Resonant waveguide grating enabled high sensitivity wide-field optical microscopy – GRATEOM

Resonant waveguide gratings (RWG) induce strong local electromagnetic fields on their top surface, which are useful in many applications. In this proposal, we will develop a high-sensitivity wide-field optical microscopy by using RWG-enhanced upconversion fluorescence (UCF) of infrared-excited rare-earth nanoparticles, as well as a new RWG-based phase-shift dark-field sensing technique based on a polarimetric approach.

First, a RWG UCF microscopy (RWG-UCFM) will be developed by using rare-earth upconversion nanoparticles (UCNPs) as biomarkers and the RWG as a substrate. The proposed RWG-UCFM can provide high contrast images because the RWG substrate can greatly enhance UCF efficiency of labelled-UCNPs thanks to the excitation resonance effect, which a strong local electromagnetic field will be built on the surface of the RWG. Comparing with other available fluorescence microscopies, RWG-UCFM provides advantages like higher sampling speed, higher signal/noise ratio, and higher spatial resolution. Especially, since the RWG-UCF excitation wavelength is at 980 nm, drawbacks like photobleaching of fluorophores, autofluorescence and large scattering from bio-tissue, which are typically penalties of traditional fluorescence microscopy, are no longer a problem for the RWG-UCFM.

The RWG-based microscopy will provide very good axial resolution (< 200 nm) thanks to evanescent wave property of local electromagnetic field built on the top of the RWG. The lateral resolution of the RWG-based microscopy is determined the lateral propagation of the guided mode resonance (GMR) mode and also the numerical aperture (NA) of the objective lens. Its lateral resolution will be typically 2 micrometres, if an objective lens of NA=0.5 is used. To improve its lateral resolution, we propose to use a structured illumination excitation, which is produced either by the interference of two excitation beams (non resonant excitation) or by using one beam resonant excited the RWG structure. With the unique multi-photon nonlinear absorption property of UCNPs, the lateral spatial resolution of the RWG-UCFM will be greatly enhanced (lateral resolution < 75 nm is expected to be obtained).

Second, a label-free wide-field polarimetric microscopy will be developed using the RWG as a substrate. The RWG will be very sensitive to the change of refractive index on its top surface as GMR occurs. We will explore the change of polarization state of an incident beam transmitting through the RWG caused by the change of refractive index. The RWG substrate will be combined with a polarization state sensing setup and a good spatial resolution optical imaging system to form a label-free wide-field RWG polarimetric microscopy (RWG-PM). The RWG-PM can provide high spatial resolution images of the interfacial refractive index distribution of a specimen on the surface of the RWG due to narrow GMR spectra linewidth, restricted propagation and evanescent field enhancement on the RWG surface.

To demonstrate the advantages of the two new proposed microscopy methods, bio-specimens will be imaged. We therefore aim to visualize chromatin structures using antibody against methylated histone H3K9me3 in OML1-P and OML1-R cells, and compare the structure images performed by our methods with those obtained by a conventional confocal microscope.