Entwicklung und Untersuchung von Sonden für Mikroskopie zur hochaufgelösten Kolokalisation

Biebricher A. Development and investigation of probes for high-resolution colocalization microscopy. Bielefeld (Germany): Bielefeld University; 2006.The presented work is centered on the advancement of high-resolution microscopy using spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM). This technique allows to circumvent the intrinsic limitation that due to the wavelike nature of the light no structures below 200nm can be resolved with conventional fluorescence microscopy. This seriously corrupts its application to the study of single biomolecule imaging within a cell since binding events take place at distances below 50nm. The high resolution can be achieved employing different types of luminescent labels which can be distinguished from each other both by their spectral and lifetime characteristics. The work is divided into two parts: In the first part, gold surfaces patterned by electron-beam lithography were tested concerning an application as substrates for a Cellular Positioning System (CPS). This system is analogous to the Globular Positioning System (GPS) in that it uses at least three differently labeled spots positioned with nm-accuracy on a surface to follow the absolute trajectories of single fluorescent molecules. The use of gold as surface for fluorescent probes has the disadvantage that strong quenching of the dyes occurs. Therefore, a modification scheme was developed which enables a controlled modification of the surface with fluorescently labeled proteins. The protein labels used are efficient spacers to reduce the quenching effect of the gold and to facilitate immobilization of further protein layers. Additionally, an organic linker was used to enhance the protein binding efficiency to the gold and to further increase the distance from the gold surface to the dyes. By controlled growing of several layers of organic linker and subsequent layers of protein, it could be demonstrated that via this method high signal to background ratios can be obtained even for structures smaller than 100 nm. In a second set of experiments, semiconductor nanocrystals were investigated concerning an application as probes for high-resolution microscopy. For this purpose, the photophysical fluctuations were observed with the aid of a SFLIM set-up. Three different samples consisting of commercially available NCs were investigated emitting at 605, 655 and 705 nm (denoted QD605, QD655 and QD705), respectively, which are made up of CdSe (QD605, QD655) or CdTe (QD705), covered with a ZnS shell and modified with strepavidin. It could be demonstrated that the set-up allows for the determination of photophysical fluctuations of single NCs with a time resolution down to 1 ms. By calibration and test measurements it could be verified, that in this fashion, photoluminescence intensity, lifetime and spectral fluctuations can be determined with high accuracy. By correlation of the observables an interrelation between spectral diffusion and lifetime fluctuations caused by changes of the radiative rate was uncovered. This novel connection was exploited to calculate radiative rate fluctuations and photoluminescence quantum yields of single NCs. In addition, a novel subpopulation of quenched emission could be uncovered indicating NC transition into a fundamentally different state. The obtained data was furthermore used to construct density plots containing all three observables which are characteristic for a certain type of NC and can also be used for NC assessment. Finally, with the aid of the developed depiction method it could be shown that anti-blinking agents lead to a significant acceleration of photophysical fluctuations. The set-up was also employed in a modified form to investigate the antibunching characteristics of the NC samples. In particular, it was demonstrated that the NC sample of type QD655 display significant biexciton emission. A novel analytical method was presented which allows for the unequivocal extraction of photons stemming from biexciton emission. This was used to calculate the biexciton decays for single NCs which was shown to vary significantly from NC to NC. For a given NC, it was also found for the first time that the relative biexciton quantum yield compared to the exciton is not constant but shows a strong increase for quenched states. This finding could be confirmed by spectrally resolved investigation of triexciton emission, which can also be used to extract the lifetime information of triexciton emission