Stereochemistry-Dependent Labeling of Organelles with a Near-Infrared-Emissive Phosphorus-Bridged Rhodamine Dye in Live-Cell Imaging.

The development of near-infrared (NIR) fluorophores that have both excellent chemical stability and photostability, as well as efficient cell permeability, is highly demanded. In this study, we present phospha-rhodamine (POR) dyes which display significantly improved performance for protein labeling. This is achieved by incorporating a 2-carboxy-3-benzothiophenyl group at the 9-position of the xanthene scaffold. The resulting cis and trans isomers were successfully isolated and structurally characterized using X-ray diffraction. The HaloTag ligand conjugates of the two isomers exhibited different staining abilities in live cells. While the cis isomer showed non-specific accumulation on the organelle membranes, the trans isomer selectively labeled the HaloTag-fused proteins, enabling the long-term imaging of cell division and the 5-color imaging of cell organelles. Molecular dynamics simulations of the HaloTag ligand conjugates within the lipid membrane suggested that the cis isomer is more prone to forming oligomers in the membrane. In contrast, the oligomerization of the trans isomer is effectively suppressed by its interaction with the lipid molecules. By taking advantage of the superior labeling performance of the trans isomer and its NIR-emissive properties, multi-color time-lapse super-resolution 3D imaging, namely super-resolution 5D-imaging, of the interconnected network between the endoplasmic reticulum and microtubules was achieved in living cells.

Structure determination using high-order spatial correlations in single-particle X-ray scattering.

Single-particle imaging using X-ray free-electron lasers (XFELs) is a promising technique for observing nanoscale biological samples under near-physiological conditions. However, as the sample's orientation in each diffraction pattern is unknown, advanced algorithms are required to reconstruct the 3D diffraction intensity volume and subsequently the sample's density model. While most approaches perform 3D reconstruction via determining the orientation of each diffraction pattern, a correlation-based approach utilizes the averaged spatial correlations of diffraction intensities over all patterns, making it well suited for processing experimental data with a poor signal-to-noise ratio of individual patterns. Here, a method is proposed to determine the 3D structure of a sample by analyzing the double, triple and quadruple spatial correlations in diffraction patterns. This ab initio method can reconstruct the basic shape of an irregular unsymmetric 3D sample without requiring any prior knowledge of the sample. The impact of background and noise on correlations is investigated and corrected to ensure the success of reconstruction under simulated experimental conditions. Additionally, the feasibility of using the correlation-based approach to process incomplete partial diffraction patterns is demonstrated. The proposed method is a variable addition to existing algorithms for 3D reconstruction and will further promote the development and adoption of XFEL single-particle imaging techniques.