The total or integrated fluorescence intensity of a through-focus series of a thin standardized uniform fluorescent or
calibration layer is shown to be suitable for image intensity correction and calibration in sectioning microscopy. This
integrated intensity can be derived from the earlier introduced SectionedImagingProperty or SIPcharts, derived from the
3D layer datasets.
By correcting the 3D image of an object with the 3D image of the standardized uniform fluorescent layer obtained under
identical conditions one is able to express the object fluorescence in units fluorescence of the calibration layer. With
object fluorescence intensities in fluorescence layer unit's or FLU's the object image intensities becomes independent of
microscope system and imaging conditions.
A direct result is that the often-appreciable lateral intensity variations present in confocal microscopy are eliminated
(shading correction). Of more general value is that images obtained with different objectives, magnifications or from
different microscope systems can be quantitatively related to each other.
The effectiveness of shading correction and relating images obtained under various microscope conditions is
demonstrated on images of standard fluorocent beads.
Expressing the object fluorescence in FLU units seems to be a promising approach for general quantification of
sectioning imaging enabling cross-correlation of imaging results over time and between imaging systems.
Layer-by-Layer or self-assembly techniques can be used to prepare Fluorescent polymer samples on glass coverslips
serving as benchmark for two-photon excitation microscopy from conventional to 4Pi set-up, or more in general
for sectioning microscopy. Layers can be realized as ultra-thin (<< 100 nm) or thin (approx. 100 nm)
characteristics coupled to different fluorescent molecules to be used for different microscopy applications. As well, stacks hosting different fluorescent molecules can be also produce. Thanks to their controllable thickness, uniformity and fluorescence properties, these polymer layers may serve as a simple and applicable standard to
directly measure the z-response of different scanning optical microscopes. In two-photon excitation microscopy z-sectioning plays a central role and uniformity of illumination is crucial due to the non-linear behaviour of emission. Since the main characteristics of a particular image formation situation can be efficiently summarized
in a Sectioned Imaging property chart (SIPchart), we think that coupling this calibration sample with SIPchart is a very important step towards quantitative microscopy. In this work we use these polymer layers to measure the z-response of confocal, two-photon excitation and 4Pi laser scanning microscopes, selecting properly ultra-thin and thin layers. Due to their uniformity over a wide region, i.e. coverslip surface, it is possible to quantify the z-response of the system over a full field of view area. These samples are also useful for monitoring photobleaching
behavior as function of the illumination intensity. Ultrathin layers are also useful to supersede the conventional
technique of calculating the derivative of the axial edges of a thick fluorescent layer. Polymer layers can be
effciently used for real time alignment of the microscope.
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