the train of short 800 nm
pulses at l P . The SFG crystal is oriented to generate the sum frequency at a

wavelength l SFG :

1
l SFG ¼

1
l F þ

1
l P :