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Successful optimization of reconstruction parameters in structured illumination microscopy – A practical guide
摘要： The impact of the different reconstruction parameters in super-resolution structured illumination microscopy (SIM) on image artifacts is carefully analyzed. These parameters comprise the Wiener filter parameter, an apodization function, zero-frequency suppression and modifications of the optical transfer function. A detailed investigation of the reconstructed image spectrum is concluded to be suitable for identifying artifacts. For this purpose, two samples, an artificial test slide and a more realistic biological system, were used to characterize the artifact classes and their correlation with the image spectra as well as the reconstruction parameters. In addition, a guideline for efficient parameter optimization is suggested and the implementation of the parameters in selected up-to-date processing packages (proprietary and open-source) is depicted.
关键词： Super resolution,Fluorescence microscopy,Structured illumination microscopy,Parameter estimation,Image reconstruction
更新于2025-09-23 15:23:52
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<i>De Novo</i> -Designed Near-Infrared Nanoaggregates for Super-Resolution Monitoring of Lysosomes in Cells, in Whole Organoids, and <i>in Vivo</i>
摘要： As the cleaners of cells, lysosomes play an important role in circulating organic matter within cells, recovering damaged organelles, and removing waste via endocytosis. Because lysosome dysfunction is associated with various diseases—lysosomal storage diseases, inherited diseases, rheumatoid arthritis, and even shock—it is vital to monitor the movement of lysosomes in cells and in vivo. To that purpose, a method of optical imaging, super-resolution imaging technology (e.g., SIM and STORM), can overcome the limitations of traditional optical imaging and afford a range of possibilities for fluorescence imaging. However, the short wavelength excitation and easy photobleaching of super-resolution fluorescence probes somewhat problematize super-resolution imaging. As described herein, we designed a low-toxicity, photostable, near-infrared small molecule fluorescence probe HD-Br for use in the super-resolution imaging of lysosomes. The interaction of lysosomes and mitochondria was dynamically traced while using the probe’s properties to label the lysosomes. Because the probe has the optimal near-infrared excitation and emission wavelengths, liver organoid 3D imaging and Caenorhabditis elegans imaging were also performed. Altogether, our findings indicate valuable approaches and techniques for super-resolution 3D and in vivo imaging.
关键词： lysosome-targeted,3D organoids imaging,C. elegans imaging,structured illumination microscopy,nanoaggregates
更新于2025-09-11 14:15:04
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Time-Resolved Structured Illumination Microscopy for Phase Separation Dynamics of Water and 2-Butoxyethanol Mixtures: Interpretation of “Early Stage” Involving Micelle-Like Structures
摘要： Phase separation dynamics of a water/2-butoxyethanol (2BE) mixture was studied with newly developed time-resolved structured illumination microscopy (SIM). Interestingly, an employed hydrophobic fluorescent probe for SIM showed spectral shifts up to 500 ns after a laser-induced temperature jump, which suggests 2BE micellar-like aggregates become more hydrophobic at the initial stage of phase separation. This hydrophobic environment in 2BE aggregates, probably due to the ejection of water molecules, continued up to at least 10 μs. Time-resolved SIM and previously-reported light scattering data clearly showed that the size of a periodic structure remained constant (ca. 300 nm) from 3 to 10 μs, and then the growth of periodic structures having the self-similarity started. We think that the former and the latter processes correspond to “early stage” (concentration growth) and “late stage” (size growth), respectively, in phase separation dynamics. Here we suggest that, in the early stage, the entity to bear 2BE phase be water-poor 2BE aggregates, and the number density of these aggregates would simply increase in time.
关键词： phase separation dynamics,hydrophobic fluorescent probe,2-butoxyethanol,structured illumination microscopy,micellar-like aggregates
更新于2025-09-10 09:29:36
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Faster, sharper, and deeper: structured illumination microscopy for biological imaging
摘要： Structured illumination microscopy (SIM) allows rapid, super-resolution (SR) imaging in live specimens. We review recent technical advances in SR-SIM, with emphasis on imaging speed, resolution, and depth. Since its introduction decades ago, the technique has grown to offer myriad implementations, each with its own strengths and weaknesses. We discuss these, aiming to provide a practical guide for biologists and to highlight which approach is best suited to a given application.
关键词： Structured illumination microscopy,resolution,biological imaging,super-resolution imaging,imaging speed,depth
更新于2025-09-09 09:28:46
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Characterization of neurite dystrophy after trauma by high speed structured illumination microscopy and lattice light sheet microscopy
摘要： Background: Unbiased screening studies have repeatedly identified actin-related proteins as one of the families of proteins most influenced by neurotrauma. Nevertheless, the status quo model of cytoskeletal reorganization after neurotrauma excludes actin and incorporates only changes in microtubules and intermediate filaments. Actin is excluded in part because it is difficult to image with conventional techniques. However, recent innovations in fluorescent microscopy provide an opportunity to image the actin cytoskeleton at super-resolution resolution in living cells. This study applied these innovations to an in vitro model of neurotrauma. New method: New methods are introduced for traumatizing neurons before imaging them with high speed structured illumination microscopy or lattice light sheet microscopy. Also, methods for analyzing structured illumination microscopy images to quantify post-traumatic neurite dystrophy are presented. Results: Human induced pluripotent stem cell-derived neurons exhibited actin organization typical of immature neurons. Neurite dystrophy increased after trauma but was not influenced by jasplakinolide treatment. The F-actin content of dystrophies varied greatly from one dystrophy to another. Comparison with existing methods: In contrast to fixation dependent methods, these methods capture the evolution of the actin cytoskeleton over time in a living cell. In contrast to prior methods based on counting dystrophies, this quantification scheme parameterizes the severity of a given dystrophy as it evolves from a local swelling to an almost-perfect spheroid that threatens to transect the neurite. Conclusions: These methods can be used to investigate genetic factors and therapeutic interventions that modulate the course of neurite dystrophy after trauma.
关键词： Traumatic brain injury,Dystrophy,Structured illumination microscopy,Human induced pluripotent stem cell derived neurons,Lattice light sheet microscopy
更新于2025-09-04 15:30:14