Tcspc system

The fluorescence lifetime of Cy3 was measured using a TCSPC system (PicoQuant) with a TCS SP8 confocal laser‐scanning microscope (Leica Microsystems) at room temperature. The fluorescently labeled Cas9 constructs (1 μM) were loaded in a glass chamber prepared by the same procedure for smFRET measurements and absorbed onto the glass surface via Neutravidin. The buffer condition was same as that of smFRET measurement: 20 mM HEPES‐KOH, pH 7.5, 100 mM KCl, 2 mM MgCl₂, 0.5 mM EGTA, 1 mM DTT, 2.5 mM TSY, 2.5 mM PCA, and 2% PCD. The Cas9 constructs on the glass surface were illuminated with a pulsed diode laser (PDL 800‐B, PicoQuant, 470 nm) through a confocal pinhole (hole size: 1 Airy unit = 0.896 μm) at a repetition rate of 20 MHz. The emission light in a range of 540–660 nm was collected through an HCX PL APO Ibd.BL 63× 1.4 NA oil objective (Leica Microsystems) in a 128 × 128 pixel format. The fluorescence lifetime data were collected on a time scale of 50 ns, resolved into 3,200 channels (i.e., 15.6 ps for each channel), and accumulated for 30 s. The data of each pixel were averaged within the whole image and then fitted with double exponential decay curves using the OriginPro 8.0J software (OriginLab). Mean lifetime (τ_mean) was calculated as ${\tau }_{\mathrm{mean}}=(A_1{\tau }_1^2+A_2{\tau }_2^2)/(A_1{\tau }_1+A_2{\tau }_2)$ where τ and A were lifetime and amplitude, respectively.