Abstract
In fluorophores, the excited state lifetime can be modulated using pump-probe excitation. By generating photoacoustic (PA) signals using simultaneous and time-delayed pump and probe excitation pulses at fluences below the maximum permissible exposure, a modulation of the signal amplitude is observed in fluorophores but not in endogenous chromophores. This provides a highly specific contrast mechanism that can be used to recover the location of the fluorophore using difference imaging. The practical challenges in applying this method to in vivo PA tomography include the typically low concentrations of fluorescent contrast agents, and tissue motion. The former results in smaller PA signal amplitudes compared to those measured in blood, while the latter gives rise to difference image artefacts that compromise the unambiguous and potentially noise-limited detection of fluorescent contrast agents. To address this limitation, a method based on interleaved pump-probe image acquisition was developed. It relies on fast switching between simultaneous and time-delayed pump-probe excitation to acquire PA difference signals in quick succession, and to minimise the effects of tissue motion. The feasibility of this method is demonstrated in tissue phantoms and in initial experiments in vivo.
Highlights
In fluorophores, the excited state lifetime can be modulated using pump-probe excitation
Fluorophores have been used as exogenous contrast agents in proof-of-principle studies to demonstrate their detection in vivo at greater depths and with higher spatial resolution than that provided by purely optical modalities[12]
The pump wavelength was set to 680 nm to coincide with the absorption peak of a NIR fluorophore (Atto[680], Atto-Tec GmbH, Germany) while the probe wavelength of 742 nm coincided with the fluorescence spectrum to induce stimulated emission (SE)
Summary
The excited state lifetime can be modulated using pump-probe excitation. The practical challenges in applying this method to in vivo PA tomography include the typically low concentrations of fluorescent contrast agents, and tissue motion The former results in smaller PA signal amplitudes compared to those measured in blood, while the latter gives rise to difference image artefacts that compromise the unambiguous and potentially noise-limited detection of fluorescent contrast agents. Since PA signals generated in vivo in weakly accumulated fluorescent contrast agents are smaller than those measured in blood and since tissue motion due to breathing or pulsatile blood flow can give rise to significant artefacts in the difference images, the unambiguous detection of fluorophores can be compromised This is noticeable if sequential image acquisition is used, i.e. the successive capture of two image data sets at different pump-probe time delays from which a difference image is obtained. This method was validated by imaging fluorophores in a motion-mimicking tissue phantom, and applied to in vivo imaging in an initial preclinical study
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