Diffusion measured by fluorescence recovery after photobleaching based on multiphoton excitation laser scanning microscopy.
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.
Schnell
Edrun Andrea
EA
The Norwegian University of Science and Technology, Department of Physics, 7491 Trondheim, Norway.
Eikenes
Live
L
Tufto
Ingunn
I
Erikson
Arne
A
Juthajan
Aphirak
A
Lindgren
Mikael
M
de Lange Davies
Catharina
C
D000465:Algorithms; D000818:Animals; D014408:Biomarkers, Tumor / Q000737:chemistry*; D045744:Cell Line, Tumor; D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D018613:Microscopy, Confocal / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D012516:Osteosarcoma / Q000737:chemistry*
D014408:Biomarkers, Tumor