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Pulsed Interleaved Excitation Fluorescence Lifetime Imaging Microscopy


The PIE-FLIM microscope is a widely applicable scanning confocal microscope. This home-built design is build around an Olympus IX 71 body. It is equipped with a 60x water objective (NA1.2) and Till Photonics Yanus IV galvo scanner unit. For widefield imaging, a triband LED source is available. 


Two quadband mirrors (405/485/560/635 and 440/510/560/635) allow us to accomodate all kinds of laser combinations for the most common fluorophores and FRET pairs. Pulsed Interleaved Excitation (PIE) is used to combine several pulsed lasers in one pulse cycle. Additionally, all incoming light is linearly polarized, enabling us to measure anisotropy at detection. Currently, the single photon detectors used, which sync to the 4-channel Time Correlated Single Photon Counting (TCSPC) device, are a mix of PicoQuant TauSPAD (high detection efficiency, low dark counts) and PicoQuant PMA hybrid (sharp IRF) detectors.


The most common applications of this microscope are: 

  • Confocal imaging


  • Time resolved fluorescence studies (lifetime)


  • Single molecule fluorescence

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