Hydraharp 400

Our experimental LiDAR data consists of two datasets captured using the asynchronous single-photon imaging technique²⁴ . The datasets were obtained from the hardware prototype consisting of a 405 nm pulsed laser (Picoquant LDH-P-C-405B), a TCSPC module (Picoquant HydraHarp 400) and a fast-gated SPAD^{66 (link)}. The laser was operated at a repetition frequency of 10 MHz for an unambiguous depth range of 15 m. Each dataset has a ground-truth photon transient cube acquired with long acquisition time without ambient light. For the face scene (Fig. 4b), we down-sampled the ground-truth data such that the average signal photon counts per pixel are 24, 2.4, and 0.8. The deer scene (Fig. 4a) was captured under strong ambient illumination (>20,000 lux) high enough to cause pileup distortion. See Table S2 for more detailed data specifications.

FCS measurements were performed on a commercial, epi-illuminated, confocal laser scanning microscope (Olympus, Tokyo, Japan, FV1200). Cy5 in aqueous solution was excited with a focused beam (338 nm, 1/e² radius) of a 638 nm laser (LDH-D-C-640 from PicoQuant GmbH, Berlin, Germany) in continuous wave. The emitted fluorescence was collected back through the microscope objective (UPlanSApo 60x/1.2 w, Olympus, Tokyo, Japan), passed through a dichroic mirror (ZT405/488/635rpc-UF2, Chroma), an emission filter (HQ720/150, Chroma, 680 nm blocking edge Brightline, Semrock, in combination with last mentioned filter or 710/40 Brightline, Semrock), and focused onto a pinhole (50 µm diameter) in the back focal plane. The fluorescence signal was finally split and directed on two avalanche photodiodes (Tau-SPAD, PicoQuant GmbH, Berlin, Germany), whose signals were collected with a data acquisition card (Hydraharp 400, Picoquant, Berlin, Germany).

FLIM data acquisition was carried out using a confocal laser scanning microscope (LSM780 inverted microscope, Zeiss) equipped additionally with a time‐correlated single‐photon counting device with picosecond time resolution (Hydra Harp 400, PicoQuant). mVenus was excited at 485 nm with a pulsed (32 MHz) diode laser at 1.2 µW at the objective (40× water immersion, C‐Apochromat, NA 1.2, Zeiss). The emitted light was collected through the same objective and detected by SPAD detectors (PicoQuant) using a narrow range bandpass filter (534/35, AHF). Images were taken at 12.5 µs pixel time and a resolution of 138 nm/pixel in a 256 × 256 pixel image. A series of 40 frames was merged into one image and analysed using the Symphotime software package (PicoQuant).

Fluorescence time traces were measured using time-correlated single-photon counting (TCSPC) using a HydraHarp 400 (PicoQuant) module attached to a MicroTime 200 confocal microscope (PicoQuant GmbH) with a 50×/0.45 NA objective (SLMPlan). An NKT SuperK Extreme pulsed (2.9–4 MHz) white light continuum laser with the SuperK Select acousto-optical tunable filter (AOTF) was used for excitation at 480 nm. A 482/12 bandpass filter was used to remove parasitic frequencies from the source. A Z488 RDC dichroic was used to reflect the excitation light into the objective, and a 488 nm notch filter was used to prevent the excitation light from reaching the detector. A detector pinhole of 150 $$\mathsf{\mu }$$ m was used. The detector used was a Single-Photon Avalanche Diode (MPD PDM Series, PicoQuant). The IRF was determined by measuring the signal from a solution of Erythrosin B in water saturated with potassium iodide [25 (link)]. The samples were prepared by placing a droplet of the neat liquid or solution on a 22 × 22 mm glass coverslip.
Fluorescence decay times were also obtained using time-gated detection during the transient absorption experiments described below.

For single-particle spectroscopy, the sample is mounted on a translation stage of a home-made optical microscope and perovskite NCs are excited at 445 nm with approximately 70 ps pulses from a laser diode through a 100×, 1.4 NA oil-immersion objective that is also used to collect PL. The interpulse duration is set to be much longer than the PL decay times in order to ensure complete relaxation of excitations between sequential laser pulses. Filtered PL signal (long pass 480 nm and bandpass 520/40 filters were used to reject scattered laser light and isolate emission from PNCs) is sent to a pair of avalanche photodiodes (PDM series, Micro Photon Devices) positioned at two arms of the standard Hanbury−Brown−Twiss (HBT) arrangement with a 50/50 beam splitter. Time-tagged, time-correlated single photon counting (TCSPC) is performed using PicoQuant HydraHarp 400 electronics. TCSPC simultaneously records photon arrival times with respect to the beginning of the measurement cycle and to the excitation laser pulse. Hence, it allows us to compile PL decay curves for any particular time segment of the PL intensity trajectory or a chosen window of the intensity distribution function while simultaneously recording second-order photon correlation functions, g²(τ). The overall system’s time resolution was better than 300 ps.

Mobility of Src molecules was determined using Imaging FCS. U2OS cells transfected with SrcFRET constructs and mCherry-vinculin were plated on fibronectin-coated glass-bottom dishes. Experiments were performed on Nikon Eclipse Ti-E microscope equipped with an H-TIRF module and a Nikon CFI HP Apo TIRF 100× Oil NA 1.49 objective. Time-lapse images (10,000 frames, 365 frames per second, ROI 32×32 pixels) were acquired with Andor iXon Ultra DU897 camera (Andor Technologies) using 488 nm and 561 nm excitation wavelengths for SrcFRET constructs and mCherry-vinculin, respectively. Data were analysed with the ImageJ software using the Imaging FCS plugin. Diffusion coefficients were calculated from relative coefficients obtained from ImFCS and calibration measurements of SrcFRET WT mobility with line-scan FCS approach. Scanning FCS was performed on Leica TCS SP8 microscope equipped with Leica HC PL APO CS2 63× Oil NA 1.4 objective and HydraHarp400 (Picoquant) TCSPC module. Acquired data were processed and analysed using in-house-developed software (Benda et al., 2015 (link)).

The TR-NIRS system was built in-house using a pulsed diode laser (LDH-P-C-810; PicoQuant, Germany) connected to a PDL 828 laser driver (PicoQuant). The laser emission was centered at 805 nm and the pulse repetition rate was set to 80 MHz. The laser output was coupled into an emission fiber ( $\varphi =400\mu \mathrm{m}$ , $\mathrm{NA}=0.22$ ; Fiberoptics Technology, Pomfret, Connecticut) that guided light to the ankle joint. Light transmitted through the ankle joint was collected with a fiber optic bundle ( $\varphi =3\mathrm{mm}$ , $\mathrm{NA}=0.55$ ; Fiberoptics Technology) that was coupled to a hybrid photomultiplier detector (PMA Hybrid; PicoQuant) and a time-correlated single photon-counting module (HydraHarp 400; PicoQuant). A three-dimensional (3-D)-printed probe-holder was used to ensure good contact between the ankle joint and the emission and detection probes. Instrument response functions (IRFs) were acquired by placing the emission and detection probes into a light-tight box containing a piece of paper, positioned 2 mm away from the emission fiber, which acted as a light diffuser to fill the numerical aperture of the detection probe.