Development of Methods for Structure and Function Determination in Living and Fixated Cells on the Single-Molecule Level Based on Coincidence Analysis and Spectrally-Resolved Fluorescence Lifetime Imaging Microscopy [SFLIM]

The proceeding evolution in molecular biology and biochemistry led to groundbreaking results in the recent years, like the mapping of the human genome. The consequence of the rising knowledge of biological structures and mechanism is that gradually smaller and infrequent units, which are not resolvable by common methods anymore, are subject to investigation. In principle there are two question in the structural exploration of biological systems: Where are the single components localized, or what distances do they have in respect to each other, and from which or how many units are they composed? To solve these questions single-molecule spectroscopy is an excellent tool. The localization of dye-labeled biomolecules is easy, as long as the distance between the single fluorophores exceeds the optical diffraction limit of about 200 nm. For distances between 1 and 10 nm the FRET-effect can be exploited. In the intermediate range of 10 to 200 nm, the so-called resolution gap, only few methods for distance determinations are available, which are usually technically demanding and limited to two dimensions. Since many biological relevant molecules, for example biomolecular machines, are exactly in this order of magnitude, it is of major importance to have a simple 3-dimensional method at hand, which closes the gap. For this purpose an algorithm based on confocal imaging microscopy has been developed, which facilitates the separation of colocalized dyes by their fluorescence lifetimes and spectral characteristics. The accuracy and applicability of the method was in this work using biological calibration compounds. Therefore DNA molecules of different lengths, whose double-stranded backbone is known to be very rigid, were terminally labeled with the dyes Bodipy 630 and Cy 5.5 and immobilized in a 3-dimensional matrix, a cell-like but homogenous inclusion reagent. Comparison with "worm-like chain" model calculations showed that the measured lengths were in good agreement with the model. Furthermore, measurements in cells were accomplished, which affirmed the suitability of the method in biological environment. Beside the localization of biomolecules more and more quantitative investigations of complex cellular units come to the fore. Often the matter is not exclusively anymore the determination of various subunits, which can be discriminated against each other by different dyes, but rather the detection of identical molecules, which assemble or are generated within a cell compartment. For example the read-out and transduction of the genetic information by polymerases, the transcription, takes place in so called transcription factories. A typical HeLa cell contains about 8.000 of such 40 to 80 nm sized centers each containing on average 8 polymerase II enzymes. The reason for the accumulation, as well as the exact number of polymerases, could not be determined so far due to a lack of suitable techniques. However, for the comprehension of the cell function it is of great importance to study these basic units. The first step in this direction, the counting of polymerase II molecules in transcription sites, ought to be conducted in the second part of this work. To be able to quantify colocalized molecules, the analysis of interphoton times deduced from antibunching experiments can be used. Therefore dyes are located in a microscopic image, subsequently singly positioned in the laser focus and the fluorescence is collected until photodestruction. Especially the carbopyronine derivatives Atto 620 and Atto 647 turned out to be best suitable for the experiments because of their high photostability and emission rate. To investigate the applicability of the method in cellular environment, dye labeled oligomers consisting of 40 thymines were incorporated into cells. It was shown that these units selectively and partly multiply hybridize to the up to 200 basepair long adenosine ends of mRNA. By coincidence it was possible to analyze up to four molecules in a single image spot. To reduce the density of the transcription centers for imaging and to enable molecule counting for the 3.000 transcription factories per nucleus, so called "cryosections", cell slices with a thickness of 100 nm, were introduced. The simplest method to label polymerase II molecules uses specific dye labeled antibodies, which singly bind to the polymerases. A fundamental requirement for the success of the experiment is a stoichiometric labeling of the antibodies with the dyes, i.e. no multiply- or unlabeled compounds are allowed to be present. Therefore a new method was developed, which allows preparing one to one labeled proteins and quantum dots by the introduction of an affine group at the dye. It could be shown that the antibodies selectively bind to their targets and first experiments with these probes towards the success of the experiment could be initiated