Longitudinal imaging of T cell-based immunotherapy with multi-spectral, multi-scale optoacoustic tomography.

Most imaging studies of immunotherapy have focused on tracking labeled T cell biodistribution in vivo for understanding trafficking and homing parameters and predicting therapeutic efficacy by the presence of transferred T cells at or in the tumour mass. Conversely, we investigate here a novel concept for longitudinally elucidating anatomical and pathophysiological changes of solid tumours after adoptive T cell transfer in a preclinical set up, using previously unexplored in-tandem macroscopic and mesoscopic optoacoustic (photoacoustic) imaging. We show non-invasive in vivo observations of vessel collapse during tumour rejection across entire tumours and observe for the first time longitudinal tumour rejection in a label-free manner based on optical absorption changes in the tumour mass due to cellular decline. We complement these observations with high resolution episcopic fluorescence imaging of T cell biodistribution using optimized T cell labeling based on two near-infrared dyes targeting the cell membrane and the cytoplasm. We discuss how optoacoustic macroscopy and mesoscopy offer unique contrast and immunotherapy insights, allowing label-free and longitudinal observations of tumour therapy. The results demonstrate optoacoustic imaging as an invaluable tool in understanding and optimizing T cell therapy.

Eigenspectra optoacoustic tomography achieves quantitative blood oxygenation imaging deep in tissues.

Light propagating in tissue attains a spectrum that varies with location due to wavelength-dependent fluence attenuation, an effect that causes spectral corruption. Spectral corruption has limited the quantification accuracy of optical and optoacoustic spectroscopic methods, and impeded the goal of imaging blood oxygen saturation (sO2) deep in tissues; a critical goal for the assessment of oxygenation in physiological processes and disease. Here we describe light fluence in the spectral domain and introduce eigenspectra multispectral optoacoustic tomography (eMSOT) to account for wavelength-dependent light attenuation, and estimate blood sO2 within deep tissue. We validate eMSOT in simulations, phantoms and animal measurements and spatially resolve sO2 in muscle and tumours, validating our measurements with histology data. eMSOT shows substantial sO2 accuracy enhancement over previous optoacoustic methods, potentially serving as a valuable tool for imaging tissue pathophysiology.

Effects of multispectral excitation on the sensitivity of molecular optoacoustic imaging.

Molecular optoacoustic (photoacoustic) imaging typically relies on the spectral identification of absorption signatures from molecules of interest. To achieve this, two or more excitation wavelengths are employed to sequentially illuminate tissue. Due to depth-related spectral dependencies and detection related effects, the multispectral optoacoustic tomography (MSOT) spectral unmixing problem presents a complex non-linear inversion operation. So far, different studies have showcased the spectral capacity of optoacoustic imaging, without however relating the performance achieved to the number of wavelengths employed. Overall, the dependence of the sensitivity and accuracy of optoacoustic imaging as a function of the number of illumination wavelengths has not been so far comprehensively studied. In this paper we study the impact of the number of excitation wavelengths employed on the sensitivity and accuracy achieved by molecular optoacoustic tomography. We present a quantitative analysis, based on synthetic MSOT datasets and observe a trend of sensitivity increase for up to 20 wavelengths. Importantly we quantify this relation and demonstrate an up to an order of magnitude sensitivity increase of multi-wavelength illumination vs. single or dual wavelength optoacoustic imaging. Examples from experimental animal studies are finally utilized to support the findings. In vivo MSOT imaging of a mouse brain bearing a tumor that is expressing a near-infrared fluorescent protein. (a) Monochromatic optoacoustic imaging at the peak excitation wavelength of the fluorescent protein. (b) Overlay of the detected bio-distribution of the protein (red pseudocolor) on the monochromatic optoacoustic image. (c) Ex vivo validation by means of cryoslicing fluorescence imaging.

Immune cell imaging using multi-spectral optoacoustic tomography.

Multispectral optoacoustic tomography (MSOT) offers the potential to image in high-resolution cells tagged with optical labels. In contrast to single wavelength imaging, multispectral excitation and spectral unmixing can differentiate labeled moieties over tissue absorption in the absence of background measurements. This feature can enable longitudinal cellular biology studies well beyond the depths reached by optical microscopy. However, the relation between spectrally resolved fluorescently labeled cells and optoacoustic detection has not been systematically investigated. Herein, we measured titrations of fluorescently labeled cells and establish the optoacoustic signal generated by these cells as a function of cell number and across different cell types. We then assess the MSOT sensitivity to resolve cells implanted in animals.

FMT-PCCT: hybrid fluorescence molecular tomography-x-ray phase-contrast CT imaging of mouse models.

The implementation of hybrid fluorescence molecular tomography (FMT) and X-ray computed tomography (CT) has been shown to be a necessary development, not only for combining anatomical with functional and molecular contrast, but also for generating optical images of high accuracy. FMT affords highly sensitive 3-D imaging of fluorescence bio-distribution, but in stand-alone form it offers images of low resolution. It was shown that FMT accuracy significantly improves by considering anatomical priors from CT. Conversely, CT generally suffers from low soft tissue contrast. Therefore utilization of CT data as prior information in FMT inversion is challenging when different internal organs are not clearly differentiated. Instead, we combined herein FMT with emerging X-ray phase-contrast CT (PCCT). PCCT relies on phase shift differences in tissue to achieve soft tissue contrast superior to conventional CT. We demonstrate for the first time FMT-PCCT imaging of different animal models, where FMT and PCCT scans were performed in vivo and ex vivo, respectively. The results show that FMT-PCCT expands the potential of FMT in imaging lesions with otherwise low or no CT contrast, while retaining the cost benefits of CT and simplicity of hybrid device realizations. The results point to the most accurate FMT performance to date.