Nano-biophotonics: new tools for chemical nano-analytics

Abstract

The nondestructive chemical analysis of biological processes in the crowded intracellular environment, at cellular membranes, and between cells with a spatial resolution well beyond the diffraction limit is made possible through Nano-Biophotonics. A number of sophisticated schemes employing nanoparticles, nano-apertures, or shaping of the probe volume in the far field have significantly extended our knowledge about lipid rafts, macromolecular complexes, such as chromatin, vesicles, and cellular organelles, and their interactions and trafficking within the cell. Here, I review some of the most recent developments in Nano-Biophotonics that already are or soon will become relevant to the analysis of intracellular processes. The pros and cons of the various techniques will be discussed and an outlook of their prospects for the near future will be provided.

Figure 1

Schematics of different Nano-Biophotonics techniques. A) Near-field optical microscopy based on tapered, metal-coated fiber probes. Typical aperture diameters range from 50 – 100 nm. The photograph shows emission from an actual near-field tip. The white scale bar is 100 µm. B) Tip-enhanced spectroscopy uses a confocal laser spot in which a gold or silver metal tip is positioned and which enhances the electromagnetic field between tip and sample. C) Single molecule localization: the intensity distribution of a single molecule can be fit with a Gaussian fit function to achieve localization of the centroid to approximately 1.5 nm. D) Plasmon-resonant particles can be detected by dark-field white light scattering, or if they are coated with Raman active molecules, by their surface-enhanced Raman response (shown in the spectrum for Rhodamine 6G molecules attached to a 60 nm silver particle). The scale bar in the electron micrograph of a gold nanoparticle is 25 μm. E) Nano-apertures restrict the probe volume of a confocal laser spot and permit single molecule analysis at micromolar concentration. The scale bar in the electron micrograph of a square nano-aperture is 50 nm. F) SHG or upconverting particles can act as nanoscale light sources. G) Principle of structured illumination microscopy. Sample features that are smaller than the wavelength of light become visible as beat frequencies in a pattern with known periodicity. H) Principle of stimulated emission depletion microscopy. Red-shifted laser beams that overlap with the emission spectrum of the excited fluorophore deplete the emission except for the very center of the excitation beam (green).