A subset of the family of fluorescent proteins are the non-fluorescent chromoproteins which are promising probe molecules for use in photoacoustic imaging and as acceptor chromophores in Förster resonance energy transfer (FRET)-based biosensors. Typical approaches for fluorescent protein optimization by screening of large libraries of variants cannot be effectively applied to chromoproteins due to their characteristic lack of fluorescence. To address this challenge, we have developed a directed evolution method to iteratively screen large libraries of protein variants on the basis of their photoacoustic signal levels. By applying this procedure to the promising Ultramarine and cjBlue chromoprotein templates, we were able to identify improved variants with a 02–04 fold increase in photoacoustic signal-to-noise ratio after only a few evolutionary steps. These improved variants enable more accurate spectral de-mixing and localization of protein-producing bacteria in vivo and serve as effective FRET acceptors for both fluorescence- and photoacoustic-based detection of protease activity.

Abstract. The metabolic rate of oxygen consumption, an important indicator of tissue metabolism, can be expressed as the change of net blood oxygen flux into and out of a tissue region per 100 g of tissue. In this work, we propose a photoacoustic and Doppler ultrasound method for imaging local blood oxygen flux of a single vessel. An imaging system for combined photoacoustic and high-frequency ultrasound microscopy is presented. This system uses a swept-scan 25-MHz ultrasound transducer with confocal dark-field laser illumination optics. A pulse-sequencer enables ultrasonic and laser pulses to be interlaced so that photoacoustic and Doppler ultrasound images are co-registered. Since the mean flow speed can be measured by color Doppler ultrasound, the vessel cross-sectional area can be measured by power Doppler or structural photoacoustic imaging, and multi-wavelength photoacoustic methods can be used to estimate oxygen saturation (sO 2 ) and total concentration of haemoglobin (C Hb ), all of the parameters necessary for oxygen flux estimation can be provided. The accuracy of the flow speed and sO 2 estimation has been investigated. In vitro sheep blood phantom experiments have been performed at different sO 2 levels and mean flow speeds. Blood oxygen flux has been estimated, and the uncertainty of the measurement has been quantified.

Abstract. The lymphatic system is responsible for fluid homeostasis and immune cell trafficking and has been implicated in several diseases, including obesity, diabetes, and cancer metastasis. Despite its importance, the lack of suitable in vivo imaging techniques has hampered our understanding of the lymphatic system. This is, in part, due to the limited contrast of lymphatic fluids and structures. Photoacoustic imaging, in combination with optically absorbing dyes or nanoparticles, has great potential for noninvasively visualizing the lymphatic vessels deep in tissues. Multispectral photoacoustic imaging is capable of separating the components; however, the slow wavelength switching speed of most laser systems is inadequate for imaging lymphatic pumping without motion artifacts being introduced into the processed images. We investigate two approaches for visualizing lymphatic processes in vivo. First, single-wavelength differential photoacoustic imaging is used to visualize lymphatic pumping in the hindlimb of a mouse in real time. Second, a fast-switching multiwavelength photoacoustic imaging system was used to assess the propulsion profile of dyes through the lymphatics in real time. These approaches may have profound impacts in noninvasively characterizing and investigating the lymphatic system.

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