Coherent Imaging of Cellular Dynamics

Mitochondrial dynamics refers to the processes of fusion, fission, and transport that aid mitochondria in accomplishing their many roles; including ATP production, oxygen sensing, and homeostasis. Due to their involvement in numerous essential cellular activity, dysfunctional mitochondria have been implicated in a wide range of human diseases. Confocal microscopy using fluorophores for molecular specificity remains the gold standard of intracellular imaging. However, fluorescent labels can be toxic to the cell upon prolonged exposure and still suffer from photobleaching. These compromise the application of confocal fluorescence microscopy for true long lasting time-lapse imaging of living samples. Optical coherence microscopy (OCM) exploits the intrinsic variation in the scattering properties of the sample to achieve fast, label-free, and highly sensitive three-dimensional imaging. Unfortunately, being label-free means OCM lacks specificity and coherence based imaging techniques have no counterpart to fluorescent markers. The invention of the photothermal optical lock-in OCM (poli-OCM) brought about the possibility of specific OCM imaging using gold nanoparticles (AuNP) as photothermal bio-markers. The use of AuNPs as specific contrast agents has substantial advantages stemming from their well-established biocompatibility and photostability. Microscopic techniques that offer fast three-dimensional imaging over extended time durations would do well in revealing previously inaccessible knowledge on mitochondria. In this work, we quantify mitochondrial dynamics based on specific poli-OCM and surface functionalization of AuNPs. In realizing mitochondria specific poli-OCM imaging, it is necessary to functionalize AuNP with mitochondria targeting capabilities. We present copolymer surface coatings that provide the AuNPs with improved stability, solubility, and cellular uptake on top of mitochondria labeling. We further optimize the utilization of these AuNP labels for poli-OCM imaging. We also demonstrated poli-OCM imaging with differently structured gold nanolabels, which could lead to the realization of multimodal imaging using a single bio-marker. The two quantification techniques we developed were based on (1) temporal autocorrelation analysis combined with a classical diffusion model and (2) single particle tracking. Autocorrelation analysis is the foundation of fluorescence correlation spectroscopy (FCS); a technique extensively used for analyzing dynamic phenomena in chemistry and biophysics. We extend this analysis to three-dimensional poli-OCM imaging allowing us to map quantified mitochondrial diffusion parameters in three dimensions within the cell. We also investigate how the size of the mitochondria with respect to the point spread function (PSF) of the poli-OCM impacts the result of our autocorrelation analysis. Single particle tracking complements our temporal autocorrelation analysis since recent advances in localization and tracking algorithms have demonstrated precision better than the size of the PSF. Finally, we demonstrate the possibility of using mitochondria specific poli-OCM imaging with the quantification techniques we developed in studying the Cockayne syndrome (CS). CS is a very rare and fatal genetic disease that has been associated with mitochondrial dysfunction. To our knowledge, no study has been conducted focusing on quantifying the effect of CS on mitochondrial dynamics

Visible spectrum extended-focus optical coherence microscopy for label-free sub-cellular tomography

We present a novel extended-focus optical coherence microscope (OCM) attaining 0.7 {\mu}m axial and 0.4 {\mu}m lateral resolution maintained over a depth of 40 {\mu}m, while preserving the advantages of Fourier domain OCM. Our method uses an ultra-broad spectrum from a super- continuum laser source. As the spectrum spans from near-infrared to visible wavelengths (240 nm in bandwidth), we call the method visOCM. The combination of such a broad spectrum with a high-NA objective creates an almost isotropic 3D submicron resolution. We analyze the imaging performance of visOCM on microbead samples and demonstrate its image quality on cell cultures and ex-vivo mouse brain tissue.Comment: 15 pages, 7 figure

3D Time-lapse Imaging and Quantification of Mitochondrial Dynamics

We present a 3D time-lapse imaging method for monitoring mitochondrial dynamics in living HeLa cells based on photothermal optical coherence microscopy and using novel surface functionalization of gold nanoparticles. The biocompatible protein-based biopolymer coating contains multiple functional groups which impart better cellular uptake and mitochondria targeting efficiency. The high stability of the gold nanoparticles allows continuous imaging over an extended time up to 3000 seconds without significant cell damage. By combining temporal autocorrelation analysis with a classical diffusion model, we quantify mitochondrial dynamics and cast these results into 3D maps showing the heterogeneity of diffusion parameters across the whole cell volume

Perspectivas clásicas y modernas de las virtudes en la empresa (II)".

Este cuaderno contiene: "En busca de la virtud: el papel de las virtudes, los valores y las fortalezas de carácter en la toma de decisiones éticas" "Participar en el bien común de la empresa" "Antes de la virtud: biología, cerebro, comportamiento y ‘sentido moral’" "La posibilidad de la virtud

Optical projection tomography for rapid whole mouse brain imaging

In recent years, three-dimensional mesoscopic imaging has gained significant importance in life sciences for fundamental studies at the whole-organ level. In this manuscript, we present an optical projection tomography (OPT) method designed for imaging of the intact mouse brain. The system features an isotropic resolution of ~50 μm and an acquisition time of four to eight minutes, using a 3-day optimized clearing protocol. Imaging of the brain autofluorescence in 3D reveals details of the neuroanatomy, while the use of fluorescent labels displays the vascular network and amyloid deposition in 5xFAD mice, an important model of Alzheimer’s disease (AD). Finally, the OPT images are compared with histological slices

A Second-Generation Device for Automated Training and Quantitative Behavior Analyses of Molecularly-Tractable Model Organisms

A deep understanding of cognitive processes requires functional, quantitative analyses of the steps leading from genetics and the development of nervous system structure to behavior. Molecularly-tractable model systems such as Xenopus laevis and planaria offer an unprecedented opportunity to dissect the mechanisms determining the complex structure of the brain and CNS. A standardized platform that facilitated quantitative analysis of behavior would make a significant impact on evolutionary ethology, neuropharmacology, and cognitive science. While some animal tracking systems exist, the available systems do not allow automated training (feedback to individual subjects in real time, which is necessary for operant conditioning assays). The lack of standardization in the field, and the numerous technical challenges that face the development of a versatile system with the necessary capabilities, comprise a significant barrier keeping molecular developmental biology labs from integrating behavior analysis endpoints into their pharmacological and genetic perturbations. Here we report the development of a second-generation system that is a highly flexible, powerful machine vision and environmental control platform. In order to enable multidisciplinary studies aimed at understanding the roles of genes in brain function and behavior, and aid other laboratories that do not have the facilities to undergo complex engineering development, we describe the device and the problems that it overcomes. We also present sample data using frog tadpoles and flatworms to illustrate its use. Having solved significant engineering challenges in its construction, the resulting design is a relatively inexpensive instrument of wide relevance for several fields, and will accelerate interdisciplinary discovery in pharmacology, neurobiology, regenerative medicine, and cognitive science

Imaging of proteins’ organization in 3D using Single Molecule Orientation and Localization Microscopy (SMOLM)

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Nonlinear imaging of 3D molecular orientation in cells and tissues by polarization splitting detection.

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Lipids−Fluorophores Interactions Probed by Combined Nonlinear Polarized Microscopy

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Lipids–Fluorophores Interactions Probed by Combined Nonlinear Polarized Microscopy

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