Micro-modulated luminescence tomography

Imaging depth of optical microscopy has been fundamentally limited to millimeter or sub-millimeter due to light scattering. X-ray microscopy can resolve spatial details of few microns deeply inside a sample but the contrast resolution is still inadequate to depict heterogeneous features at cellular or sub-cellular levels. To enhance and enrich biological contrast at large imaging depth, various nanoparticles are introduced and become essential to basic research and molecular medicine. Nanoparticles can be functionalized as imaging probes, similar to fluorescent and bioluminescent proteins. LiGa5O8:Cr3+ nanoparticles were recently synthesized to facilitate luminescence energy storage with x-ray pre-excitation and the subsequently stimulated luminescence emission by visible/near-infrared (NIR) light. In this paper, we suggest a micro-modulated luminescence tomography (MLT) approach to quantify a nanophosphor distribution in a thick biological sample with high resolution. Our numerical simulation studies demonstrate the feasibility of the proposed approach.


Introduction
Systems biology is devoted to comprehensive studies of biological components with interrelated mechanisms across resolution scales over six orders of magnitude, involving molecules, sub-cellular features, cells, organisms, and entire species [1]. Living systems are highly complicated, dynamic, and often unpredictable. To understand and manipulate these systems, quantitative measurements of interacting components and clusters are necessary using systematic and microscopic technologies such as microscopies, genomics, proteomics, bioinformatics, in vivo imaging, and computational models. Regenerative medicine utilizes principles of biology and engineering to develop and transplant engineered substitute tissues and organs [2], with various protocols for cell seeding onto porous scaffolds during incubation [3]. These constructs are then expected to restore or regenerate functionality of diseased tissues or organs. Engineered tissue growths are rather sophisticated, and as natural biological counterparts they usually recapitulate normal developmental processes [4]. Hence, systematic and microscopic technologies are critical for evaluating engineered tissue prior and post implementation. Molecular and cellular probes have versatile and sophisticated labeling capabilities, and are considered instrumental for systems biology, tissue engineering, and molecular medicine. There is a tremendous interest in biocompatible nanoparticles for in situ or in vivo molecular imaging, drug delivery, and targeted therapy [5]. Optical imaging is a primary methodology to sensitively visualize nanoparticles tagged to specific molecules and cells [6,7]. A typical example of their applications is cancer research [8,9], which employs nanoparticles to deliver drug, heat, or light to cancer cells [10]. Another example is tissue engineering. With multifunctional nanoparticles, engineered tissue constructs can not only be monitored at cellular and molecular levels but also stimulated and regularized by multiple physical means for optimal functionalities. These nanoparticle ingredients are particularly important for the paradigm shift from 2D to 3D matrices in tissue engineering.
Microscopy is the principal observational tool and has made important contributions to our understanding of biological systems and engineered tissues [11]. However, imaging depth of optical microscopy has been fundamentally limited to millimeter or sub-millimeter due to light scattering. Conventional microscopy techniques utilize visible light or electron sources [12][13][14]. Optical microscopy divides into transmission (i.e., wide-field microscopies for snapshot of 2D images in light absorption, phase contrast, or dark-field modes) and emission modes (i.e., wide-field fluorescence microscopy, confocal laser scanning microscopy, and two-photon fluorescence microscopy). These microscopic modalities are good for in vitro or in vivo studies of cultured cell/tissue samples or small animals [15]. Inherently, the resolution of optical microscopy is diffraction-limited by ~200nm with single objective techniques and ~120nm with confocal techniques. With appropriate sample preparation, stochastic information and innovative interference techniques, ~100nm resolution is achievable. Threedimensional image cubes can be obtained with optical sectioning of ~200nm lateral resolution and ~500nm axial resolution. Ultimately, multiple scattering prevents these techniques from imaging thick samples. Photoacoustic tomography permits scalable resolution at imaging depths up to ~7cm with a depth-to-resolution ratio ~200. Photoacoustic microscopy aims at millimeter imaging depth, micron-scale resolution and absorption contrast, which could be used to characterize the structure of the scaffold but cannot compete with the sensitivity of fluorescence imaging [15,16].
To enhance biological specifications, various nanoparticles are introduced and become essential to molecular medicine. Nanoparticles can be functionalized as imaging probes, similar to fluorescent and bioluminescent proteins. Unlike conventional nanoparticle probes, LiGa 5 O 8 :Cr 3+ nanoparticles were recently synthesized to facilitate the luminescence energy storage with x-ray pre-excitation and the subsequently stimulated luminescence emission by visible/near-infrared (NIR) light.
Our idea is to use a micro-modulated x-ray engraving a distribution of nanophosphors which can then have luminescence energy stored for detailed tomographic imaging deeply in tissue samples. Our goal is to reveal a distribution of LiGa 5 O 8 :Cr 3+ nanoparticles targeting specific molecular and cellular aggregates, pathways and responses in engineered tissue samples of several millimeters in size and a few microns in resolution, overcoming the imaging depth limit of all other optical microscopic methods. In this micro-modulated luminescence tomography (MLT) process, tomographic data will be acquired via nearinfrared (NIR) light stimulation and optical multiplexing. In the following section, we will describe a system design, a photon transport model, and an image reconstruction algorithm for MLT. In the third section, we report our realistic numerical simulation results. In the last section, we discuss relevant issues and conclude the paper.

System Prototyping
System Architecture: The proposed MLT system architecture consists of a micro-focus x-ray source, an x-ray zone plate, two EMCCD cameras, NIR laser stimulation sources, and a rotating stage, as shown in Fig. 1. An aluminum filter is used to have a polychromatic xray spectrum of 10kev-20kev. As a standard x-ray microscopic imaging component, the zone plate consists of radially symmetric x-ray transparent rings. The width n dr of a ring decreases with increment of its radius n r . The focal length f of a zone plate is a function of its diameter D , its outermost zone width n dr and the x-ray When an object intersects an x-ray focal line, the exposed nanophosphors in the object can be excited by x-rays. Upon NIR light stimulation, the stored x-ray energy will be released by luminescence emission from the x-ray pre-excited nanophosphors to be detected by the CCD camera. All the components will be integrated on an optical table in a light-proof box made of aluminum posts and blackened panels. The x-ray source will be mounted on a horizontally-motorized linear stage (ILS300LM, Newport) for focal plane adjustment. The xrays can be collimated into a narrow-angle cone-beam to irradiate the Fresnel zone plate. The zone plate is placed within a five axis lens positioner (IP-05A, Newport) to focus polychromatic x-rays to different foci along a line segment for well-localized x-ray excitation of nanophosphors in a phantom or sample. The object to be studied will be placed on a rotational stage (URS75BCC, Newport) on a 3D combination of linear stages (VP-25XA, VP-5ZA, Newport) for x-y-z adjustment. The EMCCD camera (iXon3 897, Andor Technology) has the sensitive 512×512 matrix with pixel size 16×16μm 2 . The two cameras are faced each other with the object in between for simultaneous data acquisition. NIR laser beams are expanded and controlled to stimulate nanoparticles for luminescence imaging.

Photon Transport Model and Reconstruction Algorithm
Photon Transport Model: MLT involves NIR light stimulation to make energy-stored nanophosphors emit luminescence photons. A light propagation model is needed to describe interactions of light photons with scattering and absorbing media, which is essential for MLT image reconstruction [20,21]. For biological samples, the diffusion approximation model, a computationally-efficient approximation to the radiative transport equation (RTE), would break down with strong absorbers, near sources, and across boundaries [22,23]. In that case, either RTE itself or an alternative photon transport model will be needed to accurately describe the photon propagation in biological tissue [23,24].
For MLT, here we propose an analytic solution of RTE assuming an infinite-space medium. Our solution can be obtained via spherical harmonics approximation [25], ( For optical imaging of biological samples, the tissue boundary must be taken into account when analyzing the photon propagation. The luminescence photon propagation in biological tissue is subject to both scattering and absorption. A significant amount of photons go across the tissue boundary and can be detected by a highly sensitive CCD camera. In this scenario, the photon propagation process can be well modeled using a semi-infinite slab. For that purpose, we use the extrapolated boundary condition, which is simple and has been shown to agree well with the MC simulation and physical measurement [26,27]. An image source is used to construct a fluence rate solution such that  