Pulse-shaping techniques can be used to image GFP-labelled drosophila embryos with high selectivity for GFP and suppression of autofluorescence. The images below show the electric field applied by a pulse-shaper and the corresponding fluorescence images of the drosophila embryo. By applying the appropriate pulse-shape we effectively tune the two photon excitation to preferentially excite GFP or autofluorescent species.Using the images obtained with the different pulse-shapes (see below), we can separate GFP and autofluorescent signals (shown on the left).

Pulse-shaping Fluorescence Microscopy

Applied electric field

Corresponding image

Learn more about pulse-shaping microscopy through our interactive pulse-shaping tutorial

Multicolor two-photon fluorescence microscopy is difficult to achieve with a conventional two-photon microscope due to the limited bandwidth of the lasers that are typically used. These lasers are tuned to excited the fluorophore of interest, but tuning is a slow process. We’ve shown that pulse-shaping an ultrabroadband laser can readily enable three-color two-photon fluorescence imaging and can be extended to even more colors if desired.

We’ve also used the pulse-shaping approach to demonstrate two-photon FRET stoichiometry to enable quantitative measurements of protein-protein interactions in tissues. This work was highlighted in an OSA Spotlight.