Pdl 800 b

The temperature-dependent steady-state photoluminescence (PL) spectra of perovskite films were measured by using a monochromator (SP-2150i, Acton), a photomultiplier tube (PMT, Acton PD471) and pulsed diode-laser head (LDH-P-C-405, PicoQuant) system. The temperature was varied from 60 K to 200 K. The time-resolved PL (TR-PL) of perovskite films was monitored at 775 nm by using a TCSPC module (PicoHarp 260, PicoQuant) with a micro channel plate photomultiplier tube (MCP-PMT, R3809U-50, Hamamatsu), a 400 nm picosecond pulsed laser (LDH-P-C-405, PicoQuant) coupled with laser-diode driver (PDL 800-B, PicoQuant).

The morphology of core-shell nanostructures was investigated using TEM (JEOL JSM2100-F) at 100 kV. A Perkin Elmer LS 55 fluorescence spectrometer was used to obtain the MEF and FRET-induced changes in photoluminescence spectra of the prepared samples. Time-resolved fluorescence spectra were measured using a Picoquant LDH-P-C-440M & PDL800-B at 20 MHz. The light source was a picosecond diode laser operating at a wavelength of 442 nm. The obtained decay curves were studied using FluoFit software (Picoquant). The excitation wavelength used for all of the PL measurements was 450 nm, which corresponds to the wavelength of the absorption maximum of CdSe QDs. Confocal images were taken using a confocal laser scanning microscope (CLSM LSM510, Carl Zeiss) with 480 nm excitation and 520 nm emission filters.

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.