Molecular specificity in photoacoustic microscopy by time-resolved transient absorption

Abstract

We have recently harnessed transient absorption, a resonant two-photon process, for ultrahigh resolution photoacoustic microscopy, achieving nearly an order of magnitude improvement in axial resolution. The axial resolution is optically constrained due to the two-photon process unlike traditional photoacoustic microscopy where the axial resolution is inversely proportional to the frequency bandwidth of the detector. As a resonant process, the arrival time of the two photons need not be instantaneous. Systematically recording the signal as a function of the delay between two pulses will result in the measurement of an exponential decay whose time constant is related to the molecular dynamics. This time constant, analogous to the fluorescence lifetime, but encompassing nonradiative decay as well, can be used to differentiate between molecular systems with overlapping absorption spectra. This is frequently the situation for closely related yet distinct molecules such as redox pairs. In order to enable the measure of the exponential decay, we have reconfigured our transient absorption ultrasonic microscopy (TAUM) system to incorporate two laser sources with precisely controlled pulse trains. The system was tested by measuring Rhodamine 6G, an efficient laser dye where the molecular dynamics are dominated by the fluorescence pathway. As expected, the measured exponential time constant or ground state recovery time, 3.3±0.7 ns, was similar to the well-known fluorescence lifetime, 4.11±0.05 ns. Oxy- and deoxy-hemoglobin are the quintessential pair whose relative concentration is related to the local blood oxygen saturation. We have measured the ground state recovery times of these two species in fully oxygenated and deoxygenated bovine whole blood to be 3.7±0.8 ns and 7.9±1.0 ns, respectively. Hence, even very closely related pairs of molecules may be differentiated with this technique.

Molecular Photoacoustic Contrast Agents: Design Principles & Applications

“…Although nonlinear RSA materials are anticipated to be the most promising dye category for generation of an enhanced nonlinear PA response it must be kept in mind that their application is very much limited to scenarios where the application of high laser fluences is feasible. While a number of studies addressing nonlinear PAI have emerged very few have involved in vivo studies , none of which have relied solely on a molecular‐based contrast agent. As such, all of the in vivo imaging applications of MPACs discussed in this review have been conducted under low laser fluence conditions where the dye either behaves as a LA or SA material.…”

Photophysical and Photoacoustic Properties of π‐Extended Curcumin Dyes. Effects of the Terminal Dimethylamino Electron‐donor and the Bridging Aryl Ring

“…It should be appreciated that high laser fluence nonlinear optical methods are not applicable to in vivo imaging, due to the American National Standards Institute (ANSI) laser fluence thresholds (46). However, in vitro high-resolution photoacoustic microscopy (PAM) is not restricted by ANSI limitations (47)(48)(49)(50)(51)(52)(53)(54)(55)(56).…”

Emerging concepts in functional and molecular photoacoustic imaging

“…Due to the pump-induced depopulation of the ground state, the second (i.e., probe) pulse experiences reduced optical absorption. The difference between the photoacoustic signals generated by the probe light in the presence and absence of the pump depends on the recovery time of S 0 from depletion, providing another molecule-specific contrast mechanism for photoacoustic imaging [29]. …”