Visualizing the native cellular organization by coupling cryofixation with expansion microscopy (Cryo-ExM)

Cryofixation has proven to be the gold standard for efficient preservation of native cell ultrastructure compared to chemical fixation, but this approach is not widely used in fluorescence microscopy owing to implementation challenges. Here, we develop Cryo-ExM, a method that preserves native cellular organization by coupling cryofixation with expansion microscopy. This method bypasses artifacts associated with chemical fixation and its simplicity will contribute to its widespread use in super-resolution microscopy.

Converting CO2 into heterocyclic compounds under accelerated performance through Fe3O4 grafted ionic liquid catalysts

Solid supported catalysts such as amines has high demand for chemical fixation of CO2 into commodity chemicals. Here, we demonstrate an accelerated platform for 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)-ionic liquids (ILs) catalyzed CO2...

Covalently Immobilized Regenerable Immunoaffinity Layer with Orientation-Controlled Antibodies Based on Z-Domain Autodisplay

A regenerable immunoaffinity layer comprising covalently immobilized orientation-controlled antibodies was developed for use in a surface plasmon resonance (SPR) biosensor. For antibody orientation control, antibody-binding Z-domain-autodisplaying Escherichia coli (E. coli) cells and their outer membrane (OM) were utilized, and a disuccinimidyl crosslinker was employed for covalent antibody binding. To fabricate the regenerable immunoaffinity layer, capture antibodies were bound to autodisplayed Z-domains, and then treated with the crosslinker for chemical fixation to the Z-domains. Various crosslinkers, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and poly (ethylene glycol)-ylated bis (sulfosuccinimidyl)suberate (BS(PEG)5), were evaluated, and DSS at a concentration of 500 μM was confirmed to be optimal. The E. coli-cell-based regenerable HRP immunoassay was evaluated employing three sequential HRP treatment and regeneration steps. Then, the Oms of E. coli cells were isolated and layered on a microplate and regenerable OM-based HRP immunoassaying was evaluated. Five HRP immunoassays with four regeneration steps were found to be feasible. This regenerable, covalently immobilized, orientation-controlled OM-based immunoaffinity layer was applied to an SPR biosensor, which was capable of quantifying C-reactive protein (CRP). Five regeneration cycles were repeated using the demonstrated immunoaffinity layer with a signal difference of <10%.

CURT1A and CURT1C mediate distinct stages of plastid conversion in Arabidopsis

The crystalline structure of prolamellar bodies (PLBs) and light-induced etioplasts-to-chloroplasts transformation have been investigated with electron microscopy methods. However, these studies suffer from chemical fixation artifacts and limited volumes of tomographic reconstruction. We have examined Arabidopsis thaliana cotyledon samples preserved by high-pressure freezing with scanning transmission electron tomography to visualize larger volumes in etioplasts and their conversion into chloroplasts. PLB tubules were arranged in a zinc blende-type lattice like carbon atoms in diamonds. Within 2 hours after illumination, the lattice collapsed from the PLB exterior and the disorganized tubules merged to form fenestrated sheets that eventually matured into lamellar thylakoids. These planar thylakoids emerging from PLBs overlapped or folded into grana stacks in PLBs’ vicinity. Since the nascent lamellae had curved membrane at their tips, we examined the localization of CURT1 proteins. CURT1A transcript was most abundant in de-etiolating cotyledon samples, and CURT1A concentrated at the peripheral PLB. In curt1a mutant etioplasts, thylakoid sheets were swollen and failed to develop stacks. In curt1c mutant, however, PLBs had cracks in their lattices, indicating that CURT1C contributes to cubic crystal growth under darkness. Our data provide evidence that CURT1A and CURT1C play distinct roles in the etioplast and chloroplast biogenesis.