Pma182

Time‐resolved PL spectroscopy and imaging measurements were performed by a home‐build system, which mainly includes fs pulsed laser, an inverted fluorescence microscope, and a spectrometer integrated with Time‐Correlated Single Photon Counting (TCSPC) detection. The excitation laser pulses were generated from a wavelength‐tunable femtosecond oscillator (Coherent Chameleon) with 200 fs pulse width and a repetition rate of 80 MHz. The excitation laser was introduced into the microscopy (Olympus IX71) and focused on the sample via an objective lens (60×). The optical image was taken by two Scientific CMOS cameras (PCO.panda 4.2 & Sony Starvis IMX226). Short‐pass filters (for two‐photon excitation) or long‐pass filters (for one‐photon excitation) were placed in the detection path to cut off the excitation laser before measuring. The sample was placed on a 3D nano‐translation stage (Physik Instrumente, P‐525) for varying the sample position precisely. The collected emission was further imported into a spectrometer (Andor Kymera‐328i) with two output ports. One port was connected with a charge‐coupled device (CCD) camera (Andor iDus420) for the spectra measurement. The other one was integrated with a photomultiplier detector (PicoQuant PMA182) which was associated with TCSPC (HydraHarp 400) for transient PL measurement.

Details of this setup have been published elsewhere^{16 (link),19 (link)}. The exciation is performed with two pulsed diode lasers from Picoquant, emiting at 405 nm (LDH-P-C-405B, FWHM 60 ps, Picoquant-Germany) and 375 nm (LDH-P-C-375B, FWHM 45 ps, Picoquant-Germany). The diodes are controlled by a driver (PDL-808 “Sepia”, PicoQuant GmbH, Berlin, Germany). The repetition frequency used for this study is 40 MHz.
A single-core fiber with a 200 μm diameter, is used to bring the excitation to the sample. The signal is then collected through a single-core optical fiber with a 365 μm diameter. Then the collected signal goes through a long pass filter (SR420, Semrock) to cut the signal from laser reflection. For spectral measurement, a spectrometer (QEPro 6500, Ocean Optics, 1.5 nm spectral resolution over a 365-950 nm spectral range) was used. For lifetime measurements a filter wheel (FW102C, Thorlabs, Newton, USA) with five filters (Semrock, New York, USA, 450 ± 10 nm, 520 ± 10 nm, 550 ± 30 nm, 620 ± 10 nm and 680 ± 10 nm) in front a photomultiplier link to a TCSPC acquisition card (PMA 182 and Time Harp 200, Picoquant, Germany) was used.

The setup of FLIM consists of a picosecond diode laser with emission wavelength of 470 nm with laser power of 5 mW (LDH470, PicoQuant, Germany) and a ∼70 ps pulse width for the excitation of o-BMVC under a scanning microscope (IX-71 and FV-300, Olympus, Japan) (23 ). The fluorescence of o-BMVC was collected using a 60X NA = 1.42 oil-immersion objective (PlanApoN, Olympus, Japan), passing through a 550/80 nm bandpass filter (Chorma, USA), and then detected using a fast PMT (PMA182, PicoQuant, Germany). The fluorescence lifetime was analyzed using a time-correlated single photon counting (TCSPC) module and software (TimeHarp-200 and SymPhoTime, PicoQuant, Germany). FLIM images were constructed from pixel-by-pixel lifetime information.