Fluoresence lifetime imaging (FLIM) was performed on an upright fluorescence microscope (A1MP; Nikon, Amsterdam, The Netherlands) equipped with a water immersion objective (25×; NA1.1; WD 2 mm; Nikon). Two-photon excitation of the pH-Lemon donor mT2 was achieved by a train of 100 fs light pulses (λexc = 880 nm; 80 MHz; Mai Tai DeepSee HP, Newport Spectra Physics; Irvine, CA). Fluorescence was detected with a GaAsP hybrid photodetector (HPM-100–40; Becker & Hickl, Berlin, Germany) after passing a bandpass filter at 445 ± 45 nm (445BP90, Omega Optical, Brattleboro, VT, USA). Fluorescence intensity decays were generated in every pixel of the image using multidimensional time-correlated single photon counting (TCSPC) employing TCSPC electronics (SPC-152; Becker & Hickl). FLIM images were generated using SPCImage 6.1 (Becker & Hickl) by plotting the amplitude weighted average fluorescence lifetime tau_ave as color-coded value. tau_mean was obtained from iterative least-squares minimizing based fitting routine of a biexponential fitting function which was reconvoluted with the instrument response function to describe the time course of the pixel fluorescence intensity decays properly.
Spcimage 6
Manufactured by Becker & Hickl
Sourced in United States
SPCImage 6.1 is a software package for data acquisition and analysis in time-resolved optical spectroscopy and microscopy applications. It provides tools for the control of hardware devices, data processing, and visualization.
Automatically generated - may contain errors
2 protocols using spcimage 6
1
Fluorescence Lifetime Imaging Microscopy Protocol
2
Two-Photon FLIM Imaging of pH-Lemon Fluorescence
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Fluoresence lifetime
imaging (FLIM) was performed on an
upright fluorescence microscope (A1MP; Nikon, Amsterdam, The Netherlands)
equipped with a water immersion objective (25×; NA1.1; WD 2 mm;
Nikon). Two-photon excitation of the pH-Lemon donor mT2 was achieved
by a train of 100 fs light pulses (λexc = 880 nm;
80 MHz; Mai Tai DeepSee HP, Newport Spectra Physics; Irvine, CA).
Fluorescence was detected with a GaAsP hybrid photodetector (HPM-100–40;
Becker & Hickl, Berlin, Germany) after passing a bandpass filter
at 445 ± 45 nm (445BP90, Omega Optical, Brattleboro, VT, USA).
Fluorescence intensity decays were generated in every pixel of the
image using multidimensional time-correlated single photon counting
(TCSPC) employing TCSPC electronics (SPC-152; Becker & Hickl).
FLIM images were generated using SPCImage 6.1 (Becker & Hickl)
by plotting the amplitude weighted average fluorescence lifetime tau_ave
as color-coded value. tau_mean was obtained from iterative least-squares
minimizing based fitting routine of a biexponential fitting function
which was reconvoluted with the instrument response function to describe
the time course of the pixel fluorescence intensity decays properly.
imaging (FLIM) was performed on an
upright fluorescence microscope (A1MP; Nikon, Amsterdam, The Netherlands)
equipped with a water immersion objective (25×; NA1.1; WD 2 mm;
Nikon). Two-photon excitation of the pH-Lemon donor mT2 was achieved
by a train of 100 fs light pulses (λexc = 880 nm;
80 MHz; Mai Tai DeepSee HP, Newport Spectra Physics; Irvine, CA).
Fluorescence was detected with a GaAsP hybrid photodetector (HPM-100–40;
Becker & Hickl, Berlin, Germany) after passing a bandpass filter
at 445 ± 45 nm (445BP90, Omega Optical, Brattleboro, VT, USA).
Fluorescence intensity decays were generated in every pixel of the
image using multidimensional time-correlated single photon counting
(TCSPC) employing TCSPC electronics (SPC-152; Becker & Hickl).
FLIM images were generated using SPCImage 6.1 (Becker & Hickl)
by plotting the amplitude weighted average fluorescence lifetime tau_ave
as color-coded value. tau_mean was obtained from iterative least-squares
minimizing based fitting routine of a biexponential fitting function
which was reconvoluted with the instrument response function to describe
the time course of the pixel fluorescence intensity decays properly.
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