Unleashing Optics and Optoacoustics for Developmental Biology

The past decade marked an optical revolution in biology: an unprecedented number of optical techniques were developed and adopted for biological exploration, demonstrating increasing interest in optical imaging and in vivo interrogations. Optical methods have become faster and have reached nanoscale resolution, and are now complemented by optoacoustic (photoacoustic) methods capable of imaging whole specimens in vivo. Never before were so many optical imaging barriers broken in such a short time-frame: with new approaches to optical microscopy and mesoscopy came an increased ability to image biology at unprecedented speed, resolution, and depth. This review covers the most relevant techniques for imaging in developmental biology, and offers an outlook on the next steps for these technologies and their applications.The work on this review article has received funding from the Deutsche Forschungsgemeinschaft (DFG), Germany (Leibniz Prize 2013; NT 3/10 1) and the Federal Ministry of Education and Research (BMBF), Photonic Science Germany, Tech2See 13N12623/ 4. J.R. acknowledges support from the European Commission FP7 CIG grant HIGH THROUGH PUT TOMO, and Spanish MINECO grant MESO IMAGING FIS2013 41802 R

Scattering correction through a space-variant blind deconvolution algorithm.

cattering 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  μm

Increasing the imaging depth through computational scattering correction.

Imaging depth is one of the most prominent limitations in light microscopy. The depth in which we are still able to resolve biological structures is limited by the scattering of light within the sample. We have developed an algorithm to compensate for the influence of scattering. The potential of algorithm is demonstrated on a 3D image stack of a zebrafish embryo captured with a selective plane illumination microscope (SPIM). With our algorithm we were able shift the point in depth, where scattering starts to blur the imaging and effect the image quality by around 30 µm. For the reconstruction the algorithm only uses information from within the image stack. Therefore the algorithm can be applied on the image data from every SPIM system without further hardware adaption. Also there is no need for multiple scans from different views to perform the reconstruction. The underlying model estimates the recorded image as a convolution between the distribution of fluorophores and a point spread function, which describes the blur due to scattering. Our algorithm performs a space-variant blind deconvolution on the image. To account for the increasing amount of scattering in deeper tissue, we introduce a new regularizer which models the increasing width of the point spread function in order to improve the image quality in the depth of the sample. Since the assumptions the algorithm is based on are not limited to SPIM images the algorithm should also be able to work on other imaging techniques which provide a 3D image volume

Three-dimensional optoacoustic mesoscopy of the tumor heterogeneity in <em>vivo</em> using high depth-to-resolution multispectral optoacoustic tomography.

Multispectral optoacoustic mesoscopy (MSOM) has been recently introduced for cancer imaging, it has the potential for high resolution imaging of cancer development in vivo, at depths beyond the diffusion limit. Based on spectral features, optoacoustic imaging is capable of visualizing angiogenesis and imaging cancer heterogeneity of malignant tumors through endogenous hemoglobin. However, high-resolution structural and functional imaging of whole tumor mass is limited by modest penetration and image quality, due to the insufficient capability of ultrasound detectors and the twodimensional scan geometry. In this study, we introduce a novel multi-spectral optoacoustic mesoscopy (MSOM) for imaging subcutaneous or orthotopic tumors implanted in lab mice, with the high-frequency ultrasound linear array and a conical scanning geometry. Detailed volumetric images of vasculature and oxygen saturation of tissue in the entire tumors are obtained in vivo, at depths up to 10 mm with the desirable spatial resolutions approaching 70&mu;m. This unprecedented performance enables the visualization of vasculature morphology and hypoxia conditions has been verified with ex vivo studies. These findings demonstrate the potential of MSOM for preclinical oncological studies in deep solid tumors to facilitate the characterization of tumor&rsquo;s angiogenesis and the evaluation of treatment strategies

Three-dimensional optoacoustic mesoscopy of the tumor heterogeneity in vivo using high depth-to-resolution multispectral optoacoustic tomography.

Optoacoustic imaging provides optical absorption contrast at ultrasonic resolutions in vivo. Previous studies showed the potential of optoacoustic imaging for oncological studies. Herein, based on a new geometry conical geometry, we show the capability of Multispectral Optoacoustic Mesoscopy (MSOM) in imaging tumor vasculature, tumor oxygenation, and the bio-distribution of contrast agents, such as gold nanoparticles, in vivo. This study shows the potential of MSOM in preclinical imaging of cancer at depths up to 10 mm and resolutions approaching 70&mu;m. These findings demonstrate the potential of MSOM to facilitate the characterization of tumor&rsquo;s angiogenesis and the evaluation of treatment strategies

Spatial heterogeneity of oxygenation and haemodynamics in breast cancer resolved in vivo by conical multispectral optoacoustic mesoscopy.

Optoacoustic imaging: Revealing the details of tumour patterns A technique that can image the entire tumour volume with high resolution may help oncologists optimize specific treatments for breast cancer. Jiao Li (Tianjin University, China), Vasilis Ntziachristos (Technical University of Munich, Germany), and colleagues have designed a multispectral optoacoustic mesoscope (MSOM) that illuminates millimetre-sized tumours with laser light of various wavelengths, and detects the ultrasound waves generated by internal absorbers, such as haemoglobin, or external nanoparticle probes. By reconstructing the ultrasound signals over multiple frequencies, the team produced 3D images of features that included the vascular network of a tumour with micrometre-scale detail. Experiments with live mice demonstrated that specific tumours could be identified through differences in spatial patterns, such as altered oxygen levels between tumour cores and peripheries. The study highlights the power of MSOM as a tool for preclinical cancer studies.The characteristics of tumour development and metastasis relate not only to genomic heterogeneity but also to spatial heterogeneity, associated with variations in the intratumoural arrangement of cell populations, vascular morphology and oxygen and nutrient supply. While optical (photonic) microscopy is commonly employed to visualize the tumour microenvironment, it assesses only a few hundred cubic microns of tissue. Therefore, it is not suitable for investigating biological processes at the level of the entire tumour, which can be at least four orders of magnitude larger. In this study, we aimed to extend optical visualization and resolve spatial heterogeneity throughout the entire tumour volume. We developed an optoacoustic (photoacoustic) mesoscope adapted to solid tumour imaging and, in a pilot study, offer the first insights into cancer optical contrast heterogeneity in vivo at an unprecedented resolution of &lt;50 mu m throughout the entire tumour mass. Using spectral methods, we resolve unknown patterns of oxygenation, vasculature and perfusion in three types of breast cancer and showcase different levels of structural and functional organization. To our knowledge, these results are the most detailed insights of optical signatures reported throughout entire tumours in vivo, and they position optoacoustic mesoscopy as a unique investigational tool linking microscopic and macroscopic observations

Selective plane illumination optical and optoacoustic microscopy for postembryonic imaging.

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&#39;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