Fig. 2.

Depletion curve and phasor analysis in M-STED microscopy. a–c Simulations of sparse foci under different conditions of acquisition. Compared with a, b was simulated with the same maximum saturation value (I_max/I_sat = 5) but lower SNR (S_max = 10), while c had lower maximum saturation value (I_max/I_sat = 2) and the same SNR. d Simulation of crowded foci under the same conditions of acquisition of a. e Simulation of filaments under the same conditions of acquisition of a. Shown are, for all the simulations, from top to bottom: first and last images of the stack (F₁ and F₈), the average variation of the fluorescence intensity as a function of the STED power (depletion curve) and the estimated k parameter (Eq. (4)), the corresponding phasor plots and the estimated positions of P_in and P_out vectors, and the final SPLIT image components F_in and F_out. Scale bars: 1 µm