FLIM imaging was performed using a Nikon Eclipse Ti A1R microscope equipped with a Time Correlated Single-Photon Counting module from PicoQuant59 . Excitation was performed using a pulsed 485nm laser (PicoQuant, LDH-D-C-485) operating at 20 MHz, and emission signal was collected through a bandpass 600/50nm filter using a gated PMA hybrid 40 detector and a TimeHarp 260 PICO board (PicoQuant). SymPhoTime 64 software (PicoQuant) was then used to fit fluorescence decay data (from full images or regions of interest) to a dual exponential model after deconvolution for the instrument response function (measured using the backscattered emission light of a 1µM fluorescein solution with 4M KI). Data was expressed as means ± standard deviation of the mean. The full width at half-maximum (FWHM) response of the instrument was measured at 176 ps.
Supplementary figure 1a shows all parameters extracted from the fits of FliptR in various GUV compositions. As similar tendencies were seen for τ1and τ2, the longest lifetime (τ1) obtained by the double exponential fits was used for all subsequent graphs.
Time correlated single photon counting module
Manufactured by PicoQuant
Sourced in Germany
The Time Correlated Single-Photon Counting (TCSPC) module is a lab equipment designed for the precise measurement of photon arrival times. It can be used to analyze the temporal characteristics of light signals, such as fluorescence decay or phosphorescence lifetimes. The module records the time difference between the excitation pulse and the detection of a single photon, enabling high-resolution time-resolved measurements.
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6 protocols using time correlated single photon counting module
1
FLIM Imaging of Membrane Probes
2
Fluorescence Decay Dynamics Analysis
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The fluorescence decay dynamics were acquired using a Nikon A1 laser scanning microscope with a PicoQuant time-correlated single photon counting module. EBr was excited by a 510 nm pulsed laser at 4 MHz repetition frequency. Laser power was 1,891 μW as measured at the back aperture of the objective. Emitted fluorescence photons were collected using a 60x water immersion objective (NA = 1.27) and directed to an avalanche photo detector (Excelitas Technologies), using a 594 long pass filter. An average of 2,000 counts were collected for every sample. Data was analysed using SymPhoTime 64 software and fitted using n-Exponential reconvolution module for 2 lifetime components. Lifetime component plots were prepared using SigmaPlot 14.
3
Membrane Tension Evaluation by FLIM
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Membrane tension was evaluated by FLIM of polarized CFTR-CTL and CFTR-KD cultures incubated for 1 h at the basal side with the FliptR fluorescent probe (1 μl/ml, SC020; Spirochrome) under controlled temperature and atmosphere (37°C, 5% CO2). FLIM imaging was performed using a Nikon Eclipse Ti A1R microscope equipped with a Time-Correlated Single-Photon Counting module from PicoQuant, as previously described (Colom et al, 2018 (link)), and a water immersion Apo LWD 40X/1.15 N.A. objective (Nikon). During acquisitions, cells were maintained at 37°C and 5% CO2 with a micro-incubator (Okolab). FliptR fluorescence lifetime was determined for at least 10 positions, always at the same height within each Transwell filter. SymPhoTime 64 software (PicoQuant) was used to fit fluorescence decay data (from full images or regions of interest) to a dual exponential model after deconvolution for the instrument response function (measured using the backscattered emission light of a 1 μM fluorescein solution with 4M KI). Data were expressed as the mean ± SD of the mean.
4
FLIM Imaging of Membrane Probes
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FLIM imaging was performed using a Nikon Eclipse Ti A1R microscope equipped with a Time Correlated Single-Photon Counting module from PicoQuant59 . Excitation was performed using a pulsed 485nm laser (PicoQuant, LDH-D-C-485) operating at 20 MHz, and emission signal was collected through a bandpass 600/50nm filter using a gated PMA hybrid 40 detector and a TimeHarp 260 PICO board (PicoQuant). SymPhoTime 64 software (PicoQuant) was then used to fit fluorescence decay data (from full images or regions of interest) to a dual exponential model after deconvolution for the instrument response function (measured using the backscattered emission light of a 1µM fluorescein solution with 4M KI). Data was expressed as means ± standard deviation of the mean. The full width at half-maximum (FWHM) response of the instrument was measured at 176 ps.
Supplementary figure 1a shows all parameters extracted from the fits of FliptR in various GUV compositions. As similar tendencies were seen for τ1and τ2, the longest lifetime (τ1) obtained by the double exponential fits was used for all subsequent graphs.
5
Fluorescence Lifetime Imaging Microscopy
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The FLIM system (LSM kit, Picoquant, Berlin, Germany) was incorporated into the Olympus IX70 and consisted of a pulsed diode laser (485 nm, FWHM = 83 ps, 20 MHz) and was equipped with a time-correlated single-photon counting module from PicoQuant (Berlin, Germany). The emission signal was collected through a band-pass emission filter centered at 510 nm (FWHM = 20 nm) using single-photon avalanche diode detectors (SPADs, PicoQuant, Berlin, Germany). Fluorescence lifetime measurements were acquired using software (SymPhoTime 64 version 2.0, PicoQuant) that controlled both the TCSPC system and the scanner system. Acquisition times were adjusted to achieve 1000 photons per pixel (with a typical acquisition time of 3 min). FLIM data were analyzed with a binning of 2 using the FLIMfit v5.1.1 software (https://flimfit.org/ , accessed on 15 January 2021), which uses a separable nonlinear least square algorithm to recover the lifetimes from the fluorescence decays [42 (link)]. The whole-cell lifetime intensity decays were fitted with the mono-exponential decay function deconvoluted with the instrument response function (IRF) to generate FLIM images showing the apparent lifetime of each pixel. The reduced chi-squared (χ2) value from the mono-exponential function is sufficient to describe the lifetime data in the present study.
6
FliptR Lifetime FLIM on Living Cells
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FLIM measurement of FliptR lifetime on living cells were performed as previously reported 23 (link) .
Briefly, HeLa cells were seeded on nanostructured SiO2 structures overnight as stated above.
Then, the culture medium was replaced with the same medium containing 1.5 µM of the FliptR probe and incubated for 10 min. FLIM imaging was performed using a Nikon Eclipse Ti A1R microscope equipped with a Time Correlated Single-Photon Counting module from PicoQuant.
Excitation was performed using a pulsed 485 nm laser (PicoQuant, LDH-D-C-485) operating at 20 MHz, and emission signal was collected through a bandpass 600/50 nm filter using a gated PMA hybrid detector and a TimeHarp 260 PICO board (PicoQuant). The duration of FLIM experiments was ~ 15s. SymPhoTime 64 software (PicoQuant) was then used to fit the fluorescence decay data (from either full images or regions of interest) to a dual exponential model after deconvolution for the instrument response function.
Briefly, HeLa cells were seeded on nanostructured SiO2 structures overnight as stated above.
Then, the culture medium was replaced with the same medium containing 1.5 µM of the FliptR probe and incubated for 10 min. FLIM imaging was performed using a Nikon Eclipse Ti A1R microscope equipped with a Time Correlated Single-Photon Counting module from PicoQuant.
Excitation was performed using a pulsed 485 nm laser (PicoQuant, LDH-D-C-485) operating at 20 MHz, and emission signal was collected through a bandpass 600/50 nm filter using a gated PMA hybrid detector and a TimeHarp 260 PICO board (PicoQuant). The duration of FLIM experiments was ~ 15s. SymPhoTime 64 software (PicoQuant) was then used to fit the fluorescence decay data (from either full images or regions of interest) to a dual exponential model after deconvolution for the instrument response function.
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