Journal: Nature Plants

Article Title: A promiscuous mechanism to phase separate eukaryotic carbon fixation in the green lineage

doi: 10.1038/s41477-024-01812-x

Figure Lengend Snippet: a , Representative immunogold TEM of the Chlorella pyrenoid after primary incubation with the RbcL antibody. A subset of gold nanoparticles are indicated by white arrowheads. b , Absolute quantification of CsRubisco holoenzyme (derived from CsRbcL) and CsLinker in vivo ( n = 3). The ratio between CsRubisco and CsLinker is indicated at the bottom (see Supplementary Fig. for details). Mean ± s.d. of 3 biological replicates. The asterisked point indicates the independent quantification from western blotting (Supplementary Fig. ). c , Left: confocal fluorescence microscopy image of droplets in the in vitro reconstitution of the Chlorella pyrenoid formed at concentrations of CsRubisco and CsLinker close to that of the chloroplast (2 μM and 4 μM, respectively). Asterisk indicates a droplet that settled between imaging of the two channels. Atto594-CsRubisco and mEGFP-CsLinker were incorporated at 0.5% and 5% molar concentrations, respectively. Single, non-repeated observation. Right: droplets can be sedimented by centrifugation and the composition of the pellet (P) relative to the supernatant (S) analysed by SDS–PAGE (repeated observation; see Supplementary Figs. and ). This experiment format was used to generate datapoints in d and e . d , Titration droplet sedimentation assays with fixed CsRubisco concentration. e , Titration droplet sedimentation assays with fixed CsLinker. Where visible in d and e , data are mean ± s.d. of 2 technical replicate experiments completed concurrently (Supplementary Fig. ). f , Average full-scale normalized half-FRAP recovery curve of Atto594-CsRubisco in droplets formed as in c . g , Average full-scale normalized half-FRAP recovery curve of mEGFP-CsLinker in droplets with the same composition. In f and g , the mean, s.e.m. and s.d. of the indicated number of technical replicates are represented by the line, the smaller shaded region and the larger shaded region, respectively. h , Time series of 0.5% (molar ratio) Atto594-CsRubisco-labelled droplets formed as in c , undergoing fusion (arrowhead) and relaxation. i , 5% mEGFP-CsLinker-labelled droplets undergoing consecutive fusions. j , Time series of a representative CsRubisco FRAP experiment, as quantified in f . The white box indicates the region bleached. k , Representative CsLinker FRAP experiment, as quantified in g . Scale bars for h – k , 1 μm. All in vitro droplet experiments in c – k were completed in a 50 mM Tris-HCl pH 8.0, 50 mM NaCl buffer.

Article Snippet: Per bombardment, 0.5 mg of 550 nm gold nanoparticles (Seashell Technologies) were incubated with 1 μg of plasmid DNA and prepared according to manufacturer instructions.

Techniques: Incubation, Quantitative Proteomics, Derivative Assay, In Vivo, Western Blot, Fluorescence, Microscopy, In Vitro, Imaging, Centrifugation, SDS Page, Titration, Sedimentation, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: 3D Super-Resolution Nuclear Q-FISH Imaging Reveals Cell-Cycle-Related Telomere Changes

doi: 10.3390/ijms25063183

Figure Lengend Snippet: Adaptive illumination supports 3D-STED FISH Nanoscopy. ( A ) In order to reduce the bleaching of telomere signals within nuclei, adaptive illumination was applied. For positions reaching sufficient confocal signal levels (left image), low-dose STED signals were measured (center: green areas) as a probing step. Only if the resulting signal again reached a threshold level was the final STED power level applied for image generation (center: blue areas). As a result, telomere spots were resolved at super-resolution while minimizing sample light dosage when imaging in multiple planes (right). ( B ) 3D STED resolution was evaluated using 40 nm fluorescent beads as a test sample. FWHM values indicate approximately the achieved optical resolution in lateral and axial imaging. Scale bars represent 20 nm for the upper image and 100 nm for the lower image. FWHM was calculated from the Gaussian fit standard deviation of intensity line profiles along the dashed lines.

Article Snippet: The red channel was also used in STED mode, supported by a 775 nm synchronized depletion laser, which was aligned at the start of each imaging session using 40 nm red fluorescent beads (Abberior Nanoparticle Set for Expert Line 595 and 775 nm).

Techniques: Imaging, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: 3D Super-Resolution Nuclear Q-FISH Imaging Reveals Cell-Cycle-Related Telomere Changes

doi: 10.3390/ijms25063183

Figure Lengend Snippet: FISH-STED determines TelC probe molecule counts per telomere based on calibration measurements. ( A ) Imaging of fluorescent DNA Origami structures with defined Abberior Star635P fluorophore was performed with standardized 3D FISH-STED settings applied to in situ telomeres. ( B ) The brightness of beads measured by automated spot analysis was stochastically defined for each type based on the spot brightness distribution and fit by a normal distribution to retrieve mean and SD values. ( C ) A factor of 0.165 was determined for the conversion of spot brightness to molecular probe numbers using linear regression. ( D ) Based on the previous brightness calibration, fluorescent probe counting per telomere spot was performed and translated into the average length of the probe-labeled sequence. In interphase, median FISH-STED spot brightness corresponded to 2.63 kbp of labeled sequence stretches. Error bars indicate SD.

Article Snippet: The red channel was also used in STED mode, supported by a 775 nm synchronized depletion laser, which was aligned at the start of each imaging session using 40 nm red fluorescent beads (Abberior Nanoparticle Set for Expert Line 595 and 775 nm).

Techniques: Imaging, In Situ, Labeling, Sequencing