Fast, long-term, super-resolution imaging with Hessian structured illumination microscopy

Abstract

To increase the temporal resolution and maximal imaging time of super-resolution (SR) microscopy, we have developed a deconvolution algorithm for structured illumination microscopy based on Hessian matrixes (Hessian-SIM). It uses the continuity of biological structures in multiple dimensions as a priori knowledge to guide image reconstruction and attains artifact-minimized SR images with less than 10% of the photon dose used by conventional SIM while substantially outperforming current algorithms at low signal intensities. Hessian-SIM enables rapid imaging of moving vesicles or loops in the endoplasmic reticulum without motion artifacts and with a spatiotemporal resolution of 88 nm and 188 Hz. Its high sensitivity allows the use of sub-millisecond excitation pulses followed by dark recovery times to reduce photobleaching of fluorescent proteins, enabling hour-long time-lapse SR imaging of actin filaments in live cells. Finally, we observed the structural dynamics of mitochondrial cristae and structures that, to our knowledge, have not been observed previously, such as enlarged fusion pores during vesicle exocytosis.

Super‐Resolution Tracking of Mitochondrial Dynamics with An Iridium(III) Luminophore

“…Highly mobile and dynamic in living cells, mitochondria are the energy‐generating organelles of cells, the disorder of which is associated with various diseases, including Alzheimer's, Parkinson's, and cancer . Recently developed super‐resolution fluorescence microscopies such as stimulated emission deletion (STED), structured illumination microscopy (SIM), and stochastic optical reconstruction microscopy (STORM), as well as other single‐molecule super‐resolution imaging techniques, are enhanced new tools for investigating the dynamics of subcellular structures, including mitochondria. At the same time, they have also added to the requirements of fluorescent dyes, which need especially low cytotoxicity and high photostability to make imaging living cells possible.…”

Principles of Hyperspectral Microscope Imaging Techniques and Their Applications in Food Quality and Safety Detection: A Review

“…For example, Huang et al. () proposed a deconvolution algorithm—Hessian matrixes—to improve the temporal resolution of structured illumination microscopy, showing great potentials for analyzing food microstructure, microbial contamination, and small molecular residues.…”

“…Many algorithms developed and used in analyzing medical images should be adopted for their applications in dealing with food-related spectral images (Litjens et al, 2017). For example, Huang et al (2018) proposed a deconvolution algorithm-Hessian matrixes-to improve the temporal resolution of structured illumination microscopy, showing great potentials for analyzing food microstructure, microbial contamination, and small molecular residues.…”

Toxicology in the Super‐Resolution Era

“…Careful attention to this mantra is especially important when performing livecell imaging (Cole, 2014). While fixed samples are less susceptible to photon damage, care still needs to be exercised to avoid photobleaching (Huang et al, 2018). More often than not, today's experiments will require some level of multiplexing.…”