https://doi.org/10.1007/s100530050148
Molecular photodynamics involved in multi-photon excitation fluorescence microscopy
School of Applied and Engineering Physics, Cornell University,
Ithaca, New York, 14853, USA
Corresponding author: a mertz@atlas.bio.espci.fr
Received:
9
March
1998
Accepted:
20
April
1998
Published online: 15 July 1998
We present a simple model for calculating the fluorescence generated by the multi-photon excitation (MPE) of molecules in solution. The model takes into account internal molecular dynamics such as ground-state depletion due to inter-system crossing (ISC), as well as external molecular dynamics associated with diffusion into and out of an excitation volume confined in 3-dimensions. Internal and external molecular dynamics are combined by using a technique of linearization of a modified diffusion equation which takes into account the possibility of concentration depletion due to photobleaching. In addition, we discuss the phenomenon of pulse saturation which effectively limits the molecular excitation rate constant in the case of short pulsed excitation. Our results are specifically applied in the context of fluorescence autocorrelation functions and single-molecule detection. In the latter case, we discuss some consequences of high-order multi-photon photobleaching. Finally, we include three appendices to rigorously define the temporal and spatial profiles of an arbitrary excitation beam, and also to discuss some properties of an exact evaluation of concentration depletion due to photobleaching.
PACS: 32.80.Wr – Other multiphoton processes / 32.50.+d – Fluorescence, phosphorescence (including quenching) / 87.64.-t – Spectroscopic and microscopic techniques in biophysics and medical physics
© EDP Sciences, Società Italiana di Fisica, Springer-Verlag, 1998
