Optoacoustic microscopy at multiple discrete frequencies

Stephan Kellnberger,Angelos Karlas,George J Tserevelakis,Hong Yang,Murad Omar,Ludwig Prade,Dominik Soliman,Vasilis Ntziachristos,Markus Seeger

doi:10.1038/s41377-018-0101-2

Abstract

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.

Highlights

Most optoacoustic implementations operate in the time domain (TD) using laser pulses that last 1–100 ns[1,2]
frequency domain (FD) imaging relies on an intensity-modulated continuous energy stream instead of photon pulses, which are used in TD implementations
frequency-domain optoacoustic microscopy (FDOM) and multiphoton microscopy FDOM excitation utilizes the same optical path as multiphoton microscopy (Fig. 1a); each modality operates in distinct spectral bands (Fig. 1b)

Summary

Objective lens

We investigated a fundamentally different approach for optoacoustic imaging by employing continuous-wave (CW) light with the intensity being modulated at multiple discrete frequencies. We hypothesized that detection of the amplitude and phase of ultrasound waves excited at multiple discrete modulation frequencies could broadly capture the spatial frequencies of the imaged object, thereby leading to high-fidelity images. To examine this hypothesis, we developed a multi-wavelength FD optoacoustic microscopy (FDOM) system that operates in the frequency range of 5–50 MHz. FDOM was implemented with multiphoton microscopy as a hybrid modality. Using modulation frequencies up to two orders of magnitude greater than the repetition rates of TD optoacoustic microscopes, we performed two- and threedimensional imaging based on ultrasound amplitude and phase measurements at multiple modulation frequencies. We discovered that the use of discrete frequencies enables the measurement of optoacoustic Doppler shifts, thereby enabling flow observations both in a microfluidic flow chamber and in tissue microvasculature in vivo

Results

20 MHz S1

Discussion

Materials and methods