Share Email Print
cover

Proceedings Paper

In-focal-plane characterization of excitation distribution for quantitative fluorescence microscopy applications
Author(s): Klaus Dietrich; Martina Brülisauer; Emine Çağin; Dietmar Bertsch; Stefan Lüthi; Peter Heeb; Ulrich Stärker; André Bernard
Format Member Price Non-Member Price
PDF $14.40 $18.00
cover GOOD NEWS! Your organization subscribes to the SPIE Digital Library. You may be able to download this paper for free. Check Access

Paper Abstract

The applications of fluorescence microscopy span medical diagnostics, bioengineering and biomaterial analytics. Full exploitation of fluorescent microscopy is hampered by imperfections in illumination, detection and filtering. Mainly, errors stem from deviations induced by real-world components inducing spatial or angular variations of propagation properties along the optical path, and they can be addressed through consistent and accurate calibration. For many applications, uniform signal to noise ratio (SNR) over the imaging area is required. Homogeneous SNR can be achieved by quantifying and compensating for the signal bias. We present a method to quantitatively characterize novel reference materials as a calibration reference for biomaterials analytics. The reference materials under investigation comprise thin layers of fluorophores embedded in polymer matrices. These layers are highly homogeneous in their fluorescence response, where cumulative variations do not exceed 1% over the field of view (1.5 x 1.1 mm). An automated and reproducible measurement methodology, enabling sufficient correction for measurement artefacts, is reported. The measurement setup is equipped with an autofocus system, ensuring that the measured film quality is not artificially increased by out-of-focus reduction of the system modulation transfer function. The quantitative characterization method is suitable for analysis of modified bio-materials, especially through patterned protein decoration. The imaging method presented here can be used to statistically analyze protein patterns, thereby increasing both precision and throughput. Further, the method can be developed to include a reference emitter and detector pair on the image surface of the reference object, in order to provide traceable measurements.

Paper Details

Date Published: 26 June 2017
PDF: 9 pages
Proc. SPIE 10333, Optical Methods for Inspection, Characterization, and Imaging of Biomaterials III, 103330L (26 June 2017); doi: 10.1117/12.2270316
Show Author Affiliations
Klaus Dietrich, NTB Interstaatliche Hochschule für Technik Buchs (Switzerland)
Martina Brülisauer, NTB Interstaatliche Hochschule für Technik Buchs (Switzerland)
Emine Çağin, NTB Interstaatliche Hochschule für Technik Buchs (Switzerland)
Dietmar Bertsch, NTB Interstaatliche Hochschule für Technik Buchs (Switzerland)
Stefan Lüthi, NTB Interstaatliche Hochschule für Technik Buchs (Switzerland)
Peter Heeb, NTB Interstaatliche Hochschule für Technik Buchs (Switzerland)
Ulrich Stärker, Volpi AG (Switzerland)
André Bernard, NTB Interstaatliche Hochschule für Technik Buchs (Switzerland)


Published in SPIE Proceedings Vol. 10333:
Optical Methods for Inspection, Characterization, and Imaging of Biomaterials III
Pietro Ferraro; Simonetta Grilli; Monika Ritsch-Marte; Christoph K. Hitzenberger, Editor(s)

© SPIE. Terms of Use
Back to Top