Spcimage software

An LSM 710 (Carl Zeiss, Germany) fluorescence confocal laser-scanning microscope equipped with a femtosecond Ti:Sa laser with a repetition rate of 80 MHz and pulse duration of 140 fs, and an FLIM module based on time-correlated single photon counting - Simple Tau 152 TCSPC (Becker & Hickl GmbH, Germany), were used to obtain one- and two-photon fluorescence and FLIM images of the cultured cells. A water immersion objective С-Apochromat 40x/1.2 NA W Korr was used for image acquisition. During image acquisition, the cells were maintained at 37 °C and 5% CO₂.
For two-photon fluorescence microscopy and FLIM of tumors in vivo an MPTflex (JenLab GmbH, Germane) multiphoton tomograph, equipped with a tunable 80 MHz, 200 fs MaiTai Ti:Sa laser and a TCSPC-based FLIM module (Becker & Hickl GmbH, Germany) were used. The images were acquired through a 40x/1.3 NA oil immersion EC Plan-Neofluar objective.
ImageJ 1.39p software (NIH, USA) was used for fluorescence image processing. Analysis of the FLIM data was performed using SPCImage software (Becker & Hickl GmbH, Germany).

Cells were imaged in 8-well plates on a inverted TCS-SP2 microscope (Leica Microsystems, Germany) and SPC-150 TCSPC boards (Becker & Hickl, Germany). FMR-1 and FMR-2 were excited using a picosecond-pulsed (90 ps optical pulse width, 20 MHz repetition rate) 467 nm diode laser (Hamamatsu, Japan) through a 63X 1.2 NA water objective, with a 485 nm dichroic mirror and a 500 long pass filter. All imaging was done at room temperature. Each FLIM image was acquired over 900 s to ensure as little noise as possible (much shorter acquisition rates are possible). Lifetime histograms and FLIM images were analysed using SPCImage software (Becker & Hickl, Germany).

Two-photon in vivo imaging was performed with a Dermainspect (JenLab GmbH, Jena, Germany) device equipped with a tunable femtosecond Ti:sapphire laser (Mai Tai XF, Spectra Physics, USA). The laser was operated at 760 nm and generated 100-fs pulses at a repetition rate of 80 MHz. The 410–680 nm bandpass filter was used to detect autofluorescence.
FLIM images were processed in the SPCImage software (Becker&Hickl, Berlin, Germany) incorporated into the Dermainspect system. Fluorescence decay in each pixel was fitted with a sum of two exponentials (fast and slow) with a fixed shift value, and the intensity threshold was chosen depending on the image quality. The obtained lifetime (τ₁ and τ₂) and amplitude (a₁ and a₂) values were further exported and used for the evaluation of lifetime distributions and image segmentation. The average lifetime was defined as τ_m = (a₁τ₁ + a₂τ₂)/(a₁ + a₂). All the images were built using the ImageJ software. The utilized MPT-FLIM system has been previously presented in details elsewhere^{75 (link)}.