Abstract
Selective two-photon excitation of fluorescent probe molecules using phase-only modulated ultrashort 15-fs laser pulses is demonstrated. The spectral phase required to achieve the maximum contrast in the excitation of different probe molecules or identical probe molecules in different micro-chemical environments is designed according to the principles of multiphoton intrapulse interference (MII). The MII method modulates the probabilities with which specific spectral components in the excitation pulse contribute to the two-photon absorption process due to the dependence of the absorption on the power spectrum of E2(t) [1-3]. Images obtained from a number of samples using the multiphoton microscope are presented.
Highlights
Two-photon microscopy has provided researchers with unique possibilities for fluorescence imaging and photochemistry
Two-photon microscopy has been shown to be amenable to multiple probe staining, whereby two-photon transitions excite different probe molecules that emit at different wavelengths [6,7], and for functional imaging of living cells [8,9,10,11]
Selective excitation can be used to enhance contrast and to achieve functional imaging of samples stained with fluorescent probes sensitive to their microscopic chemical environment
Summary
Two-photon microscopy has provided researchers with unique possibilities for fluorescence imaging and photochemistry. It offers attractive advantages, including higher resolution, background-free signal, lower background scattering, better penetration in thick samples, and reduced photon-induced damage [4,5,6], which arise from the basic physical principle that the absorption depends on the square of the excitation intensity. Two-photon microscopy has been shown to be amenable to multiple probe staining, whereby two-photon transitions excite different probe molecules that emit at different wavelengths [6,7], and for functional imaging of living cells [8,9,10,11]. Selective excitation can be used to enhance contrast and to achieve functional imaging of samples stained with fluorescent probes sensitive to their microscopic chemical environment
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