Photoacoustic imaging of fluorophores using pump-probe excitation

Abstract

A pump-probe technique for the detection of fluorophores in tomographic PA images is introduced. It is based on inducing stimulated emission in fluorescent molecules, which in turn modulates the amount of thermalized energy, and hence the PA signal amplitude. A theoretical model of the PA signal generation in fluorophores is presented and experimentally validated on cuvette measurements made in solutions of Rhodamine 6G, a fluorophore of known optical and molecular properties. The application of this technique to deep tissue tomographic PA imaging is demonstrated by determining the spatial distribution of a near-infrared fluorophore in a tissue phantom. 1983). 32. E. Zhang, J. Laufer, and P. Beard, "Backward-mode multiwavelength photoacoustic scanner using a planar Fabry-Perot polymer film ultrasound sensor for high-resolution three-dimensional imaging of biological tissues," Appl.

Photoacoustic pump-probe tomography of fluorophores in vivo using interleaved image acquisition for motion suppression

“…However, for low concentrations, such as those typically found in vivo , pump-probe excitation is likely to result in multiple excitation-relaxation cycles per fluorophore molecule, which in turn increase the local temperature and pressure, and hence PA signal amplitude. The difference signal amplitude therefore depends on the ratio of fluorophore concentration and excitation fluence, which is discussed in more detail elsewhere30.…”

“…While pump-probe difference imaging was first demonstrated in tissue phantoms30, challenges to its in vivo application remain. 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.…”

Emerging concepts in functional and molecular photoacoustic imaging

“…This mechanism has been applied to extend the scope of photoacoustic imaging from chromophores to fluorophores [28]. As shown in the mid row of Fig.…”

“…Recently, the feasibility of stimulated emission-based photoacoustic imaging was experimentally demonstrated in a 6-mm-thick tissue phantom consisting of polymer capillaries filled with Atto680 and murine blood [28]. The pump and probe wavelengths were selected to be 680 and 742 nm, respectively, matching the excitation and emission peaks of Atto680.…”

“…(C) Stimulated emission-based photoacoustic images of a tissue phantom consisting of polymer capillaries filled with Atto680 (white arrows) and murine blood (red arrows) acquired using both simultaneous and delayed pump-probe pulses. The differential image completely eliminates the blood signals and highlights the transient absorption signal from Atto680 (adapted from [28]). (D) ground-state depletion-based isotropic-resolution PAM of fixed red blood cells in a volume of 50 μm × 50 μm × 20 μm.…”