Fig. 6: 12.5-Gfps polarization anisotropy imaging of fluorescein, 4 K, 20 K molecules and their combinations, excited by a single NIR femtosecond laser pulse via the two-photon process.

a Schematic of a single cuvette with one type of fluorescence molecule and a single pattern. BBO: beta barium borate, BPF: band-pass filter, CyL: cylindrical lens, HWP: half-wave plate, P0: linear polarizer, LPF: long-pass filter. b Schematic of dual cuvettes side-by-side with two types of fluorescence molecules and two patterns. In (a) and (b), the red and yellow beams represent the fluorescence excitation and emission, respectively. A long-pass filter is employed to select the 800 nm light for two-photon fluorescence, replacing the short-pass filter used in one-photon fluorescence in Fig. 2a, b. Figure 6o shows the averaged anisotropy lifetimes for single and dual cuvette measurements of the same FL, 4 K, and 20 K samples as in 1 P excitation. In (c–n), results of (c–e) fluorescein molecule, (f–h) 4 K molecule, (i–k) 20 K molecule in the single-cuvette configuration, and (l–n) fluorescein and 20 K molecules in the dual-cuvette configuration. In (c), (f), (i), and (l), Left \(y\)-axis: evolutions of normalized spatially integrated intensities from both \(x\)- and \(y\)-polarization channels (red and blue solid lines). Right \(y\)-axis: evolutions of spatially averaged anisotropy. The green solid lines are the measured anisotropies, and the black dashed lines represent exponential fits. The circles and arrows group the plots for either the left- or the right-\(y\) axis. In (l), top plots are for 20 K molecule and bottom plots are for fluorescein. d, g, j, and m Polarization anisotropy evolutions, over the first 4.5 ns. Only 8 representative snapshots are shown for clarity. e, h, k and n Anisotropy lifetime maps of the imaged samples. o Spatially averaged anisotropy lifetimes of three types of molecules. Error bars represent their standard deviations over space.