The fluorescence lifetime images were recorded using DCS-120 confocal laser scanning FLIM system based on a TCSPC module ( SPC-150 , Becker & Hickl , Berlin , Germany ) . The scanner was attached to an inverted microscope ( ECLIPSE TE2000 - E , Nikon , Japan ) . Single photon excited fluorescence from the sample was collected through a Plan APO 100×/NA1.4 oil immersion objective . The signals were detected by Hybrid Photon Detectors HPM-100 - 40 ( Becker & Hickl , Berlin , Germany ) that were connected to a TCSPC module ( Becker & Hickl , Berlin , Germany ) . The excitation source was a picosecond super continuum ( 400 - 650 nm ) laser with an acousto - optic tunable filter ( AOTF ) ( SC400 - 4 , Fianium , UK ) . The AlexaFluor 546 and Alexa Fluor 647 fluorescence were excited by 545 and 640 nm , respectively . In order to avoid significant pile - up error in all our measurements , the peak count rate never exceeded the recommended 10 % of the excitation rate . The fluorescence signals were collected using band - pass filters ET590/50 for AlexaFluor 546 , and ET700/75 for AlexaFluor546 and AlexaFluor647 ( Chroma Technology Corp. , Bellows Falls , USA ) . All images were acquired at 256 × 256 pixels . The signal acquisition time was around 60 s. The number of nuclei analyzed for each particular experiment was not less than 10 . The analysis of the FLIM data , including lifetime and FRET efficiency calculation was performed using SPCImage ( Becker & Hickl , Berlin , Germany ) as described in the manufacturer 's protocols 13 .