The Development Of Compact CW-STED System And Investigation On Fluorescent Probe Characteristics

In the research of life sciences and other fields,optical microscopy is the most widely used detection technique due to its non-invasive and low damage characteristics.Limited by Abbe’s diffraction limit,the spatial resolution of traditional optical microscope is over 200nm.However,many biological and biomedical targets,e.g.microtubules,has featured scales below 100 nm,which is beyond the capability of traditional optical methods.Therefore,breaking the limit of the optical diffraction has become the top priority of developing novel optical detection techniques.At present,stimulated emission depletion microscopy(STED)has played an important role in biomedical research,with its features like far-field super-resolution detection,multi-dimensional imaging in vivo and the capability of detecting targets with fluorescent markers.In the PhD research,a compact STED system based on continuous wave(CW)lasers at wavelengths of 405 nm/532 nm was built and used to comprehensively study spectroscopy as well as fluorescence dynamics for novel fluorescent markers,e.g.fluorescent dye and quantum dots.Besides,different biological organizations have also been imaged with different fluorescent markers.The investigations are summarized in the following.1.Construction and optimization of the compact STED system based on the 405 nm/532nm CW laser(CW-STED).By applying fast response acousto-optic modulators,precisely controlling the laser beams can be realized on the order of nanoseconds.Therefore,the influence of photobleaching can be significantly inhibited.By optimizing the synchronization of acousto-optic modulator,single photon counter and piezoelectric translation stage,high-speed scanning imaging can be achieved.A method for calibration of focal spots of excitation and depletion beams is optimized.The CW STED system has been optimized and integrated into the compact system in order to significantly enhance working stability.2.Study the fluorescence quenching rate of Coumarin 102 in CW-STED system.First,based on fluorescence lifetime,quenching rate and vibrational relaxation rate,a fluorescence dynamic model for CW-STED which is statistical equilibrium was established.Then,the suppression factor of Coumarin 102 was measured with the CW-STED system,to evaluate the fluorescence quenching rate on the basis of the fluorescence dynamic model.Experimental results indicated that due to the presence of aggregation-caused quenching(ACQ),quenching rate of Coumarin 102 dye increases with the increasing concentrations.A depletion induced quenching phenomenon(DIQ)was also found in which fluorescence quenching rate significantly increases with the increasing power of the depletion beam.The DIQ phenomenon can be attributed to the enhanced collision caused by local temperature increment due to the increased power of depletion beam.DIQ can affect suppression effect and inhibit the improvement of spatial resolution of CW-STED system by increasing power of depletion beam.3.Study the STED properties of new quantum dots.The 405 nm/532 nm CW-STED system is rarely used in practice.One of the primary reasons is the lack of applicable fluorescent marker with low saturation intensity.In the 405 nm/532 nm CW-STED system,the depletion characteristics of three carbon quantum dots and two CdSe quantum dots are systematically investigated.In different organic solvents,due to the influence of the polarity of solvent molecules,the depletion curves and saturation intensity of carbon quantum dots vary apparently.The saturation intensity of the CDs-2 in the alcohol solvent is the smallest.In the 492/10 nm fluorescent band,the CDs-2 exhibits an ultra-low saturation intensity of 0.4 MW/cm~2,making it a potential STED fluorescent marker for 405nm/532 nm STED system.The CdSe quantum dots exhibits a phenomenon of up-conversion luminescence,which is potentially applied for fluorescence difference microscopy.4.Super-resolution fluorescent imaging of biological targets.This manuscript describes the preparation procedures of various quantum dots,mouse brain cell slices labeled by Coumarin 102 and neuroblastoma cell slices labeled by Atto 390 Phalloidin in detail.In the405 nm confocal fluorescence microscope,180 nm resolution has been achieved using the CDs-2 carbon dot.The CdSe quantum dots have strong fluorescence emission and up-conversion luminescence phenomenon,which can be used to develop new probes for fluorescence difference microscopy.The Coumarin 102 dye has a certain biological compatibility but cannot specifically label proteins.The Atto 390 Phalloidin can be specifically combined with F actin in neuroblastoma cells by immunofluorescence labeling.With the Atto 390 Phalloidin,the 405 nm/532 nm CW STED system achieves a spatial resolution of 120 nm.The investigations provide additional options for STED in life science research.