No abstract

Cell-penetrating peptides (CPPs) have been extensively studied during the past decade, because of their ability to promote the cellular uptake of various cargo molecules, e.g., oligonucleotides and proteins. In a recent study of the uptake of several analogues of penetratin, Tat(48-60) and oligoarginine in live (unfixed) cells [Thorén et al. (2003) Biochem. Biophys. Res. Commun. 307, 100-107], it was found that both endocytotic and nonendocytotic uptake pathways are involved in the internalization of these CPPs. In the present study, the membrane interactions of some of these novel peptides, all containing a tryptophan residue to facilitate spectroscopic studies, are investigated. The peptides exhibit a strong affinity for large unilamellar vesicles (LUVs) containing zwitterionic and anionic lipids, with binding constants decreasing in the order penetratin > R(7)W > TatP59W > TatLysP59W. Quenching studies using the aqueous quencher acrylamide and brominated lipids indicate that the tryptophan residues of the peptides are buried to a similar extent into the membrane, with an average insertion depth of approximately 10-11 A from the bilayer center. The membrane topology of the peptides was investigated using an assay based on resonance energy transfer between tryptophan and a fluorescently labeled lysophospholipid, lysoMC, distributed asymmetrically in the membranes of LUVs. By determination of the energy transfer efficiency when peptide was added to vesicles with lysoMC present exclusively in the inner leaflet, it was shown that none of the peptides investigated is able to translocate across the lipid membranes of LUVs. By contrast, confocal laser scanning microscopy studies on carboxyfluorescein-labeled peptides showed that all of the peptides rapidly traverse the membranes of giant unilamellar vesicles (GUVs). The choice of model system is thus crucial for the conclusions about the ability of CPPs to translocate across lipid membranes. Under the conditions used in the present study, peptide-lipid interactions alone cannot explain the different cellular uptake characteristics exhibited by these peptides.

We present a simple method for the optical manipulation and spectroscopy of colloidal silver nanoparticles in aqueous solution using optical tweezers combined with dark-field microscopy. Because of their localized plasmon resonances, single trapped metal nanoparticles can be used as efficient near-field optical probes, with potential applicability in surface-enhanced spectroscopy, near-field microscopy, and biochemical sensing schemes. As a proof of principle, we study the near-field optical interaction between a trapped and an immobilized Ag nanoparticle.Gold and silver nanoparticles are objects of active research because of their documented or proposed importance in various nanooptics applications such as surface-enhanced spectroscopy, 1 biochemical sensors, near-field scanning optical microscopy (NSOM), 2 and nanophotonics devices. 3 The fascinating optical properties originate from localized surface plasmon (LSP) resonances, a class of surface modes that involves the collective excitation of conduction electrons in response to an incident electromagnetic field. Gold and silver nanoparticles are chemically stable and typically exhibit LSPs in the visible wavelength range, where they may cause a tremendous increase in various optical cross-sections and an associated enhancement of the electromagnetic fields near the particle surface. The latter effect is exploited in surfaceenhanced Raman scattering (SERS), which enables molecular vibration spectroscopy with unsurpassed detection sensitivity. 4 The resonance frequencies strongly depend on particle shape and size 5 as well as on the optical properties of the material within the near-field of the particle. Metal nanoparticles can thus be used as efficient optical probes, as demonstrated by recent scattering-type NSOM experiments based on immobilized 6 and optically trapped 7,8 gold particles.In most nanooptics applications, there is a clear demand for nanoparticles with optimized LSP characteristics. For example, the LSP should coincide with the wavelength of the excitation laser and have a high quality factor in SERS and NSOM, whereas many biochemical sensing applications require nanoparticles for which the LSP shift due to a change in the surrounding refractive index is maximized. The need for optimized LSP performance is of course even more critical in applications that utilize or focus on a single isolated nanoparticle or particle cluster, such as single-molecule SERS, 9,10 NSOM, or single-particle biosensing. 11 In this letter, we demonstrate a straightforward method for the simultaneous optical trapping and spectroscopy of individual nanoparticles in solution using a single-beam gradient trap, or "optical tweezers", combined with dark-field microscopy. The technique thus allows us to select particles with desired LSP characteristics from heterogeneous colloidal solutions and manipulate these particles at will. As a simple illustration of the potential of this approach, we record the variation in LSP spectra when two separate silver nanoparticles are brought...

Here we present the first demonstration of the physical association between membranes involved in MCSs: by using optical imaging and manipulation, strong attracting forces between ER and chloroplasts are revealed. We used Arabidopsis thaliana expressing green fluorescent protein in the ER lumen and observed leaf protoplasts by confocal microscopy. The ER network was evident, with ER branch end points apparently localized at chloroplast surfaces. After rupture of a protoplast using a laser scalpel, the cell content was released. ER fragments remained attached to the released chloroplasts and could be stretched out by optical tweezers. The applied force, 400 pN, could not drag a chloroplast free from its attached ER, which could reflect protein-protein interactions at the ER-chloroplast MCSs. As chloroplasts rely on import of ER-synthesized lipids, we propose that lipid transfer occurs at these MCSs. We suggest that lipid transfer at the MCSs also occurs in the opposite direction, for example to channel plastid-synthesized acyl groups to supply substrates for ER-localized synthesis of membrane and storage lipids.

We will demonstrate how optical tweezers can be combined with a microfluidic system to create a versatile microlaboratory. Cells are moved between reservoirs filled with different media by means of optical tweezers. We show that the cells, on a timescale of a few seconds, can be moved from one reservoir to another without the media being dragged along with them. The system is demonstrated with an experiment where we expose E. coli bacteria to different fluorescent markers. We will also discuss how the system can be used as an advanced cell sorter. It can favorably be used to sort out a small fraction of cells from a large population, in particular when advanced microscopic techniques are required to distinguish various cells. Patterns of channels and reservoirs were generated in a computer and transferred to a mask using either a sophisticated electron beam technique or a standard laser printer. Lithographic methods were applied to create microchannels in rubber silicon (PDMS). Media were transported in the channels using electroosmotic flow. The optical system consisted of a combined confocal and epi-fluorescence microscope, dual optical tweezers and a laser scalpel.