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New Becker+Hickl ultra-fast detectors improve NADH FLIM

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Image: NADH Lifetime image, amplitude-weighted lifetime of double-exponential fit

NADH FLIM is based on the separation of the fluorescence decay components of the bound and the unbound fraction of NAD(P)H. The amplitudes and the decay times of the components are used to derive information on the metabolic state of the cells or the tissue. The separation of the decay components and the accuracy of the amplitudes and lifetimes improves substantially by using the ultra-fast HPM-100-06 and HPM-100-07 hybrid detectors. The IRF width in combination with the SPC-150N and SPC-150NX TCSPC modules is less than 20 ps. An IRF this fast does not interfere with the fluorescence decay. The usual deconvolution process in the data analysis virtually becomes a simple curve fitting, and the decay parameters are obtained at unprecendented accuracy.

A lifetime image of the amplitude-weighted lifetime of a double-exponential fit is shown below. The decay data in a selected spot of 9x9 pixels is shown on the right. Due to the fast response of the detector-TCSPC combination the rise of the fluorescence occurs almost instantaneously. Images of the amplitude ratio, a1/a2 (unbound/bound ratio), and of the fast (t1, unbound NADH) and the slow decay component (t2, bound NADH) are shown further below.

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Decay curve in selected spot, 9x9 pixel area.

 

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Images of the amplitude ratio, a1/a2 (unbound/bound ratio), and of the fast (t1, unbound NADH) and the slow decay component (t2, bound NADH). FLIM data format 512x512 pixels, 1024 time channels. Time-channel width 10ps. HPM 100 06 detector, SPC-150N TCSPC module, Zeiss LSM 880 NLO, two-photon excitation at 750 nm.

For further details please see application note ‘New ultra-fast detectors improve NADH FLIM’, please click here to download.

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