Cell biochemistry studied by single-molecule imaging

Abstract

Over the last decade, there have been remarkable developments in live-cell imaging. We can now readily observe individual protein molecules within living cells and this should contribute to a systems level understanding of biological pathways. Direct observation of single fluorophores enables several types of molecular information to be gathered. Temporal and spatial trajectories enable diffusion constants and binding kinetics to be deduced, while analyses of fluorescence lifetime, intensity, polarization or spectra give chemical and conformational information about molecules in their cellular context. By recording the spatial trajectories of pairs of interacting molecules, formation of larger molecular complexes can be studied. In the future, multicolour and multiparameter imaging of single molecules in live cells will be a powerful analytical tool for systems biology. Here, we discuss measurements of single-molecule mobility and residency at the plasma membrane of live cells. Analysis of diffusional paths at the plasma membrane gives information about its physical properties and measurement of temporal trajectories enables rates of binding and dissociation to be derived. Meanwhile, close scrutiny of individual fluorophore trajectories enables ideas about molecular dimerization and oligomerization related to function to be tested directly.

Novel Fluorescent Core–Shell Nanocontainers for Cell Membrane Transport

“…The desired product 4 (429 mg, 92.0%) was purified by column chromatography (silica gel, CH 2 Cl 2 /petroleum ether (2:1)) as a red powder. 1 H NMR (300 MHz, CD 2 Cl 2 , 300 K, δ ppm): 8.06 (s, 4H), 7.52 (s, 4H), 7.45 (t, J ) 7.7 Hz, 2H), 7.42 (d, J ) 7.9 Hz, 4 H), 7.40-6.60 (m, 88H), 2.68 (m, 4 H, CH), 1.91 (s, 24H, CH 3 ), 1.90 (s, 24H, CH 3 ), 1.05 (d, J ) 6.6 Hz, 24H, CH 3 ). 13 (5).…”

“…The final purification was done by column chromatography (silica gel, CH 2 Cl 2 /petroleum ether (3:1)) to yield product 7 (188.8 mg, 88.0%) as a red powder. 1 (12). A. N-(2,6-Diisopropylphenyl)-9-bromo-perylene-3,4-dicarboxy-imide (8) (2.2 g, 3.9 mmol) and 4-methoxyphenylboronic acid (9) (500 mg, 3.3 mmol) were dissolved in 200 mL of toluene under argon atmosphere, then 30 mL of 1 M aqueous potassium carbonate solution and tetrakis(triphenylphosphine)palladium(0) (Pd(PPh 3 ) 4 ) (380 mg, 0.33 mmol) were added.…”

“…Column chromatography (silica gel, CH 2 Cl 2 ) afforded the desired product 5 as a red powder (267 mg, 90.0%). 1 (7). PDI-labeled first-generation dendrimer bearing eight ethynyl groups (2) (100 mg, 0.0308 mmol) and 2-bromo-2-methylpropionic acid 4-(3-oxo-2,4,5-triphenylcyclopenta-1,4-dienyl)-phenyl ester (6) (270 mg, 0.492 mmol) were dissolved in 5 mL of o-xylene and sealed in a 8 mL glass tube.…”

“…After evaporation of the solvent, the residue was purified by column chromatography (silica gel, CH 2 Cl 2 ) to afford N-(2,6-diisopropylphenyl)-9-(4-methoxyphenyl)-perylene-3,4-dicarboxy-imide (10, 1.38 g, 60.3%) as a purple powder. 1 (20 mL) under argon at 0°C. After stirring for 24 h, the solvent was removed under reduced pressure and the residue purified by column chromatography (silica gel, CH 2 Cl 2 ) to afford N-(2,6-diisopropylphenyl)-9-(4-hydroxy-phenyl)-perylene-3,4-dicarboxy-imide (11, 1.20 g, 95%) as a purple powder.…”

Single-molecule fluorescence imaging by total internal reflection fluorescence microscopy (IUPAC Technical Report)

“…Photobleaching (and subsequent recovery) can also be used to investigate the stoichiometry of multi-protein complexes and the turnover of subunits, as was recently done for the bacterial flagellar motor [32]. TIRF is also a popular route to single-molecule detection in cells [17,33,34]. Furthermore, as mentioned above, TIRF excitation may be used in a number of forms of super-resolution microscopy.…”