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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. [ABSTRACT FROM AUTHOR]

The article titled "Inside Cover: Stereochemistry-Dependent Labeling of Organelles with a Near-Infrared-Emissive Phosphorus-Bridged Rhodamine Dye in Live-Cell Imaging" presents a new photostable near-infrared labeling agent developed by Masayasu Taki, Yasushi Okada, Florence Tama, Shigehiro Yamaguchi, and their team. The compounds, which are stereoisomers based on phosphorus-bridged rhodamine, have different staining abilities in live cells. The trans-isomer can permeate organelle membranes and label target proteins, while the cis-isomer accumulates nonspecifically on lipid bilayers. The authors of the article are Qian Wu, Masayasu Taki, Yoshiki Tanaka, Manish Kesherwani, Quan Manh Phung, Sawako Enoki, Yasushi Okada, Florence Tama, and Shigehiro Yamaguchi. [Extracted from the article]

The article titled "Innentitelbild: Stereochemistry-Dependent Labeling of Organelles with a Near-Infrared-Emissive Phosphorus-Bridged Rhodamine Dye in Live-Cell Imaging" presents a new photostable near-infrared (NIR) labeling agent developed by Masayasu Taki, Yasushi Okada, Florence Tama, Shigehiro Yamaguchi, and their colleagues. The compounds, which are stereoisomers based on phosphorus-bridged rhodamine, have different staining abilities in live cells. The trans-isomer can permeate organelle membranes and label target proteins, while the cis-isomer accumulates nonspecifically on lipid bilayers. The authors of the article are Qian Wu, Masayasu Taki, Yoshiki Tanaka, Manish Kesherwani, Quan Manh Phung, Sawako Enoki, Yasushi Okada, Florence Tama, and Shigehiro Yamaguchi. [Extracted from the article]

Summary Membrane proteins play important roles in various cellular functions. To analyze membrane proteins, nanodisc technology using membrane scaffold proteins allows single membrane protein units to be embedded into the lipid bilayer disc without detergents. Recent advancements in high-speed atomic force microscopy (HS-AFM) have enabled us to monitor the real-time dynamics of proteins in solution at the nanometer scale. In this study, we report HS-AFM imaging of membrane proteins reconstituted into nanodiscs using two membrane protein complexes, SecYEG complex and MgtE dimer. The observed images showed single particles of membrane protein-embedded nanodiscs in an end-up orientation whereby the membrane was fixed parallel to the supporting solid surface and in a side-on orientation whereby the membrane plane was vertically fixed to the solid surface, enabling the elucidation of domain fluctuations in membrane proteins. This technique provides a basic method for the high-resolution imaging of single membrane proteins by HS-AFM. Graphical Abstract Highlights • Membrane protein-embedded nanodiscs were used for high-speed (HS)-AFM observation • Nanodiscs in an end-up and side-on orientation were observed by HS-AFM in real time • HS-AFM combined with nanodiscs is a powerful tool for membrane protein dynamics Haruyama et al. established a procedure for the observation of membrane proteins in nanodiscs by high-speed AFM in real time. Using the two-dimensional crystal of streptavidin as well as mica as a solid support surface, dynamic structural changes of membrane proteins in the lipid environment were visualized from two distinct angles. [ABSTRACT FROM AUTHOR]