RESUMO
In diabetes, pancreatic ß-cells play a key role. These cells are clustered within structures called islets of Langerhans inside the pancreas and produce insulin, which is directly secreted into the blood stream. The dense vascularization of islets of Langerhans is critical for maintaining a proper regulation of blood glucose homeostasis and is known to be affected from the early stage of diabetes. The deep localization of these islets inside the pancreas in the abdominal cavity renders their in vivo visualization a challenging task. A fast label-free imaging method with high spatial resolution is required to study the vascular network of islets of Langerhans. Based on these requirements, we developed a label-free and three-dimensional imaging method for observing islets of Langerhans using extended-focus Fourier domain Optical Coherence Microscopy (xfOCM). In addition to structural imaging, this system provides three-dimensional vascular network imaging and dynamic blood flow information within islets of Langerhans. We propose our method to deepen the understanding of the interconnection between diabetes and the evolution of the islet vascular network.
RESUMO
AIMS/HYPOTHESIS: It is generally accepted that structural and functional quantitative imaging of individual islets would be beneficial to elucidate the pathogenesis of type 1 diabetes. We here introduce functional optical coherence imaging (FOCI) for fast, label-free monitoring of beta cell destruction and associated alterations of islet vascularisation. METHODS: NOD mouse and human islets transplanted into the anterior chamber of the eye (ACE) were imaged with FOCI, in which the optical contrast of FOCI is based on intrinsic variations of the index of refraction resulting in a faster tomographic acquisition. In addition, the phase sensitivity allows simultaneous label-free acquisition of vascularisation. RESULTS: We demonstrate that FOCI allows longitudinal quantification of progressive autoimmune insulitis, including the three-dimensional quantification of beta cell volume, inflammation and vascularisation. The substantially increased backscattering of islets is dominated by the insulin-zinc nanocrystals in the beta cell granules. This translates into a high specificity for the functional beta cell volume of islets. Applying FOCI to a spontaneous mouse model of type 1 diabetes, we quantify the modifications of the pancreatic microvasculature accompanying the progression of diabetes and reveal a strong correlation between increasing insulitis and density of the vascular network of the islet. CONCLUSIONS/INTERPRETATION: FOCI provides a novel imaging technique for investigating functional and structural diabetes-induced alterations of the islets. The label-free detection of beta cell volume and infiltration together with vascularisation offers a unique extension to study ACE-transplanted human islets. These results are contributing to a deeper understanding of human islet transplant rejection and label-free in vivo monitoring of drug efficacy.
Assuntos
Diabetes Mellitus Tipo 1/patologia , Animais , Modelos Animais de Doenças , Genótipo , Humanos , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/patologia , Transplante das Ilhotas Pancreáticas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos KnockoutRESUMO
The identification of a beta-cell tracer is a major quest in diabetes research. However, since MRI, PET and SPECT cannot resolve individual islets, optical techniques are required to assess the specificity of these tracers. We propose to combine Optical Coherence Microscopy (OCM) with fluorescence detection in a single optical platform to facilitate these initial screening steps from cell culture up to living rodents. OCM can image islets and vascularization without any labeling. Thereby, it alleviates the need of both genetically modified mice to detect islets and injection of external dye to reveal vascularization. We characterized Cy5.5-exendin-3, an agonist of glucagon-like peptide 1 receptor (GLP1R), for which other imaging modalities have been used and can serve as a reference. Cultured cells transfected with GLP1R and incubated with Cy5.5-exendin-3 show full tracer internalization. We determined that a dose of 1 µg of Cy5.5-exendin-3 is sufficient to optically detect in vivo the tracer in islets with a high specificity. In a next step, time-lapse OCM imaging was used to monitor the rapid and specific tracer accumulation in murine islets and its persistence over hours. This optical platform represents a versatile toolbox for selecting beta-cell specific markers for diabetes research and future clinical diagnosis.
Assuntos
Carbocianinas/farmacologia , Diabetes Mellitus/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/análise , Células Secretoras de Insulina/citologia , Tomografia de Coerência Óptica/métodos , Animais , Linhagem Celular , Cricetulus , Feminino , Corantes Fluorescentes , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Camundongos , Camundongos Endogâmicos ICR , PeptídeosRESUMO
Quantitative three-dimensional blood flow imaging is a valuable technique to investigate the physiology of the brain. Two-photon microscopy (2PM) allows quantification of the local blood flow velocity with micrometric resolution by performing repeated line scans, but prohibitively long measurement times would be required to apply this technique to full three-dimensional volumes. By multiplexing the image acquisition over depth, Fourier domain optical coherence tomography (FDOCT) enables quantification of blood flow velocities with a high volume acquisition rate, albeit at a relatively low spatial resolution. Extended-focus optical coherence microscopy (xfOCM) increases the lateral resolution without sacrificing depth of field and therefore combines the high volume acquisition rate of FDOCT with a resolution comparable to 2PM. Here, we demonstrate high-resolution quantitative imaging of the blood flow velocity vector's magnitude in the adult murine brain with xfOCM.
Assuntos
Circulação Cerebrovascular , Tomografia de Coerência Óptica/métodos , Animais , Encéfalo/irrigação sanguínea , CamundongosRESUMO
We introduce photothermal optical lock-in Optical Coherence Microscopy (poli-OCM), a volumetric imaging technique, which combines the depth sectioning of OCM with the high sensitivity of photothermal microscopy while maintaining the fast acquisition speed inherent to OCM. We report on the detection of single 40 nm gold particles with a 0.5 µm lateral and 2 µm axial resolution over a 50 µm depth of field and the three-dimensional localization of gold colloids within living cells. In combination with intrinsic sample contrast measured with dark-field OCM, poli-OCM offers a versatile platform for functional cell imaging.
Assuntos
Ouro/química , Imageamento Tridimensional/métodos , Nanopartículas Metálicas/química , Microscopia/métodos , Fenômenos Ópticos , Temperatura , Sobrevivência Celular , Dimetilpolisiloxanos/química , Células HeLa , Humanos , Razão Sinal-RuídoRESUMO
We demonstrate label-free imaging of cerebral ß-amyloidosis ex vivo and in a living mouse model of Alzheimer's disease using extended-focus Fourier domain optical coherence microscopy (xfOCM). xfOCM provides 3D, high-resolution images of individual ß-amyloid plaques in the brain parenchyma and vasculature and requires no staining of the alzheimeric sample under investigation. xfOCM also opens the possibility to perform minimally invasive studies of ß-amyloid pathology in vivo, without the use of labeling methods, which potentially confound experimental findings.
Assuntos
Peptídeos beta-Amiloides/química , Angiopatia Amiloide Cerebral/patologia , Modelos Animais de Doenças , Tomografia de Coerência Óptica/métodos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Animais , Angiopatia Amiloide Cerebral/genética , Angiopatia Amiloide Cerebral/metabolismo , Análise de Fourier , Humanos , Camundongos , Camundongos Transgênicos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Neuroimagem/instrumentação , Neuroimagem/métodos , Placa Amiloide/genética , Placa Amiloide/patologia , Coloração e Rotulagem , Tomografia de Coerência Óptica/instrumentaçãoRESUMO
Diabetes is characterized by hyperglycemia that can result from the loss of pancreatic insulin secreting ß-cells in the islets of Langerhans. We analyzed ex vivo the entire gastric and duodenal lobes of a murine pancreas using extended-focus Optical Coherence Microscopy (xfOCM). To identify and quantify the islets of Langerhans observed in xfOCM tomograms we implemented an active contour algorithm based on the level set method. We show that xfOCM reveals a three-dimensional islet distribution consistent with Optical Projection Tomography, albeit with a higher resolution that also enables the detection of the smallest islets (≤ 8000 µm(3)). Although this category of the smallest islets represents only a negligible volume compared to the total ß-cell volume, a recent study suggests that these islets, located at the periphery, are the first to be destroyed when type I diabetes develops. Our results underline the capability of xfOCM to contribute to the understanding of the development of diabetes, especially when considering islet volume distribution instead of the total ß-cell volume only.