Two-stage focus-hold system for rapid ultra-sensitive read-out of large-area biochips

Jaroslaw Jacak, Jan Hesse, Clemens Hesch, Gerhard Schütz

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)251-254
Number of pages4
JournalJOURNAL OF MICROSCOPY-OXFORD
Volume234
Issue number3
DOIs
Publication statusPublished - Jun 2009

Keywords

  • Fluorescence
  • Focus-hold system
  • Microarray
  • Single-molecule microscopy
  • Oligonucleotide Array Sequence Analysis/methods
  • Time Factors
  • Data Collection/methods

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