Fluorescence Lifetime Imaging System For Viscosity Detection In Microenvironment And Its Application

Traditional fluorescence microscopy can only qualitatively provide the spatial distribution of fluorescent molecules depending on the differences of fluorescent spectra or fluorescence intensity.Comparably,fluorescence lifetime imaging microscopy(FLIM)shows unique advantages,for example,fluorescence lifetime is independent of excitation light intensity,photobleaching and concentration of probes,only dependent on the energy level of fluorescent probes.The fluorescence lifetime can reflect the local environment surrounding the fluorescent probe,and the quantitative measurement of microenvironmental parameters can be attainable.Fluorescence lifetime can be acquired by various methods,such as time correlated single photon counting based on time-domain FLIM technique(TCSPC-FLIM),and time-gate based FLIM.Among these microscopies,TCSPC-FLIM is presently most well-developed and widely used method in fluorescence lifetime imaging microscopy.This microscopy possesses high sensitivity for single photon detection,temporal resolution and signal-to-noise ratio,displaying obvious advantages over other techniques.Therefore,it can be used for quantitative imaging and measurement of biochemical and physical parameters in the microenvironment of cells or other samples,including viscosity,oxygen pressure,temperature,ion concentration,molecular polarity,p H,etc.In biological microenvironment,fluid viscosity largely determines the transportation of intracellular masses,signal transduction,interactions between biological macromolecules and diffusion of metabolites.Abnormal changes of cellular viscosity become a hallmark of occurrence of diseases or early pathological state.In the process of endoplasmic reticulum,microviscosity also exerts great functions in the endoplasmic reticulum autophagy,which is a highly selective form of autophagy.This process is relevant to the maintenance of cellular homeostasis of unfolded proteins.Endoplasmic reticulum autophagy plays an important role in the quality control of endoplasmic reticulum proteins.Many pathophysiological processes and seriousdiseases can cause protein folding or misfolding in endoplasmic reticulum,such as tumorigenesis,neurodegenerative diseases,diabetes mellitus,etc.The accumulation of unfolded or misfolded proteins in endoplasmic reticulum may induce endoplasmic reticulum stress response.Endoplasmic reticulum autophagy is able to alleviate endoplasmic reticulum stress and maintain cellular normal functions.Therefore,a comprehensive understanding of microviscosity in endoplasmic reticulum autophagy can provide a promising method for the diagnosis and precaution of such diseases.With above considerations,an imaging system capable of measuring both fluorescence intensity and fluorescence lifetime will be of great helpful for the detection of microenvironmental parameters.In this thesis,a confocal-FLIM imaging system based on TCSPC FLIM technology are constructed.And based on the home-built FLIM system,the feasibility of dual-mode microenvironment detection and the accuracy of local microenvironment viscosity change detection during endoplasmic reticulum autophagy are studied.The major work in this thesis shown as follows:1.Based on laser scanning confocal microscopy and TCSPC technique,a confocal-FLIM imaging system was built,and the reliability of the system was calibrated by the FLIM imaging of a standard specimen(Convallaria rhizome slide).In the meanwhile,the analytical method of fluorescence lifetime data was introduced in detail.2.Study on the optical features of fluorescent probes with two emission peaks,a dual-mode microenvironment viscosity detection method is proposed.This method can detect microenvironment viscosity by ratiometrically fluorescent imaging and fluorescence lifetime imaging.The results obtained by each mode are mutually supportive,which can further improve the detection accuracy and precision.3.With the bifunctional fluorescent probe,we proved the feasibility of the probes in ER autophagy process.The exponential function relationship between viscosity and lifetime was obtained from above results.Finally,local viscosity change during endoplasmic reticulum autophagy was successfully detected on confocal-FLIM imaging system.The innovation involved in this thesis are itemed as follows:1.According to the principle of laser scanning confocal microscopy and TCSPC technique,a setup of confocal-FLIM imaging system was built,which features with low cost,easy upgrade and extension in multifunctional measurement;and it can be further updated or administered at any time in view of hardware damage or aging.2.A dual-mode of viscosity imaging method was proposed to image microviscosity ratiometrically fluorescent imaging and fluorescence lifetime imaging,of which the results are mutually calibrated and can further improve the viscosity detection accuracy and precision.3.Local viscosity changes during endoplasmic reticulum autophagy were successfully detected on our home-built Confocal-FLIM imaging system.This application can provide a quantitative method for detecting dynamic autophagy in living cells.At the same time,the fluorescent probe is expected to be a useful and reliable tool for studying endoplasmic reticulum autophagy.