Techniques and Applications of Mesoscopic Fluorescence Imaging

There is increasing interest towards visualization of tissue level with deep penetration depth in bioscience and medical research, mesoscopic imaging exchanges resolution for penetration depth, which offers hundreds of micrometer resolution and up to several millimeter penetration depth. By introducing fluorescence dyes or with intrinsic fluorescence, much higher contrast of images can be recorded compared to reflection imaging. To assess the characteristics of fluorescence imaging system, in the first part of this thesis I will discuss 3D printing technique as a novel phantom fabrication method which enables the fabrication of optically realistic and morphologically complex tissue-simulating phantoms for the development and evaluation of optical imaging products. In the second part, I will discuss about the techniques and applications of Angled fluorescence laminar optical tomography (aFLOT), a modified fluorescence tomographic imaging technique based on 3D reconstructions. To extend the capability of aFLOT to acquire more bioinformation besides, its availability for quantification and statistics has been studied. Some technical improvements of aFLOT system performance has also been taken by incorporating algorithms for imaging processing. In addition, examples of biomedical applications have been discussed to demonstrate the capability of aFLOT system in both bioscience and medical research field

Diffuse reflectance spectroscopy for determination of optical properties and chromophore concentrations of mice internal organs in the range of 350 nm to 1860 nm

The development of photomedical modalities for diagnostics and treatment has created a need for knowledge of the optical properties of the targeted biological tissues. These properties are essential to plan certain procedures, since they determine the light absorption, propagation and penetration in tissues. One way to measure these properties is based on diffuse reflectance spectroscopy (DRS). DRS can provide light absorption and scattering coefficients for each wavelength through a non-invasive, fast and in situ interrogation, and thereby tissue biochemical information. In this study, reflectance measurements of ex vivo mice organs were investigated in a wavelength range between 350 and 1860 nm. To the best of our knowledge, this range is broader than previous studies reported in the literature and is useful to study additional chromophores with absorption in the extended wavelength range. Also, it may provide a more accurate concentration of tissue chromophores when fitting the reflectance spectrum in this extended range. In order to extract these concentrations, optical properties were calculated in a wide spectral range through a fitting routine based on an inverse Monte-Carlo look-up table model. Measurements variability was assessed by calculating the Pearson correlation coefficients between each pair of measured spectra of the same type of organ

On The Development of a Dynamic Contrast-Enhanced Near-Infrared Technique to Measure Cerebral Blood Flow in the Neurocritical Care Unit

A dynamic contrast-enhanced (DCE) near-infrared (NIR) method to measure cerebral blood flow (CBF) in the neurocritical care unit (NCU) is described. A primary concern in managing patients with acquired brain injury (ABI) is onset of delayed ischemic injury (DII) caused by complications during the days to weeks following the initial insult, resulting in reduced CBF and impaired oxygen delivery. The development of a safe, portable, and quantitative DCE-NIR method for measuring CBF in NCU patients is addressed by focusing on four main areas: designing a clinically compatible instrument, developing an appropriate analytical framework, creating a relevant ABI animal model, and validating the method against CT perfusion. In Chapter 2, depth-resolved continuous-wave NIR recovered values of CBF in a juvenile pig show strong correlation with CT perfusion CBF during mild ischemia and hyperemia (r=0.84, p\u3c0.001). In particular, subject-specific light propagation modeling reduces the variability caused by extracerebral layer contamination. In Chapter 3, time-resolved (TR) NIR improves the signal sensitivity to brain tissue, and a relative CBF index is be both sensitive and specific to flow changes in the brain. In particular, when compared with the change in CBF measured with CT perfusion during hypocapnia, the deconvolution-based index has an error of 0.8%, compared to 21.8% with the time-to-peak method. To enable measurement of absolute CBF, a method for characterizing the AIF is described in Chapter 4, and the theoretical basis for an advanced analytical framework—the kinetic deconvolution optical reconstruction (KDOR)—is provided in Chapter 5. Finally, a multichannel TR-NIR system is combined with KDOR to quantify CBF in an adult pig model of ischemia (Chapter 6). In this final study, measurements of CBF obtained with the DCE-NIR technique show strong agreement with CT perfusion measurements of CBF in mild and moderate ischemia (r=0.86, p\u3c0.001). The principle conclusion of this thesis is that the DCE-NIR method, combining multidistance TR instrumentation with the KDOR analytical framework, can recover CBF values that are in strong agreement with CT perfusion values of CBF. Ultimately, bedside CBF measurements could improve clinical management of ABI by detecting delayed ischemia before permanent brain damage occurs

Near-Infrared Quantum Dots For Bioimaging And Targeting Applications

<p>Luminescent semiconductor nanocrystals or quantum dots (QDs) offer attractive characteristics as a new class of fluorescent probes for molecular, cellular and in vivo imaging. While traditional cadmium-containing QDs have been widely used in biomedical research, diagnostics, and drug delivery, the cytotoxicity arising from the release of Cd2+ ions caused by the degradation of the surface coating is deemed to be a shortfall of cadmium-based QDs for long-term cellular and in vivo imaging. Here we report a direct synthesis of silver-doped zinc selenide QDs in water with near-infrared tunable fluorescence emissions, coinciding with the biological window of transmission to offer high signal-to-noise for fluorescence imaging of cells and small animals. Glutathione, which carries both carboxyl and amino groups, serves as a stabilizing ligand and offers the flexibility of decorating the surface of the QDs with moieties such as proteins, peptides and DNA. The cytotoxicity of the as-synthesized QDs was evaluated on macrophage (RAW 264.7) cells and human mesenschymal stem cells using MTS cell viability assay. The results indicated that the silver- doped ZnSe QDs possess low cytotoxicity. In vivo biodistribution study shows that these bare QDs are different from conventional QDs, it traversed through systemic route and could accumulate in the stomach of nude mice. These QDs were conjugated to monoclonal CD44v6 antibody and tested with human gastric adenocarcinoma cell line (AGS). The results indicated the feasibility of modifying the surface properties of these QDs for efficient targeting applications. The QDs were also conjugated to heparin and used to formulate nanocomplexes with chitosan to encapsulate tumor necrosis factor-alpha. Quantitative imaging analysis revealed in vivo trafficking kinetics of the nanocomplexes to the lymph nodes after subcutaneous administration into nude mice. This study demonstrates the potential of incorporation of near-infrared-emitting QDs in nanocarrier drug delivery that allows in vivo trafficking of the biodistriution events and will be of greatly improve the development new drug nanocarrier formulations.</p>Dissertatio