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Multi-spot live-image autofocusing for high-throughput microscopy of fluorescently stained bacteria


Zeder, M; Pernthaler, J (2009). Multi-spot live-image autofocusing for high-throughput microscopy of fluorescently stained bacteria. Cytometry Part A, 75(9):781-788.

Abstract

Screening by automated high-throughput microscopy has become a valuable research tool. An essential component of such systems is the autonomous acquisition of focused images. Here we describe the implementation of a high-precision autofocus routine for imaging of fluorescently stained bacteria on a commercially available microscope. We integrate various concepts and strategies that together substantially enhance the performance of autonomous image acquisition. These are (i) nested focusing in brightfield and fluorescence illumination, (ii) autofocusing by continuous life-image acquisition during movement in z-direction rather than at distinct z-positions, (iii) assessment of the quality and topology of a field of view (FOV) by multi-spot focus measurements and (iv) acquisition of z-stacks and application of an extended depth of field algorithm to compensate for FOV unevenness. The freely provided program and documented source code allow ready adaptation of the here presented approach to various platforms and scientific questions.

Screening by automated high-throughput microscopy has become a valuable research tool. An essential component of such systems is the autonomous acquisition of focused images. Here we describe the implementation of a high-precision autofocus routine for imaging of fluorescently stained bacteria on a commercially available microscope. We integrate various concepts and strategies that together substantially enhance the performance of autonomous image acquisition. These are (i) nested focusing in brightfield and fluorescence illumination, (ii) autofocusing by continuous life-image acquisition during movement in z-direction rather than at distinct z-positions, (iii) assessment of the quality and topology of a field of view (FOV) by multi-spot focus measurements and (iv) acquisition of z-stacks and application of an extended depth of field algorithm to compensate for FOV unevenness. The freely provided program and documented source code allow ready adaptation of the here presented approach to various platforms and scientific questions.

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37 citations in Web of Science®
35 citations in Scopus®
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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Department of Plant and Microbial Biology
Dewey Decimal Classification:580 Plants (Botany)
Language:English
Date:2009
Deposited On:19 Jan 2010 18:18
Last Modified:05 Apr 2016 13:46
Publisher:Wiley-Blackwell
ISSN:0196-4763
Additional Information:The definitive version is available at www3.interscience.wiley.com
Publisher DOI:10.1002/cyto.a.20770
PubMed ID:19658173
Permanent URL: http://doi.org/10.5167/uzh-27879

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