Fig. 1. Schematic experimental setup for selective two-photon microscopy. Femtosecond laser pulses are compressed and sent to the pulse shaper. The modulated beam is then focused on the microscope slide with the specimen. The two-photon induced fluorescence is collected by a microscope objective and imaged on a CCD.

Fig. 2. (2.02 MB) Experimental results and theoretical predictions of selective two-photon excitation of HTPS solutions at different pH. For this image the pulses were shaped with α=1.5π, γ=20 fs, and δ scanned from 0 to 4π. The movie shows the changes in the measured (black dots) and calculated (green line) contrast ratio as a function of the phase function parameter δ. (a) The laser spectrum (black line) and phase (green line) of the laser pulse. (b) Calculated power spectrum (green) of E²(t) phase modulated pulse at specific phase δ and for TL pulse (black line). Absorption spectra of HTPS at pH 10 (red line) and pH 6 (blue line) (c) Measured (black dots) and predicted (green line and big green dot) contrast ratio.

Fig. 3. (1.28 MB) Experimental demonstration of pH-sensitive selective two-photon microscopy. The sample being imaged has an acidic (left side of the frame at pH 6) and a basic (right side of the frame at pH 10) region, both labeled with HPTS. (a) Image of the sample obtained with transform-limited pulses. The diagrams on the right show the spectrum of the 21-fs laser pulses, centered at 842 nm, and the spectral phase of the pulse (blue dashed line or red dotted line, that maximize pH 6 or pH 10 fluorescence, respectively). (b) Image of the same sample and location obtained with pulses that have been optimized for selective excitation of HPTS in an acidic micro-environment. Notice that only the left region shows significant two-photon excitation. For this image α=1.5π, γ=20 fs, and δ=0.75π. (c) Image of the same sample and location obtained with pulses that have been optimized for selective excitation of HPTS in a basic micro-environment. Notice that only the right region shows significant two-photon excitation. For this image α=1.5π, γ=20 fs, and δ=0.25π. The movie shows an experiment where the phase function parameter δ is scanned from 0 to 4π. Selective two-photon excitation from the two pH regions in the sample is observed at specific values of δ.

Fig. 4. (2.4 MB) Selective two-photon microscopy of pieces of PMMA doped with different fluorescent probes. (a) Image showing two-photon induced fluorescence from both pieces top-C540 doped PMMA and bottom-R6G doped PMMA, obtained with 17 fs transform-limited pulses centered at 790 nm. (b) Image obtained with pulses optimized for selective C540 excitation. For this image the pulses were shaped with α=1.5π, γ=20 fs, and δ=0.31π. (c) Image obtained with pulses optimized for selective R6G excitation. For this image the pulses were shaped with α=1.5π, γ=20 fs, and δ=0.74π. The movie shows an experiment where the phase function parameter δ is scanned from 0 to 2π. Selective two-photon excitation from the two PMMA pieces (left is C540 and right is R6G) is observed at specific values of δ.

Fig. 5. (738 kB) Selective two-photon microscopy of 10 µm blue and 15 µm green fluorescent polystyrene microspheres. (a) Image showing two-photon induced fluorescence from both microspheres, obtained with 15 fs transform-limited pulses centered at 790 nm. (b) Image obtained with pulses optimized for selective excitation of the blue microsphere. For this image the pulses were shaped with α=2.5π, γ=10 fs, and δ=0.75π. (c) Image obtained with pulses optimized for selective excitation of the green microsphere. For this image the pulses were shaped with α=2.5π, γ=10 fs, and δ=1.25π. The movie shows an experiment where the phase function parameter δ is scanned from 0 to 2π. Selective two-photon excitation from the two microspheres is observed at specific values of δ.