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In a tunable 3D structured illumination microscopy (3D-SIM) based on an illumination system comprised by a multi-slit array and a Fresnel biprism, the 3D structured illumination (SI) pattern depends on the design of the slit array. In this work, we present an extension of SPARCOM to the more challenging three-dimensional scenario, where we recover a volume from a set of recorded frames, rather than an image. However, SPARCOM has only been formulated for the two-dimensional setting, where the sample is assumed to be an infinitely thin single-layer, and thus is unsuitable to most biological specimens. One of these methods is the sparsity-based approach for super-resolution microscopy from correlation information of high emitter-density frames, dubbed SPARCOM, which utilizes sparsity in the correlation domain while assuming that the blinking emitters are uncorrelated over time and space, yielding both high temporal and spatial resolution. In recent years, a variety of approaches have been suggested to reduce imaging times by allowing the use of higher emitter densities. Thus, this concept in its classical form has low temporal resolution, limiting its application to slow-changing specimens.
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However, the low emitter density concept requires lengthy imaging times to achieve full coverage of the imaged specimen on the one hand, and minimal overlap on the other. In the single-molecule localization microscopy (SMLM) approach, a sequence of diffraction-limited images, produced by a sparse set of emitting fluorophores with minimally overlapping point- spread functions is acquired, allowing the emitters to be localized with high precision by simple post-processing. In conclusion, transgenic rats provide a platform to visualize cAMP signals in-vitro and in-situ.īreakthroughs in the field of chemistry have enabled surpassing the classical optical diffraction limit by utilizing photo-activated fluorescent molecules. Tissues including lungs, heart, mesentery, kidney, expressed the H187 FRET sensor. Samples were excited using 405 nm and 561 nm lasers and emission was collected from 424 nm -724 nm at 10 nm intervals. Hyperspectral z-stacks of tissues and vessels were acquired using a Nikon A1R confocal microscope equipped with 20X multi-immersion objective and a 32 channel PMT detector. In current studies, we utilize hyperspectral imaging and analysis approaches to characterize the distribution of FRET signals in different tissues and vessels harvested from transgenic rats. We developed a transgenic rat model that expresses the H187 FRET sensor. In many cell types including PMVECs, transient transfection of FRET sensors results in a low transfection efficiency (1-5%), which limits the possibilities of studying spatial distribution of FRET signals in many cells simultaneously. In previous studies, we used 4-dimensional (x, y, z, and λ) hyperspectral imaging and analysis approaches to measure agonist – induced distribution of cAMP signals in cultured pulmonary microvascular endothelial cells. However, FRET sensors inherently offer low signal-to-noise ratios, making cAMP measurements challenging, especially in 3 spatial dimensions. Fӧrster resonance energy transfer (FRET) based cAMP sensors have become a standard approach to visualize cAMP signals in cells.
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