Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy

G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, A. Diaspro

Research output: Contribution to journalArticlepeer-review

Abstract

Layer-by-layer technique is used to adsorb a uniform ultrathin layer of fluorescently labelled polyelectrolytes on a glass cover slip. Due to their thickness, uniformity and fluorescence properties, these ultrathin layers may serve as a simple and applicable standard to directly measure the z-response of different scanning optical microscopes. In this work we use ultrathin layers to measure the z-response of confocal, two-photon excitation and 4Pi laser scanning microscopes. Moreover, due to their uniformity over a wide region, i.e. cover slip surface, it is possible to quantify the z-response of the system over a full field of view area. This property, coupled with a bright fluorescence signal, enables the use of polyelectrolyte layers for representation on sectioned imaging property charts: a very powerful method to characterize image formation properties and capabilities (z-response, off-axis aberration, spherical aberration, etc.) of a three-dimensional scanning system. The sectioned imaging property charts method needs a through-focus dataset taken from such ultrathin layers. Using a comparatively low illumination no significant bleaching occurs during the excitation process, so it is possible to achieve long-term monitoring of the z-response of the system. All the above mentioned properties make such ultrathin layers a suitable candidate for calibration and a powerful tool for real-time evaluation of the optical sectioning capabilities of different three-dimensional scanning systems especially when coupled to sectioned imaging property charts.

Original languageEnglish
Pages (from-to)88-95
Number of pages8
JournalJournal of Microscopy
Volume225
Issue number1
DOIs
Publication statusPublished - Jan 2007

Keywords

  • 4Pi microscopy
  • Confocal microscopy
  • Optical sectioning
  • Shift-variant system
  • Two-photon excitation microscopy
  • Ultrathin uniform fluorescent PE layer
  • Z-response

ASJC Scopus subject areas

  • Instrumentation

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