TY - JOUR
T1 - Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy
AU - Vicidomini, G.
AU - Schneider, M.
AU - Bianchini, P.
AU - Krol, S.
AU - Szellas, T.
AU - Diaspro, A.
PY - 2007/1
Y1 - 2007/1
N2 - 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.
AB - 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.
KW - 4Pi microscopy
KW - Confocal microscopy
KW - Optical sectioning
KW - Shift-variant system
KW - Two-photon excitation microscopy
KW - Ultrathin uniform fluorescent PE layer
KW - Z-response
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U2 - 10.1111/j.1365-2818.2007.01718.x
DO - 10.1111/j.1365-2818.2007.01718.x
M3 - Article
C2 - 17286698
AN - SCOPUS:33846833462
VL - 225
SP - 88
EP - 95
JO - The Microscopic Journal and Structural Record
JF - The Microscopic Journal and Structural Record
SN - 0022-2720
IS - 1
ER -