Studies On The New Standards For Characterizing The Response Function Of Time Resolved Fluorescence Instruments

As the development of physical and electronic technology, the development of time-resolved fluorescence technology has been promoted. As a result, kinds of standard instrument with capability of ultrafast time-resolved measurement are appearing one after another. Compared with the traditional steady-state fluorescence technique, time-resolved fluorescence can provide information of lifetime and the distribution and proportion of different fluorescence lifetime component in the life sciences, analytical chemistry, nano-science and other fields which has a wide range of applications. We all know that it is the most common method of improving the time resolution by measuring instrument response function and fitting the experimental data by convolution algorithm, so in this study, we use NaI to quench fluorescein sodium as a new method to obtain a short segment lifetime fluorescence emission in yellow and green wave band, which can be used to measure instrument response function of time-resolved fluorescence. We use this method of measuring instrument response function to fit the fluorescence lifetime of yellow fluorescent protein, green fluorescent protein fluorescence which has obtained satisfactory results. This method not only solves the problem of repeatedly measurement of IRF with scattering sample in different wavelength but also avoid the problem that the different colors of the IRF and fluorescence emission may cause a significant distortion (i.e. color effects) thus fail to properly resolve fluorescence intensity decays. The main study results are as follows:1. We obtained a good instrument response function by using NaI quenched fluorescein sodium which can used as an instrument response function standard in yellow and green wave band. And we improve the intensity of the fluorescent signal by improving the solubility of the dye. 2. We measured an accurate fluorescence lifetime of17ps for quenched fluorescein sodium with femtosecond frequency upconversion apparatus, this lifetime is much less than the response time of the photo detector which prove that this IRF standard can be used as an effective standard of the instrument response time measurements.3. We have measured the fluorescence decay spectra and lifetime of yellow fluorescent protein, green fluorescent protein with this instrument response function gained by new method of measurement. As a result, it improved time resolution of fluorescence imaging which lay a good foundation for the future study in the cell fluorescence imaging detection.