Photoacoustic microscopy (PAM) is an imaging modality well suited to mapping vasculature and other strong absorbers in tissue. However, one of the primary drawbacks to PAM when used for high-resolution imaging is the relatively poor axial resolution due to the inverse dependence on the transducer bandwidth. While submicron lateral resolution PAM can be achieved by tightly focusing the excitation light, the axial resolution is fundamentally limited to 10s of microns for typical transducer frequencies. Here we present a multiphoton PAM technique called transient absorption ultrasonic microscopy (TAUM), which results in a completely optically resolved voxel with an experimentally measured axial resolution of 1.5 microns. This technique is demonstrated by imaging individual red blood cells in three dimensions in blood smear and ex vivo tissues. To the best of our knowledge, this is the first demonstration of fully resolved, volumetric photoacoustic imaging of erythrocytes.

Optical coherence tomography (OCT) is a medical imaging technique that provides tomographic images at micron scales in three dimensions and high speeds. The addition of molecular contrast to the available morphological image holds great promise for extending OCT’s impact in clinical practice and beyond. Fundamental limitations prevent OCT from directly taking advantage of powerful molecular processes such as fluorescence emission and incoherent Raman scattering. A wide range of approaches is being researched to provide molecular contrast to OCT. Here we review those approaches with particular attention to those that derive their molecular contrast directly from modulation of the OCT signal. We also provide a brief overview of the multimodal approaches to gaining molecular contrast coincident with OCT.

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.

Photoacoustic microscopy (PAM) is a hybrid imaging modality that combines optical illumination with ultrasonic detection to achieve absorption contrast imaging of endogenous and exogenous chromophores. Optical resolution PAM achieves high lateral-resolution by tightly focusing the excitation light; however the axial resolution is still dependent upon the bandwidth of the ultrasonic transducer. As a result, PAM images have highly asymmetric voxels with submicron lateral resolution and axial resolution typically limited to tens of microns. We have previously reported on a resonant multiphoton approach to PAM called transient absorption ultrasonic microscopy (TAUM), which enables high axial resolution by frequency encoding the photoacoustic signal at the overlap of a pump and a probe beam. This approach enables photoacoustic imaging with subcellular resolution on par with other multiphoton microscopy techniques. Here, we report on an innovation that enables TAUM imaging with a much less sophisticated optical system than previously reported. If we allow the time delay between the pump and probe to collapse to zero, the pump and probe optical paths can be combined. An amplitude modulator in the single beam path is sufficient to encode the TAUM signal at the second harmonic of the modulation frequency. The resulting system is essentially a standard optical resolution PAM system that incorporates an amplitude modulator and utilizes a Fourier post processing algorithm to improve the axial resolution by approximately an order of magnitude. A prototype system based on this approach has been assembled and tested on fixed bovine erythrocytes.

A four dimensional data set of the cardiac cycle of a zebrafish embryo was acquired using postacquisition synchronization of real time photoacoustic b-scans. Utilizing an off-axis photoacoustic microscopy (OA-PAM) setup, we have expanded upon our previous work with OA-PAM to develop a system that can sustain 100 kHz line rates while demodulating the bipolar photoacoustic signal in real-time. Real-time processing was accomplished by quadrature demodulation on a Field Programmable Gate Array (FPGA) in line with the signal digitizer. Simulated data acquisition verified the system is capable of real-time processing up to a line rate of 1 MHz. Galvanometer-scanning of the excitation laser inside the focus of the ultrasonic transducer enables real data acquisition of a 200 by 200 by 200 pixel, volumetric data set across a 2 millimeter field of view at a rate of 2.5 Hz.