Genomic research is nowadays based on high throughput analytical techniques. Microarray assays are commonly used to determine DNA content of heterogeneous mixtures up to full genome scale. For low amounts of sample material this method, however, requires time consuming and error prone PCR based amplification steps. Here, we present an assay with the ability to characterize the cDNA content of a low number of cells using ultra-sensitive fluorescence microscopy.
For detection, a newly developed chip reader was used. The instrument is based on a modified fluorescence microscope with single dye sensitivity. The highly sensitive CCD detector is operated in TDI mode, which allows avoiding overhead times for sample positioning and signal integration. This enabled the scanning of areas of 1x0.2cm2 within 50 seconds at a pixel size of 200nm. At this resolution, single dye molecules can be reliably detected with an average signal to background noise ratio of ~42. For DNA hybridization experiments, oligonucleotides were covalently linked to a newly developed aldehyde surface. Subsequently, fluorescence labeled complementary oligonucleotides were hybridized at various concentrations. Down to femto-molar oligonucleotide concentrations, specific signals were detected. At 10fM concentration signals of individual specifically hybridized oligonucleotide molecules were resolvable. This assay provides the conceptual basis for expression profiling of low amounts of sample material without signal amplification.