Two-photon Fluorescence with Diode Laser Excitation

1 Two-photon Fluorescence with Diode Laser Excitation 

Two-photon laser scanning microscopes and microscopes for two-photon single molecule experiments use femtosecond lasers. Two-photon fluorescence excitation with a picosecond diode laser cannot be expected to work with the same efficiency. The average intensity is only a few mW. Moreover, the pulse duration is of the order of 100 ps, so that the peak power is many orders of magnitude lower than for a fs Ti Sapphire laser. The relative excitation efficiency of different lasers can be estimated as follows  

The factor k depends on the two-photon absorption cross section of the dye at the laser wavelength, on the pulse shape, and on the spatial energy distribution in the focus. Because the effective excitation depends on the reciprocal pulse width, not on its square, the prospects are not too bad for detecting diode-laser-excited two-photon fluorescence. It has in fact been demonstrated that two-photon excitation can be achieved even with CW lasers [228]. An experiment to demonstrate two-photon excitation by diode lasers is shown in Fig. 5.134. 

The laser emits at a wavelength of 785 run. The average power of the laser was adjusted to approximately 10 mW. The pulse width was determined by TCSPC and was about 300 ps FWHM. 

Diode lasers emit a small amount of light at wavelengths very different from the nominal emission wavelength. To block emission below 650 nm, a 695 nm filter glass was put into the excitation beam. The fluorescence light was detected by a Hamamatsu H7422P-40 photomultiplier module. The single-photon pulses 

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