Abstract
In non-degenerate two-photon microscopy (ND-TPM), the required energy for fluorescence excitation occurs via absorption of two photons of different energies derived from two synchronized pulsed laser beams. ND-TPM is a promising imaging technology offering flexibility in the choice of the photon energy for each beam. However, a formalism to quantify the efficiency of two-photon absorption (TPA) under non-degenerate excitation, relative to the resonant degenerate excitation, is missing. Here, we derive this formalism and experimentally validate our prediction for a common fluorophore, fluorescein. An accurate quantification of non-degenerate TPA is important to optimize the choice of photon energies for each fluorophore.
Highlights
Fluorescence microscopy is an essential tool in biomedical imaging due to the availability of a wide spectrum of fluorescent dyes and probes [1]
The energies of the two photons do not need to be the same: the sum of their energies must be equal to the total energy required for the ground state to excited state transition. This feature allows for non-degenerate two-photon excitation (ND-TPE), where excitation occurs via simultaneous absorption of two photons of different energies derived from two laser beams
For the selected tuning range, the contribution of the IR to D-TPE was insignificant relative to the near infrared (NIR) beam contribution, because of the extremely low D-two-photon absorption cross-section (TPACS) in this range
Summary
Fluorescence microscopy is an essential tool in biomedical imaging due to the availability of a wide spectrum of fluorescent dyes and probes [1]. The energies of the two photons do not need to be the same: the sum of their energies must be equal to the total energy required for the ground state to excited state transition This feature allows for non-degenerate two-photon excitation (ND-TPE), where excitation occurs via simultaneous absorption of two photons of different energies derived from two laser beams. A more sensitive, low excitation power method for quantification of ND-TPACS is needed Toward this end, we extend the two-photon fluorescence excitation technique [20] to allow for ND-TPE. We experimentally validate our theoretical prediction: we apply the non-degenerate two-photon fluorescence excitation technique to measure ND-TPACS of a well-characterized fluorophore, fluorescein [20, 21], throughout our available tuning range and compare them to the corresponding D-TPACS. We confirm that the fluorescence generated by ND-TPE is linearly proportional to the excitation power of each beam [8, 25]
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