Quantifying fluorescence signals in confocal image stacks deep in turbid media

S. Beer; U. Maeder; T. Bergmann; J. M. Burg; M. Fiebich; F. Runkel

doi:10.1117/12.907875

2 February 2012 Quantifying fluorescence signals in confocal image stacks deep in turbid media

S. Beer, U. Maeder, T. Bergmann, J. M. Burg, M. Fiebich, F. Runkel

Author Affiliations +

Proceedings Volume 8227, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIX; 82271G (2012) https://doi.org/10.1117/12.907875
Event: SPIE BiOS, 2012, San Francisco, California, United States

Abstract

When confocal depth stacks are taken, the collected signal (normally the fluorescence signal), decays dependent of the depth of the confocal slice in the turbid medium. This decay is caused by scattering and absorption of the exciting light and of the fluorescence light. As the attenuation parameters, i.e. scattering and absorption coefficients, are normally unknown when observing a new sample, a method is proposed to compensate for the attenuation of the involved light by correcting the fluorescence signal using the attenuation behavior of the sample measured directly on the spot where the fluorescence stack is taken. The method works without any a priori knowledge about the optical properties of the sample. Using this self-reference technique, a confocal fluorescence depth stack can be created where the signal intensity is not dependent on the scattering and absorption caused intensity decay. The proposed method is tested on fluorescent beads embedded in scattering and absorbing hydrogel phantoms.

Citation Download Citation

S. Beer, U. Maeder, T. Bergmann, J. M. Burg, M. Fiebich, and F. Runkel "Quantifying fluorescence signals in confocal image stacks deep in turbid media", Proc. SPIE 8227, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIX, 82271G (2 February 2012); https://doi.org/10.1117/12.907875

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KEYWORDS

Signal attenuation

Confocal microscopy

Luminescence

Particles

Scattering

Absorption

Skin

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