Overdriven laser diode optoacoustic microscopy.

Laser diodes are small and inexpensive but don't afford the pulse energy and beam profile required for optoacoustic (photoacoustic) microscopy. Using two novel modulation concepts, i.e. overdriving continuous-wave laser diodes (CWLD) and frequency-wavelength multiplexing (FWM) based on illumination pulse-trains, we demonstrate concurrent multi-wavelength optoacoustic microscopy with signal-to-noise ratios of > 17 dB, < 2 µm resolution at repetition rates of 1 MHz. This unprecedented performance based on an adaptable trigger engine allowed us to contrast FWM to wavelength alternating acquisition using identical optical components. We showcase this concept's superiority over conventional optoacoustic microscopes by visualizing vascular oxygenation dynamics and circulating tumor cells in mice. This work positions laser diodes as a technology allowing affordable, tunable, and miniaturizable optoacoustic microscopy.

Frequency wavelength multiplexed optoacoustic tomography.

Optoacoustics (OA) is overwhelmingly implemented in the Time Domain (TD) to achieve high signal-to-noise ratios by maximizing the excitation light energy transient. Implementations in the Frequency Domain (FD) have been proposed, but suffer from low signal-to-noise ratios and have not offered competitive advantages over time domain methods to reach high dissemination. It is therefore commonly believed that TD is the optimal way to perform optoacoustics. Here we introduce an optoacoustic concept based on pulse train illumination and frequency domain multiplexing and theoretically demonstrate the superior merits of the approach compared to the time domain. Then, using recent advances in laser diode illumination, we launch Frequency Wavelength Multiplexing Optoacoustic Tomography (FWMOT), at multiple wavelengths, and experimentally showcase how FWMOT optimizes the signal-to-noise ratios of spectral measurements over time-domain methods in phantoms and in vivo. We further find that FWMOT offers the fastest multi-spectral operation ever demonstrated in optoacoustics.

In Vivo Three-Dimensional Raster Scan Optoacoustic Mesoscopy Using Frequency Domain Inversion.

Optoacoustic signals are typically reconstructed into images using inversion algorithms applied in the time-domain. However, time-domain reconstructions can be computationally intensive and therefore slow when large amounts of raw data are collected from an optoacoustic scan. Here we considered a fast weighted ω-k (FWOK) algorithm operating in the frequency domain to accelerate the inversion in raster-scan optoacoustic mesoscopy (RSOM), while seamlessly incorporating impulse response correction with minimum computational burden. We investigated the FWOK performance with RSOM measurements from phantoms and mice in vivo and obtained 360-fold speed improvement over inversions based on the back-projection algorithm in the time-domain. This previously unexplored inversion of in vivo optoacoustic data with impulse response correction in frequency domain reconstructions points to a promising strategy of accelerating optoacoustic imaging computations, toward video-rate tomography.

Low-cost single-point optoacoustic sensor for spectroscopic measurement of local vascular oxygenation.

Optical sensors developed for the assessment of oxygen in tissue microvasculature, such as those based on near-infrared spectroscopy, are limited in application by light scattering. Optoacoustic methods are insensitive to light scattering, and therefore, they can provide higher specificity and accuracy when quantifying local vascular oxygenation. However, currently, to the best of our knowledge, there is no low-cost, single point, optoacoustic sensor for the dedicated measurement of oxygen saturation in tissue microvasculature. This work introduces a spectroscopic optoacoustic sensor (SPOAS) for the non-invasive measurement of local vascular oxygenation in real time. SPOAS employs continuous wave laser diodes and measures at a single point, which makes it low-cost and portable. The SPOAS performance was benchmarked using blood phantoms, and it showed excellent linear correlation (R2=0.98) with a blood gas analyzer. Subsequent measurements of local vascular oxygenation in living mice during an oxygen stress test correlated well with simultaneous readings from a reference instrument.

Optoacoustic microscopy at multiple discrete frequencies.

Optoacoustic (photoacoustic) sensing employs illumination of transient energy and is typically implemented in the time domain using nanosecond photon pulses. However, the generation of high-energy short photon pulses requires complex laser technology that imposes a low pulse repetition frequency (PRF) and limits the number of wavelengths that are concurrently available for spectral imaging. To avoid the limitations of working in the time domain, we have developed frequency-domain optoacoustic microscopy (FDOM), in which light intensity is modulated at multiple discrete frequencies. We integrated FDOM into a hybrid system with multiphoton microscopy, and we examine the relationship between image formation and modulation frequency, showcase high-fidelity images with increasing numbers of modulation frequencies from phantoms and in vivo, and identify a redundancy in optoacoustic measurements performed at multiple frequencies. We demonstrate that due to high repetition rates, FDOM achieves signal-to-noise ratios similar to those obtained by time-domain methods, using commonly available laser diodes. Moreover, we experimentally confirm various advantages of the frequency-domain implementation at discrete modulation frequencies, including concurrent illumination at two wavelengths that are carried out at different modulation frequencies as well as flow measurements in microfluidic chips and in vivo based on the optoacoustic Doppler effect. Furthermore, we discuss how FDOM redefines possibilities for optoacoustic imaging by capitalizing on the advantages of working in the frequency domain.

Continuous wave laser diodes enable fast optoacoustic imaging.

Pulsed laser diodes may offer a smaller, less expensive alternative to conventional optoacoustic laser sources; however they do not provide pulse rates faster than a few tens of kHz and emit at wavelengths only within the near-infrared region. We investigated whether continuous wave (CW) laser diodes, which are available in visible and near-infrared regions, can be good optoacoustic light sources when overdriven with a peak current >40-fold higher than the CW absolute maximum. We found that overdriven CW diodes provided ∼10 ns pulses of ∼200 nJ/pulse and repetition rates higher than 600 kHz without being damaged, outperforming many pulsed laser diodes. Using this system, we obtained images of phantoms and mouse ear and human arm in vivo, confirming their use in optoacoustic imaging and sensing.