The ability to perform optical sectioning is one of the great advantages of laser-scanning microscopy. This introduces, however, a number of difficulties due to the scanning process, such as lower frame rates due to the serial acquisition process. Here we show that by introducing spatiotemporal pulse shaping techniques to multiphoton microscopy it is possible to obtain full-frame depth resolved imaging completely without scanning. Our method relies on temporal focusing of the illumination pulse. The pulsed excitation field is compressed as it propagates through the sample, reaching its shortest duration at the focal plane, before stretching again beyond it. This method is applied to obtain depth-resolved twophoton excitation fluorescence (TPEF) images of drosophila egg-chambers with nearly 105 effective pixels using a standard Ti:Sapphire laser oscillator.
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| http://dx.doi.org/10.1364/opex.13.001468 | DOI Listing |
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Patterned two-photon illumination by spatiotemporal shaping of ultrashort pulses.
Opt Express
December 2008
Neurophysiology and New Microscopies Laboratory, Wavefront engineering microscopy group, CNRS UMR8154, INSERM S603, Paris Descartes University, 45, rue des Saints Pères, 75270 Paris Cedex 06, France.
Multiphoton excitation by temporally focused pulses can be combined with spatial Fourier-transform pulse shaping techniques to enhance spatial control of the excitation volume. Here we propose and demonstrate an optical system for the generation of such spatiotemporally engineered light pulses using a combination of spatial control by a two-dimensional reconfigurable light modulator, with a dispersive optical setup for temporal focusing. We show that although the properties of a holographic beam significantly differ from those of plane-wave illumination used in previous temporal focusing realizations, this leads only to a slightly reduced axial resolution.
View Article and Find Full Text PDFThe ability to perform optical sectioning is one of the great advantages of laser-scanning microscopy. This introduces, however, a number of difficulties due to the scanning process, such as lower frame rates due to the serial acquisition process. Here we show that by introducing spatiotemporal pulse shaping techniques to multiphoton microscopy it is possible to obtain full-frame depth resolved imaging completely without scanning.
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