Imaging Receptor-Mediated Endocytosis with a Polymeric

Jul 31, 2007 - College of Pharmacy, The Ohio State UniVersity, Columbus, Ohio 43210, and Weldon School of Biomedical. Engineering, Purdue UniVersity, ...
10 downloads 11 Views 1MB Size
9980

J. Phys. Chem. B 2007, 111, 9980-9985

Imaging Receptor-Mediated Endocytosis with a Polymeric Nanoparticle-Based Coherent Anti-Stokes Raman Scattering Probe Ling Tong,† Yanhui Lu,‡ Robert J. Lee,‡ and Ji-Xin Cheng*,†,§ Department of Chemistry, Purdue UniVersity, West Lafayette, Indiana 47906, DiVision of Pharmaceutics, College of Pharmacy, The Ohio State UniVersity, Columbus, Ohio 43210, and Weldon School of Biomedical Engineering, Purdue UniVersity, West Lafayette, Indiana 47906 ReceiVed: May 7, 2007; In Final Form: June 19, 2007

Coherent anti-Stokes Raman scattering (CARS) microscopy was used to visualize receptor-mediated endocytosis and intracellular trafficking with the aid of a CARS probe. The probe was made of 200-nm polystyrene particles encapsulated in folate-targeted liposomes. By tuning (ωp - ωs) to 3045 cm-1, which corresponds to the aromatic C-H stretching vibration, the polystyrene nanoparticles with a high density of aromatic C-H bonds were detected with a high signal-to-noise ratio, while the epi-detected CARS signal from cellular organelles was cancelled by the destructive interference between the resonant contribution from the aliphatic C-H vibration and the nonresonant contribution. Without any photobleaching, the CARS probe allowed single-particle tracking analysis of intracellular endosome transport. No photodamage to cells was observed under the current experimental conditions. These results show the advantages and potential of using a CARS probe to study cellular processes.

Introduction With the development of fluorescent probes and realization of three-dimension (3D) resolution by confocal detection1 or two-photon excitation,2 fluorescence microscopy has become a powerful and widely used bio-imaging tool.3 However, most fluorophores are prone to photobleaching, which makes continuous observation of biological processes difficult. Recently, semiconductor quantum dots were developed as bright and photostable fluorophores to alleviate the photobleaching problem.4,5 The big challenge for quantum dots is the cytotoxicity due to the release of Cd2+ or Se2- ions. Deleterious effects of quantum dots on embryo development6 and cell viability7 have been observed. Therefore, development of novel imaging probes is still greatly needed. In this paper, we report a coherent anti-Stokes Raman scattering (CARS) probe for 3D imaging of cellular processes without photobleaching. CARS is a third-order nonlinear optical process in which a pump field Ep (ωp) and a Stokes field Es (ωs) interact with a sample and generate an anti-Stokes field Eas at the frequency of ωas ) (ωp - ωs) + ωp. The CARS signal can be significantly enhanced when (ωp - ωs) is tuned to a Raman-active vibrational band, which provides the vibrational contrast. Besides the molecular selectivity, CARS microscopy offers several other advantages summarized as follows. The CARS frequency is higher than the excitation frequencies and can thus be detected in the presence of one-photon-induced fluorescence. The temporally and spatially overlapping pump and Stokes laser pulses are tightly focused into a sample to generate a signal in a small excitation volume (