Figure 1.

Demonstration of wide-field three-photon excitation at 1300 nm. (a–c) Effect of increasing spot size. Sample consists of a drop of quantum dots on a glass microscope slide. Color scale is arbitrary, but all images share the same scale. Circles of diameter 50, 100, 150 and 200 μm are provided as a guide to the eye, and a yellow contour indicates where the intensity drops below 25% of peak. (a) × 2.5 beam expander, 0.7 s exposure. (b) × 3.5 beam expander, 1.5 s exposure. (c) × 5 beam expander, 10 s exposure. (d) Fixed HeLa cells stained with QDot 605 quantum dots. Power at the sample is 80 mW, integration time is 10 s, the scale bar is 100 μm and flat-field correction is applied to compensate for non-uniform excitation. (e) Excitation and emission spectra of the 525 nm quantum dots. Arrows at 650 and 433 nm indicate the location on the one-photon absorption spectrum corresponding to two- and three-photon excitation, respectively. (f) Fluorescence intensity as a function of average power, demonstrating three-photon excitation.