We formulate a simple strategy for mitigation of laser-induced damage through pulse shaping and demonstrate
experimentally the effect of laser pulse duration on the degree of optically induced damage for two-photon microscopy
imaging. We use a broadband Ti:Sapphire laser source, aided with a shaper, and adjust both the laser pulse duration and
energy to maintain constant two-photon excitation efficiency. The damage is assessed by the dynamics of two-photon
excited autofluorescence intensity and sample morphology during prolonged laser exposure. We observe that for a 5-μm
layer of skin tissue the damage rate is independent of the pulse shape, which suggests that the primary damage
(bleaching) mechanism stems from the two-photon excitation itself. For optically thick dried blood samples, taken as
another example, the data suggests that the damage is driven by
one-photon absorption. In the later case, it is favorable
to use shorter laser pulses to mitigate photodamage while maintaining adequate intensity of two-photon excited
autofluorescence.
High-order dispersion of ultrashort laser pulses (with ~100 nm bandwidth) is shown to account for significant reduction
of two-photon excitation fluorescence and second harmonic generation signal produced at the focal plane of a laser-scanning
two-photon microscope. The second- and third-order corrections recover 20-40% of the signal intensity
expected for a transform-limited laser pulse, while the rest depends on the proper compensation of higher-order terms. It
can be accomplished through the use of a pulse shaper by measuring and correcting all nonlinear spectral phase
distortions.
An adaptive pulse shaper controlled by multiphoton intrapulse interference phase scanning (MIIPS) was used, together with a prism-pair, to measure and cancel high-order phase distortions introduced by a high-numerical-aperture objective and other dispersive elements of a two-photon laser-scanning microscope. The delivery of broad-bandwidth (~100 nm), sub-12-fs pulses was confirmed by interferometric autocorrelation measurements at the focal plane. A comparison of two-photon imaging with transform-limited and second-order-dispersion compensated laser pulses of the same energy showed a 6-to-11-fold improvement in the two-photon excitation fluorescence signal when applied to cells and tissue, and up to a 19-fold improvement in the second harmonic generation signal from a rat tendon specimen.
Shorter pulses, in theory, should be favorable in nonlinear microscopy and yield stronger signals. However, shorter
pulses are much more prone to chromatic dispersion when passing through the microscope objective, which significantly
broadens its pulse duration and cancels the expected signal gain. In this paper, multiphoton intrapulse interference phase
scan (MIIPS) was used to compensate chromatic dispersion introduced by the 1.45 NA objective. The results show that
with MIIPS compensation, the increased signal is realized. We also find that third and higher order dispersion
compensation, which cannot be corrected by prism pairs, is responsible for an additional factor of 4.7 signal gain.
Ultrashort <15 fs pulses are shown to provide higher fluorescence intensity, deeper sample penetration, and
single laser selective excitation. To realize these advantages chromatic dispersion effects must be
compensated. We use multiphoton intrapulse interference phase scan (MIIPS) to measure and then
eliminate high-order distortions on pulses with a bandwidth greater than 100nm FWHM. Once
compensated, the transform limited pulses deliver higher signal intensity, and this translates into deeper
optical penetration depth with a high signal-to-noise ratio. By using a pulse shaper and taking advantage of
the broad spectrum of the ultrafast laser, selective excitation of different cell organelles is observed due to
the difference in nonlinear optical susceptibility of different chromophores without the use of an emission
filter wheel.
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