Thermosensitive photothermal agents (AuNRs@pNIPAAm) for accurate temperature control were prepared by covalent binding pNIPAAm to gold nanorods. Stretched pNIPAAm collapses when temperature increases around low critical solution temperature, inducing a redshift of LSPR wavelength. To obtain a high-throughput LSPR scattering spectral mapping of AuNRs@pNIPAAm particles, we demonstrate a spatiotemporal resolution plasmonic spectroscopy (SRPS) which provides the LSPR wavelength of multiple AuNRs@ pNIPAAm particles simultaneously at millisecond level (limited by camera frame rate).
SRPS is performed on a home-made two-channel highly inclined and laminated optical (HILO) sheet dark-field microscope. Nanoparticles with LSPR wavelength of ~750 nm are incubated with cells. Two continuous lasers (with wavelength of 730 and 785 nm, avoid the broadband scattering background from biological environment) are combined and modulated in order to generate a pulse beam. This beam is then directed onto the cover-slide with an appropriate incident angle to generate a HILO sheet, which is suitable for high spatial resolution imaging of nanoparticles inside cell samples. Another 808 nm laser is focused on the sample to heat nanoparticles.
Scattering light of nanoparticles is collected by the same objective, which is recorded by a CMOS camera. CMOS camera and the pulse laser are synchronized in order to capture the dark-field images from two wavelength lasers periodically, which generates a two-channel image sequence. With this configuration, we measure the scattering intensity of nanoparticles illuminated by two lasers simultaneously, which can be further converted to LSPR wavelength image.
We described here two plasmonic-based nanoprobes with purpose of imaging dynamic biologic process of single tumor cells. At first, we proposed a multi-modified core-shell gold@silver nanorods for real-time monitoring the entire autophagy process at single-cell level. Autophagy is vital for understanding the mechanisms of human pathologies, developing novel drugs and exploring approaches for autophagy controlling. The plasmon resonance scattering spectra of the nanoprobes was superoxide radicals (O2•-)-dependent, a major indicator of cell autophagy, and suitable for real-time monitoring at single-cell level. More importantly, with the introduction of ‘relay probe’ operation, two types of O2•--regulating autophagy processes were successfully traced from the beginning to the end, and the possible mechanism was also proposed. According to our results, intracellular O2•- level controlled the autophagy process by mediating the autolysosome generation. Different starvation approaches can induce different autophagy processes, such as diverse steady state time-consuming. In addition, a plasmonic-based nanothermometer was prepared via dense thermosensitive polymer (pNIPAAm) capping on gold nanorods, of which the plasmon resonance spectra was linearly dependent on adjacent temperature. In this work, the white light transmitted dark-field illuminator was replaced by a laser total internal reflection dark-field microscope (LTIR-DFM) system in order to overcome the low-throughput and inexorable biological scattering background of DFM, as well as interference from mechanic noise, nanoprobe direction, optical system drift, etc. With this nanothermometer, we have successfully captured temporal biological thermal process (thermogenesis) occurred in single tumor cells, providing a new potential strategy for in-situ cellular analysis.
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