Share Email Print

Journal of Biomedical Optics

Diffusion measured by fluorescence recovery after photobleaching based on multiphoton excitation laser scanning microscopy
Author(s): Edrun A. Schnell; Live Eikenes; Ingunn Tufto; Arne Erikson; Aphirak Juthajan; Mikael Lindgren; Catharina de Lange Davies
Format Member Price Non-Member Price
PDF $20.00 $25.00
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

Fluorescence recovery after photobleaching (FRAP) is a widely used method to measure diffusion. The technique is normally based on one-photon excitation, which limits diffusion to two dimensions due to extended photobleaching in the axial direction. Multiphoton excitation, on the other hand, creates a well-defined focal volume. In the present work, FRAP based on a scanning laser beam and two-photon excitation is used to measure diffusion of macromolecules in solution and gels, as well as in the extracellular matrix in multicellular spheroids and tumor tissue in dorsal chambers. The bleaching profile is determined experimentally in immobilized gels, and for small scanning areas (approximately twice the lateral radius of the laser beam) a Gaussian bleaching distribution is found. In addition, the bleaching profile is determined theoretically based on the convolution of the Gaussian point spread function and a circular scanning area. The diffusion coefficient is determined by fitting a mathematical model based on a Gaussian laser beam profile to the experimental recovery curve. The diffusion coefficient decreases with increasing complexity of the sample matrix and increasing the amount of collagen in the gels. The potential of using two-photon laser scanning microscopes for noninvasive diffusion measurements in tissue is demonstrated.

Paper Details

Date Published: 1 November 2008
PDF: 12 pages
J. Biomed. Opt. 13(6) 064037 doi: 10.1117/1.3042274
Published in: Journal of Biomedical Optics Volume 13, Issue 6
Show Author Affiliations
Edrun A. Schnell, Norwegian Univ. of Science and Technology (Norway)
Live Eikenes, Norwegian Univ. of Science and Technology (Norway)
Ingunn Tufto, Norwegian Univ. of Science and Technology (Norway)
Arne Erikson, Norwegian Univ. of Science and Technology (Norway)
Aphirak Juthajan, Norwegian Univ. of Science and Technology (Norway)
Mikael Lindgren, Norwegian Univ. of Science and Technology (Norway)
Catharina de Lange Davies, Norwegian Univ. of Science and Technology (Norway)

© SPIE. Terms of Use
Back to Top