Single-particle imaging reveals intraflagellar transport–independent transport and accumulation of EB1 in \u3cem\u3eChlamydomonas\u3c/em\u3e flagella

The microtubule (MT) plus-end tracking protein EB1 is present at the tips of cilia and flagella; end-binding protein 1 (EB1) remains at the tip during flagellar shortening and in the absence of intraflagellar transport (IFT), the predominant protein transport system in flagella. To investigate how EB1 accumulates at the flagellar tip, we used in vivo imaging of fluorescent protein–tagged EB1 (EB1-FP) in Chlamydomonas reinhardtii. After photobleaching, the EB1 signal at the flagellar tip recovered within minutes, indicating an exchange with unbleached EB1 entering the flagella from the cell body. EB1 moved independent of IFT trains, and EB1-FP recovery did not require the IFT pathway. Single-particle imaging showed that EB1-FP is highly mobile along the flagellar shaft and displays a markedly reduced mobility near the flagellar tip. Individual EB1-FP particles dwelled for several seconds near the flagellar tip, suggesting the presence of stable EB1 binding sites. In simulations, the two distinct phases of EB1 mobility are sufficient to explain its accumulation at the tip. We propose that proteins uniformly distributed throughout the cytoplasm like EB1 accumulate locally by diffusion and capture; IFT, in contrast, might be required to transport proteins against cellular concentration gradients into or out of cilia

The fishes of ascension Island, central Atlantic Ocean - new records and an annotated checklist

A checklist of the fishes of Ascension Island is presented. The species Rhincodon typus, Alopias superciliosus, Isurus oxyrinchus, Carcharhinus obscurus, Galeocerdo cuvier, Sphyrna lewini, Hexanchus griseus, Manta birostris, Gymnothorax vicinus, Hippocampus sp., Epinephelus itajara, Cookeolus japonicus, Apogon pseudomaculatus, Phaeoptyx pigmentaria, Remora albescens, Caranx bartholomaei, Carangoides ruber, Decapterus tabl, Seriola dumerili, Thalassoma sanctaehelenae, Cryptotomus sp., Ruvettus pretiosus, Acanthocybium solandri, Auxis rochei, Auxis thazard, Euthynnus alletteratus, Katsuwonus pelamis, Thunnus alalunga, Thunnus obesus, Xiphias gladius, Istiophorus platypterus, Kajikia albida, Makaira nigricans, Tetrapturus pfluegeri, Hyperoglyphe perciformis, Schedophilus sp., Cantherhines macrocerus, Sphoeroides pachygaster and Diodon eydouxii are recorded for the first time from Ascension Island. We have recognized two previous records as identification errors and indicate 11 other records as doubtful. Including the 40 new records, we now list 173 fish species from Ascension Island, of which 133 might be considered 'coastal fish species'. Eleven of these (8.3%) appear to be endemic to the island and a further 16 species (12%) appear to be shared endemics with St Helena Island.Darwin Initiative [EIDCF012]info:eu-repo/semantics/publishedVersio

Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones

Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10–30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples

120nm resolution in thick samples with structured illumination and adaptive optics

μLinear Structured Illumination Microscopy (SIM) provides a two-fold increase over the diffraction limited resolution. SIM produces excellent images with 120nm resolution in tissue culture cells in two and three dimensions. For SIM to work correctly, the point spread function (PSF) and optical transfer function (OTF) must be known, and, ideally, should be unaberrated. When imaging through thick samples, aberrations will be introduced into the optical system which will reduce the peak intensity and increase the width of the PSF. This will lead to reduced resolution and artifacts in SIM images. Adaptive optics can be used to correct the optical wavefront restoring the PSF to its unaberrated state, and AO has been used in several types of fluorescence microscopy. We demonstrate that AO can be used with SIM to achieve 120nm resolution through 25m of tissue by imaging through the full thickness of an adult C. elegans roundworm. The aberrations can be corrected over a 25μm × 45μm field of view with one wavefront correction setting, demonstrating that AO can be used effectively with widefield superresolution techniques

Multicolor 3D Super-resolution Imaging by Quantum Dot Stochastic Optical Reconstruction Microscopy

We demonstrate multicolor three-dimensional super-resolution imaging with quantum dots (QSTORM). By combining quantum dot asynchronous spectral blueing with stochastic optical reconstruction microscopy and adaptive optics, we achieve three-dimensional imaging with 24 nm lateral and 37 nm axial resolution. By pairing two short-pass filters with two appropriate quantum dots, we are able to image single blueing quantum dots on two channels simultaneously, enabling multicolor imaging with high photon counts

25^th Anniversary of STED Microscopy and the 20^th Anniversary of SIM: Feature introduction

This feature issue commemorating 25 years of STED microscopy and 20 years of SIM is intended to highlight the incredible progress and growth in the field of superresolution microscopy since Stefan Hell and Jan Wichmann published the article Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy in Optics Letters in 1994.ImPhys/Imaging Physic

Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones.

Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow &lt;20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10-30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples