Summary We report here the development of a method for holding the focal plane in a fluorescence-based biochip scanner. The fast read-out of large (multiple cm2) 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. Various long- and short-term effects disturb the often multiple hour-long data-acquisitioning process and cause blurred or unusable image data. Traditional focus-hold systems were often limited in terms of range, reaction time, sensitivity or demanded a large number of additional components. Our system uses the back-reflected illumination beam always present in total internal reflection fluorescence 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-mm2 glass slides with single-molecule sensitivity over the whole array. Single-fluorescence dyes are imaged as diffraction-limited spots.
- Focus-hold system
- Single-molecule microscopy
- Oligonucleotide Array Sequence Analysis/methods
- Time Factors
- Data Collection/methods