Fluorescence instrument response standards in two-photon time-resolved spectroscopy

Rafal Luchowski, Mariusz Szabelski, Pabak Sarkar, Elisa Apicella, Krishna Midde, Sangram Raut, Julian Borejdo, Zygmunt Gryczynski, Ignacy Gryczynski

Research output: Contribution to journalArticlepeer-review

15 Scopus citations


We studied the fluorescence properties of several potential picosecond lifetime standards suitable for two-photon excitation from a Ti : sapphire femtosecond laser. The fluorescence emission of the selected tiuorophores (rose bengal, pyridine 1, and LDS 798) covered the visible to near-infrared wavelength range from 550 to 850 nm. We suggest that these compounds can be used to measure the appropriate instrument response functions needed for accurate deconvolution of fluorescence lifetime data. Lifetime measurements with multiphoton excitation that use scatterers as a reference may fail to properly resolve fluorescence intensity decays. This is because of the different sensitivities of photodetectors in different spectral regions. Also, detectors often lose sensitivity in the near-infrared region. We demonstrate that the proposed references allow a proper reconvolution of measured lifetimes. We believe that picosecond lifetime standards for two-photon excitation will find broad applications in multiphoton spectroscopy and in fluorescence lifetime imaging microscopy (FLIM).

Original languageEnglish
Pages (from-to)918-922
Number of pages5
JournalApplied Spectroscopy
Issue number8
StatePublished - Aug 2010


  • FLIM
  • Fluorescence lifetime imaging microscopy
  • IRF
  • Instrument response function
  • Lifetime standards
  • Two-photon excitation


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