Two-Stage Focus Hold System for Rapid Ultra-sensitive Readout of Large Area Biochips

We report here the development of a method for holding the focal plane in a fluorescence based biochip scanner. The fast readout of large (multiple cm 2 ) glass slides as used in modern chip technology imposes severe constraints on the focal system. The limited focal depth of high-NA objectives together with the demand for single molecule sensitivity challenges traditional focus hold systems (FHS). Various long and short term effects disturb the often multiple hour long data acquisitioning process and cause blurred or unusable image data. Traditional FHS's were often limited in terms of range, reaction time, sensitivity or demanded a large number of additional components. Our system utilizes the back reflected illumination beam always present in total internal reflection fluorescence (TIRF) microscopy to generate an error proportional electrical signal, which in turn drives an actuator correcting the objective – sample distance. The latter consists of a fast but range-limited piezo drive attached to the objective and a slower motor coupled to the microscope's z-drive. With this combination fast reaction times and virtually unlimited correction distances are possible. We show the applicability by scanning DNA microarrays on 27×18 mm 2 glass slides with single molecule sensitivity over the whole array. Single fluorescence dyes are imaged as diffraction limited spots.