Whole-body optical imaging of post-embryonic stage model organisms is a challenging and long sought-after goal. It requires a combination of high-resolution performance and high-penetration depth. Optoacoustic (photoacoustic) mesoscopy holds great promise, as it penetrates deeper than optical and optoacoustic microscopy while providing high-spatial resolution. However, optoacoustic mesoscopic techniques only offer partial visibility of oriented structures, such as blood vessels, due to a limited angular detection aperture or the use of ultrasound frequencies that yield insufficient resolution. We introduce 360° multi orientation (multi-projection) raster scan optoacoustic mesoscopy (MORSOM) based on detecting an ultra-wide frequency bandwidth (up to 160 MHz) and weighted deconvolution to synthetically enlarge the angular aperture. We report unprecedented isotropic in-plane resolution at the 9–17 μm range and improved signal to noise ratio in phantoms and opaque 21-day-old Zebrafish. We find that MORSOM performance defines a new operational specification for optoacoustic mesoscopy of adult organisms, with possible applications in the developmental biology of adulthood and aging.

Intravital imaging of large specimens is intrinsically challenging for postembryonic studies. Selective plane illumination microscopy (SPIM) has been introduced to volumetrically visualize organisms used in developmental biology and experimental genetics. Ideally suited for imaging transparent samples, SPIM can offer high frame rate imaging with optical microscopy resolutions and low phototoxicity. However, its performance quickly deteriorates when applied to opaque tissues. To overcome this limitation, SPIM optics were merged with optical and optoacoustic (photoacoustic) readouts. The performance of this hybrid imaging system was characterized using various phantoms and by imaging a highly scattering ex vivo juvenile zebrafish. The results revealed the system's enhanced capability over that of conventional SPIM for high‐resolution imaging over extended depths of scattering content. The approach described here may enable future visualization of organisms throughout their entire development, encompassing regimes in which the tissue may become opaque.

Scattering within biological samples limits the imaging depth and the resolution in microscopy. We present a prior and regularization approach for blind deconvolution algorithms to correct the influence of scattering to increase the imaging depth and resolution. The effect of the prior is demonstrated on a three-dimensional image stack of a zebrafish embryo captured with a selective plane illumination microscope. Blind deconvolution algorithms model the recorded image as a convolution between the distribution of fluorophores and a point spread function (PSF). Our prior uses image information from adjacent z-planes to estimate the unknown blur in tissue. The increased size of the PSF due to the cascading effect of scattering in deeper tissue is accounted for by a depth adaptive regularizer model. In a zebrafish sample, we were able to extend the point in depth, where scattering has a significant effect on the image quality by around 30???m.

Zebrafish animal models have traditionally been used in developmental biology studies but have recently become promising models of cancer, tissue regeneration and metabolic disorders, as well as efficient platforms for functional genomics and phenotype-based drug discovery. Most studies of zebrafish have examined only the embryonic or larval stages of development, yet many questions in developmental biology and biomedicine require analysis of adults, when zebrafish are large and opaque. Conventional microscopy methods are highly sensitive to light scattering and therefore cannot be applied to zebrafish older than a few weeks. We describe a novel multi-modality system that can observe zebrafish from the larval stage to adulthood. Using a hybrid platform for concurrent selective plane illumination microscopy (SPIM) and optoacoustic mesoscopy we show continuous imaging of fish growth over 47 days of development at a similar object size-to-resolution ratio. Using multiple wavelength illumination over the visible and short-wavelength infrared regions, we reveal that the optoacoustic method can follow GFP-based contrast used in SPIM, enabling molecular imaging interrogation in adult fish. Moreover we optoacoustically reveal many other features of zebrafish based on optical contrast not present in SPIM, including contrast from endogenous blood, water and lipids. The hybrid method presented can extend optical imaging to adult zebrafish employed as model systems for studying long-term processes in development, cancer, diabetes and other disorders.

Zebrafish are common model organisms in developmental biology, but have recently emerged as imaging targets of research in cancer, tissue regeneration, metabolic disorders, functional genomics, and phenotype-based drug discovery. Conventionally, zebrafish are studied during the first few days of development using optical microscopy methods. However, optical methods are not suited for imaging at later stages, since the fish become opaque. To address needs to visualize beyond the first days of development, a novel multimodality system for observing zebrafish from larval stage to adulthood is developed. Using a hybrid platform for concurrent selective plane illumination microscopy (SPIM) and optoacoustic mesoscopy, fish (ex vivo) at stages of development up to 47 days at a similar object size-to-resolution ratio are imaged. Using multiple wavelength illumination over the visible and short-wavelength infrared regions, it is demonstrated that the optoacoustic method can follow GFP-based contrast used in SPIM, enabling molecular imaging interrogation in adult fish. Moreover, the optoacoustic modality reveals zebrafish features based on optical contrast absent in SPIM, including contrast from endogenous blood, water, and lipids. It is discussed how the hybrid system method can enable the study of zebrafish in a wider range of applications and over time-scales not possible currently when using optical microscopy.