Advancement of single molecule microarray scanner towards imaging cytometry

Single molecule sensitive fluorescence microscopy is currently one of the major techniques in biological and medical research. New applications in the area of cytometry demand fast, stable, and reliable methods to acquire large amounts of (image) data for successful statistical analysis. In this work I addressed two major limitations: restricted time reference in multi-color scanning and blurred scan images. After a general survey over fluorescence microscopy I give a description of limiting technical parameters various commercial and scientific biochip-readers suffer from. An in-depth discussion of relevant physical and technical aspects leads to the concept of an advanced fluorescence scanner whose implementation details are also provided. The above mentioned limited time-reference of 'classical' scanning was addressed by a multi-color alternating excitation approach. Through rapid switching between excitation wavelengths, synchronized sample movement, and image readout multiple tightly time-referenced scan-images in different excitation channels can be obtained. This method provides single molecule sensitivity combined with sharp mapping of moving particles (D ∼0.5μm2/s) as found in lipid bilayers or living cells. Also a localization precision of point light sources down to ∼10 nm is shown. Furthermore the fast imaging of large glass-slides as used in a number of biochip-applications usually suffers from blurring. Thus, a stable method for keeping the specimen in the focal plane is required. The implemented novel approach utilizes the totally reflected excitation laser beam as error indicator. Together with a two-stage (discrete coarse and analog fine) objective z adjustment a fast and sensitive control loop is formed which keeps an initially set sample-objective distance within the limited focal depth of a high-NA objective (usually some hundreds of nanometers) while scanning a multiple cm2 biochip with single molecule sensitivity. Finally, this thesis contains two applications of the new technologies. Firstly, the method was optimized to enable micropatterning studies for measuring protein-protein interaction in the live cell plasma membrane. Microstructured surfaces functionalized with fluorophore-labeled antibodies were brought in contact with living cells. The redistribution of the associated membrane-proteins was recorded on large chip-areas with the new scanner. Secondly, to successfully apply fluorescence imaging in cytometry it is also necessary to discriminate mobile fluorophores used as markers in cells against static (dirt) particles. Through the introduction of asynchronous time-delay and integration (TDI) mode we could record the time course of the fluorescence signal. Combined with rotating the polarization excitation a characterization of absorption dipoles is possible. This allowed e.g. distinguishing proteins with average labeling of 0.55 versus 2.89 fluorophores. (author)