Direct Imaging of Transmembrane Dynamics of Single Nanoparticles with Darkfield Microscopy: Improved Orientation Tracking at Cell Sidewall
2014-04-01T00:00:00Z (GMT) by
Investigation of the cellular internalization processes of individual nanoparticles (NPs) is of great scientific interest with implications to drug delivery and NP biosafety. Herein, by using dual-channel polarization darkfield microcopy (DFM) and single gold nanorods (AuNRs) as orientation probes, we developed a method that is capable of monitoring AuNR orientation dynamics during its transmembrane process. With annular oblique illumination and a birefringent prism to split AuNR plasmonic scattering into two channels of orthogonal polarizations, the AuNR azimuth and polar angles are obtained from their intensity difference and intensity sum. By placing the focal plane of the microscope objective at the elevated cell sidewall rather than at the flat cell top, interference from cellular background is reduced and the signal-to-noise ratio of AuNR orientation sensing is improved significantly, especially for AuNRs inserting into the membrane at a large out-of-plane angle. As a result, we were able to capture the complete membrane-crossing dynamics of single AuNRs. This powerful method could be utilized to obtain valuable insights on NP endocytosis mechanisms of various cells.
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