Mehmet S. Ozturk

Mesoscopic fluorescence molecular tomography of reporter genes in bioprinted thick tissue

Abstract. Three-dimensional imaging of thick tissue constructs is one of the main challenges in the field of tissue engineering and regenerative medicine. Optical methods are the most promising as they offer noninvasive, fast, and inexpensive solutions. Herein, we report the use of mesoscopic fluorescence molecular tomography (MFMT) to image function and structure of thick bioprinted tissue hosted in a 3-mm-thick bioreactor. Collagen-based tissue assembled in this study contains two vascular channels formed by green fluorescent protein- and mCherry-expressing cells. Transfected live cell imaging enables us to image function, whereas Flash Red fluorescent bead perfusion into the vascular channel allows us to image structure. The MFMT optical reconstructions are benchmarked with classical microscopy techniques. MFMT and wide-field fluorescence microscopy data match within 92% in area and 84% in location, validating the accuracy of MFMT reconstructions. Our results demonstrate that MFMT is a well-suited imaging modality for fast, longitudinal, functional imaging of thick, and turbid tissue engineering constructs.

Mesoscopic Fluorescence Tomography of a Photosensitizer (HPPH) 3D Biodistribution in Skin Cancer

RATIONALE AND OBJECTIVES Photodynamic therapy (PDT) is a promising strategy for treating cancer. PDT involves three components: a photosensitizer (PS) drug, a specific wavelength of drug-activating light, and oxygen. A challenge in PDT is the unknown biodistribution of the PS in the target tissue. In this preliminary study, we report the development of a new approach to image in three dimensions the PS biodistribution in a noninvasive and fast manner. MATERIALS AND METHODS A mesoscopic fluorescence tomography imaging platform was used to image noninvasively the biodistribution of 2-[1-hexyloxyethyl]-2 devinyl pyropheophorbide-a (HPPH) in preclinical skin cancer models. Seven tumors were imaged and optical reconstructions were compared to nonconcurrent ultrasound data. RESULTS Successful imaging of the HPPH biodistribution was achieved on seven skin cancer tumors in preclinical models with a typical acquisition time of 1 minute. Two-dimensional fluorescence signals and estimated three-dimensional PS distributions were located within the lesions. However, HPPH distribution was highly heterogeneous with the tumors. Moreover, HPPH distribution volume and tumor volume as estimated by ultrasound did not match. CONCLUSIONS The results of this proof-of-concept study demonstrate the potential of MFMT to image rapidly the HPPH three-dimensional biodistribution in skin cancers. In addition, these preliminary data indicate that the PS biodistribution in skin cancer tumors is heterogeneous and does not match anatomical data. Mesoscopic fluorescence molecular tomography, by imaging fluorescence signals over large areas with high spatial sampling and at fast acquisition speeds, may be a new imaging modality of choice for planning and optimizing of PDT treatment.

Mesoscopic Fluorescence Molecular Tomography for Evaluating Engineered Tissues

Optimization of regenerative medicine strategies includes the design of biomaterials, development of cell-seeding methods, and control of cell-biomaterial interactions within the engineered tissues. Among these steps, one paramount challenge is to non-destructively image the engineered tissues in their entirety to assess structure, function, and molecular expression. It is especially important to be able to enable cell phenotyping and monitor the distribution and migration of cells throughout the bulk scaffold. Advanced fluorescence microscopic techniques are commonly employed to perform such tasks; however, they are limited to superficial examination of tissue constructs. Therefore, the field of tissue engineering and regenerative medicine would greatly benefit from the development of molecular imaging techniques which are capable of non-destructive imaging of three-dimensional cellular distribution and maturation within a tissue-engineered scaffold beyond the limited depth of current microscopic techniques. In this review, we focus on an emerging depth-resolved optical mesoscopic imaging technique, termed laminar optical tomography (LOT) or mesoscopic fluorescence molecular tomography (MFMT), which enables longitudinal imaging of cellular distribution in thick tissue engineering constructs at depths of a few millimeters and with relatively high resolution. The physical principle, image formation, and instrumentation of LOT/MFMT systems are introduced. Representative applications in tissue engineering include imaging the distribution of human mesenchymal stem cells embedded in hydrogels, imaging of bio-printed tissues, and in vivo applications.

High-Resolution Mesoscopic Fluorescence Molecular Tomography Based on Compressive Sensing

Mesoscopic fluorescence molecular tomography (MFMT) is new imaging modality aiming at 3-D imaging of molecular probes in a few millimeter thick biological samples with high-spatial resolution. In this paper, we develop a compressive sensing-based reconstruction method with l1-norm regularization for MFMT with the goal of improving spatial resolution and stability of the optical inverse problem. Three-dimensional numerical simulations of anatomically accurate microvasculature and real data obtained from phantom experiments are employed to evaluate the merits of the proposed method. Experimental results show that the proposed method can achieve 80 μm spatial resolution for a biological sample of 3 mm thickness and more accurate quantifications of concentrations and locations for the fluorophore distribution than those of the conventional methods.

3D Bioprinting and 3D Imaging for Stem Cell Engineering

Three-dimensional (3D) bio-printing, a technology to create 3D tissue through layer-by-layer approach, offers great capacity to engineer tissue with desired cells, growth factors and biomaterial scaffolds in spatial patterns to mimic the native tissue architecture. With its flexibility and power, the 3D bio-printing technology can also be used to control stem cell fate and creating 3D stem cell niches. Meanwhile, 3D bio-printed tissues often incorporate thick opaque scaffold, dense population of cells, and are often large in size (1–100 mm). Thus, there are significant difficulties in visualizing the biological events within thick tissue constructs using current microscopic techniques. To elucidate the interaction of stem cells with the microenvironment in tissue engineering applications, it is necessary to develop novel molecular imaging techniques to non-invasively observe stem cell fate, cell-cell interactions, and structural features of an engineered tissue in real time. In this chapter, we review the usage of bio-printing technologies in stem cell and tissue engineering application, and the most recent development in the optical molecular imaging techniques for thick tissue imaging.

Dental imaging using laminar optical tomography and micro CT

Dental lesions located in the pulp are quite difficult to identify based on anatomical contrast, and, hence, to diagnose using traditional imaging methods such as dental CT. However, such lesions could lead to functional and/or molecular optical contrast. Herein, we report on the preliminary investigation of using Laminar Optical Tomography (LOT) to image the pulp and root canals in teeth. LOT is a non-contact, high resolution, molecular and functional mesoscopic optical imaging modality. To investigate the potential of LOT for dental imaging, we injected an optical dye into ex vivo teeth samples and imaged them using LOT and micro-CT simultaneously. A rigid image registration between the LOT and micro-CT reconstruction was obtained, validating the potential of LOT to image molecular optical contrast deep in the teeth with accuracy, non-invasively. We demonstrate that LOT can retrieve the 3D bio-distribution of molecular probes at depths up to 2mm with a resolution of several hundred microns in teeth.

Improving mesoscopic fluorescence molecular tomography through data reduction

Mesoscopic fluorescence molecular tomography (MFMT) is a novel imaging technique that aims at obtaining the 3-D distribution of molecular probes inside biological tissues at depths of a few millimeters. To achieve high resolution, around 100-150μm scale in turbid samples, dense spatial sampling strategies are required. However, a large number of optodes leads to sizable forward and inverse problems that can be challenging to compute efficiently. In this work, we propose a two-step data reduction strategy to accelerate the inverse problem and improve robustness. First, data selection is performed via signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) criteria. Then principal component analysis (PCA) is applied to further reduce the size of the sensitivity matrix. We perform numerical simulations and phantom experiments to validate the effectiveness of the proposed strategy. In both in silico and in vitro cases, we are able to significantly improve the quality of MFMT reconstructions while reducing the computation times by close to a factor of two.

Biofabrication and 3D localization of multilayered cellular constructs using Laser Direct-Write and Mesoscopic Fluorescent Molecular Tomography

Recent advances in tissue engineering applications demand micro-scale precision in both design and evaluation. Herein, we aim to meet these challenges through a combination of two innovative techniques: gelatin-based Laser Direct-Write (LDW) and Mesoscopic Fluorescence Molecular Tomography (MFMT). LDW was used to precisely pattern cell-loaded, alginate microbeads, in layer-by-layer fashion, to construct thick, multilayered cellular constructs. Then, MFMT was utilized to deeply assess and 3D-model cellular localization. The combination of these two techniques may provide the accuracy and precision needed for the analysis of tissue engineering in 3D.

Longitudinal Volumetric Assessment of Glioblastoma Brain Tumor in 3D Bio-Printed Environment by Mesoscopic Fluorescence Molecular Tomography

3D optical reconstruction of in-vitro Glioblastoma brain tumors was evaluated longitudinally with Mesoscopic Fluorescence Molecular Tomography. Tumor response to the Temozolomide, a clinical drug, were evaluated through volumetric change.

SNR characterization of Mesoscopic Fluorescence Molecular Tomography with EMCCD camera

Progress in bio-printing techniques has accentuated the need for non-invasive imaging modalities with high sensitivity, high resolution, and imaging depth of a few millimeters. Mesoscopic Fluorescence Molecular Tomography (MFMT) is a promising imaging modality for 3D localization and quantification of fluorescent labeled cells in thick scaffolds. Here we report on the characterization of our second generation MFMT system which uses an Electron-Multiplying charge coupled device (EMCCD). More precisely, we report on the effects of EM gain and deep cooling of the camera on signal-to-noise ratio (SNR) and hence the sensitivity. Experimental results showed that these parameters, when used in conjunction, increased SNR by at least 3 fold.