Univ.-Prof. Dr. Peter Hinterdorfer
July 20, 2004
Within the presented work, a new type of fluorescence reader has been developed. The instrument is specifically designed for fast scanning of biochips at the sensitivity of single dye molecules. It is based on a conventional epi-microscope, which is equipped with a high precision xy-stage that allows for sample positioning with an accuracy in the 100nm range. To ensure high light collection efficiency, objectives with high numerical aperture are used. Their short focal depth makes a good focussing essential for high quality imaging. For this purpose, a focus hold system has been implemented into the intrument. It utilises the reflection of a laser beam from the sample surface as control for a feed-back loop to the focussing unit. Fluorophores are excited by lasers, which allow for selective and efficient excitation of the fluorescent dyes cyanin 3 (Cy3) and cyanin 5 (Cy5). Proper beam shaping of the lasers prevents fluorescence excitation and photobleaching in areas that are not imaged onto the detector. Fluorescence light is detected on a CCD camera, which is operated in time delayed integration (TDI) mode. In this mode, sample shift and CCD readout are synchronised and overhead times during image acquisition minimised. Using this operation scheme, an area of 5x5mm2 can be imaged within 12 minutes at a pixel size of 200x200nm2 with single molecule sensitivity.
The function of the setup has been tested by measuring fluorescent signals from a lipid bilayer (dipalmitoyl-phosphatidycholine - DPPC) containing trace amounts of Cy5 labelled lipids (dipalmitoyl-phosphatidylethanolamine - DPPE-Cy5). Cy5 labelled lipids are clearly visible as diffraction limited spots with a signal to noise ratio of ~37. The constant peak width along the scan demonstrates proper function of the focus hold system and synchronisation of CCD camera and xy-stage.
One application of the system represents live cell screening. Here, the expression of the potassium channel KV1.3 was measured on a population of Jurkat cells. The ion channels of these T-lymphocytes are specifically addressed using Cy5 labelled Hongotoxin HgTX1-A19C-Cy5 under saturating incubation conditions. The number of KV1.3 channels was determined by directly counting single molecule signals on each cell. A broad distribution of 0 to 400 channels per cell was found.
The applicability of the microscope in bioanalysis is shown on a biochip-based protein detection system. In a sandwich fluorescent linked immunosorbent assay (sandwich FLISA) the human interleukine hIL-2 was detected down to concentrations of 5pg/ml without signal amplification.
Additionally, an experiment, which combines for the first time optical and electrical single molecule detection, is presented. The effect of Förster resonance energy transfer (FRET) is utilised as a sensitive distance sensor in the nanometre range between Cy5 and Cy3 labelled gramicidin monomers. Occurrences of optically observed FRET events between individual pairs of gramicidin monomers have been correlated with simultaneously recorded electrical currents through the respective ion channels. The results demonstrate the feasibility of correlated optical and electrical measurements on single ion channels.