Optical interrogation of biological tissues offers great variety of intrinsic probing mechanisms as well as highly specific contrast approaches based on tissue-specific expression of fluorescent proteins and extrinsically administered molecular biomarkers. Yet, most of the important living organisms and tissues remain inaccessible by the current optical imaging techniques due to complications arising from intense light scattering in tissues. Our new imaging approach, termed Multispectral Optoacoustic Tomography (MSOT), "listens" to ultrasonic vibrations created by ultrashort flashes of light at multiple wavelengths in order to attain high resolution tomographic views through several millimeters to centimeters of tissues, being independent of photon scattering.

Optical Imaging Enables Biological Discovery

For centuries, biological discovery was based on optical imaging, in particular microscopy but also several chromophoric assays and photographic approaches. The compelling advantages of light have more recently driven the development of powerful classes of fluorescent tags that can stain functional and molecular processes in-vivo. The most widely acknowledged technology is naturally the 2008 Nobel-prize awarded fluorescent protein, which offers perhaps the most versatile tool for biological fluorescence imaging [1]. Fluorescent proteins attain the ability to tag cellular motility and sub-cellular process, from gene expression and signaling pathways to protein function and interactions, merging optimally with post-genomic "-omics" investigations and interrogating biology at the systems level. At the organ and organism level, optical biological imaging has traditionally focused on studying life on dead specimens, i.e. through histology or immunohistochemistry, on thin sections that yield minimal photon scattering. The need to study evolution, function and disease in unperturbed environments and over time, has however entrusted modern optical visualization with the task of in vivo application.

Scattering Sets Limits for Light Penetration and Spatial Resolution

The underlying physical barrier for high-resolution (the so-called diffraction limited) optical imaging is the significant light diffusion.

When imaging with light through tissue, photons interfere with cellular interfaces and organelles leading to multiple scattering events within the specimen under investigation [2]. The detected light therefore loses information on its origin and propagation path, blurring the images and destroying spatial resolution. Even state-of-the-art multiphoton microscopy is usually limited to superficial imaging up to a depth of 0.5-1 mm in most living tissues. Macroscopic optical imaging has recently evolved as an alternative method for imaging large diffuse specimen and utilizes fully diffusive photons, typically from structures that are larger than 1 cm. In its more advanced form, techniques like Fluorescence Molecular Tomography (FMT) illuminates the sample under investigation at multiple projections and utilizes mathematical models of photon propagation in tissues, in combination with capturing diffusive photons propagating through tissue, to reconstruct the underlying imaging contrast, albeit with much lower resolution than in microscopy [3]. Several different implementations, developed over the past years, have been successfully used to three-dimensionally image bio-distribution of fluorochromes in entire animals, molecular pathways of cancer and cardiovascular decease, offering quantitative imaging. Optical tomography in diffuse objects, however, has been developed and today applied in tissues with dimensions that are normally larger than 1 cm and usually offers low spatial resolution on the order of 1 mm.

Multi-Spectral Optoacoustic Tomography (MSOT)

Optoacoustic (or photoacoustic) tomography is a hybrid imaging modality that has recently demonstrated unprecedented high-resolution imaging of chromophore distribution and vasculature deep in tissues of small animals. Optoacoustic imaging relies on detection of ultrasonic signals induced by absorption of pulsed light, effect whose initial discovery by Alexander Graham Bell dates back to 1880. However, it was not until the last decade that potential of optoacoustics was successfully explored for tissue imaging [4]. Since scattering of ultrasonic waves in biological tissues is extremely weak, as compared to that of light, biomedical optoacoustic imaging combines high optical absorption contrast with good spatial resolution limited only by ultrasonic diffraction. Unlike in pure optical imaging, the spatial resolution here is not determined nor limited by light diffusion, therefore such performance cannot be achieved by any other optical imaging technology developed so far. Originally, optoacoustic imaging of tissues targeted endogenous tissue contrast, primarily resolving oxy- and deoxy-hemoglobin and different vascular structures [4]. However, high contrast images were also obtained from tissues with low blood content, such as fat and bones (fig. 1) [5].
While good quality anatomical optoacoustic images can be generated by measurements at single light wavelength, we use multi-wavelength illumination and spectral processing in order to identify certain molecular markers in the presence of a highly heterogeneous background. Molecules with absorption spectra that are different than the ones of background tissue are best suited for MSOT imaging. In figure 2 it is demonstrated how the three-dimensional distribution of mCherry fluorescent protein is resolved deep within the brain of an adult zebrafish with spatial resolution of about 38 micron [6]. The fish measures 6 mm in diameter and exhibits fully diffusive behavior thus, apparently, it was so far not accessible by any of the existing optical imaging methods.