Supercritical angle fluorescence (SAF) microscopy.

We explore a new confocal microscope for the detection of surface-generated fluorescence. The instrument is designed for high resolution imaging as well as for the readout of large biochips. Special feature is the separated collection of two different fluorescence emission modes. One optical path covers the emission into the glass at low surface angles, the other captures high angles, exceeding the critical angle of the water/glass interface. Due to the collection of the supercritical angle fluorescence (SAF) the confocal detection volume is strictly confined to the interface, whereas the low angles collect much deeper from the aqueous analyte solution. Hence the system can deliver information about surfacebound and unbound fraction of fluorescent analyte simultaneously.