Photonic Crystal Enhanced Microscopy

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Introduction

Photonic Crystal Enhanced Microscopy (PCEM) is an imaging technique that enables quantification of cell-surface interactions without cytotoxic Staining agent or temporally-unstable fluorophores. PCEM uses a photonic crystal biosensor surface as a substrate for cellular attachment that can be visualized using standard microscopy methods. The photonic crystal surface acts as a high efficiency resonant reflector at certain wavelengths and this wavelength can be modulated by the density of the material that is attached to it. A spectroscopic scanning system and noncoherent illumination allow for the possibility of this innovation to function.

Photonic Crystal-Enhanced Microscopy (PCEM) is a powerful multimode imaging approach that combines label-free (LF) and enhanced-fluorescence (EF) imaging modalities for quantification and visualization of the interactions between cells and their substrate.

Current cellular imaging modalities such as fluorescence microscopy require labeling with fluorophores. However, the illumination of these fluorophores typically causes photobleaching and cellular damage. PCEM presents a method that allows for imaging of cellular surfaces while avoiding the side effects of traditional fluorescence imaging modalities.

Recently, PCEM has been applied to a wide range of cellular applications. These include long-term monitoring of cell attachment over substantial time scales (hours to days) without disturbing the extracellular environment. PCEM also allows for the observation of attachment and chemotaxis processes of dental stem cells. Identification of single-cell movement and filopodial extension may provide future insight into numerous cellular processes such as tumor cell metastasis and stem cell differentiation.

Concept

Figure illustrate the detection system that can be built upon.  A laser light source can be used along with a polarization fiber to provide a linearly-polarized beam. This light source then is collimated using a condenser lens to yield a beam with the desired diameter.  A half-wave plate is then used to adjust the electric field polarization to be perpendicular to the PC grating lines. The output beam is focused by a cylindrical lens to form a linear beam at the back focal plane of the objective lens with a 50/50 beamsplitter. In the sample incubator path, light beam is pass through a objective lens, the rotated line shaped beam then is use to illuminate the PC. The reflected light is projected to a imaging spectrometer with a narrow sit aperture.

Optical schematic of PC-enhanced microscope

Some System can achieve 0.6µm spatial resolution. Spatial and spectral resolution depend on the optical setup and photonic crystal chosen for the desired application.

Time-lapse label-free images of cellular attachment of mHAT9a cells

Photonic Crystal Production

the process begins with depositing a thin layer of liquid UV epoxy polymer between a Si wafer template and a glass substrate. The epoxy then is converted to a solid with UV light exposure. The template is peeled away and the grating pattern is transferred to the glass. finally A thin layer of sputter-deposited TiO2 film is applied over the grating structure.

(a) Schematic diagram of the photonic crystal (PC) biosensor. A PC sensor is comprised of a replica molded polymer grating overcoated with a high refractive index thin film of TiO2. Inset: Photo of a PC fabricated upon a glass cover slip. (b) Scanning electron micrograph of the PC surface. Reproduced with permission from

Applications

• Label Free Imaging

PCEM is a label free method that has been primarily used to understand and visualize cell interactions with their surface. Short time lapse movies of STEM cell attachment have been generated using PCEM technology. PCEM has been used to image cell attachment and distribution over long periods of times ranging from hours to days without disturbing or harming the cell culture. The attachment of dental cells and cell movements were also identified. Additionally the movements of single cells and filopodial extension were distinguishable using PCEM. Future applications of PCEM include monitoring of tumor cell metastasis and STEM cell differentiation.

• Photo Crystal Enhanced Fluorescence

PCEM has also been combined with fluorescence tags to yield another modality: photo crystal enhanced fluorescence (PCEF). PCEF is a surface-specific fluorescence imaging modality that measures changes in cell-substrate gap distances in the adhesion of live cells. The substrate in PCEF is a photonic crystal. The vertical distance of cell elements from the substrate and spatial information concerning cell attachment to the substrate is quantified by fluorescent tagging of cellular components. Numerical calculations are then compared to PCEF results for the final determination of the spatial properties of the cells attaching to the substrate. In a proof-of-concept study 3T3 fibroblast cells were cultured on a fibronectin layered photonic crystal with the nucleus or cell membrane fluorescently labelled. A benefit of this technology is that coupling prisms, coupling fluids, or advanced microscope objectives are not required as they are for the majority types of microscopic imaging. PCEF generates a fluorescence enhancement factor image that uses the distance of the fluorescence emission from the surface to image cells and generate maps of the surface interactions of cellular components at the single-cell level.

• Label-Free PCEM for Nanoparticles

Additionally, PCEM has been used for the detection of metallic and dielectric nanoparticle attachment to a photonic crystal surface. These particles were detected by means of light scattering and resonant properties, such as wavelength shifting and absorption quenching. PCEM was able to detect surface adsorbed TiO₂ or gold single nanoparticles. In this method, the photonic crystal resonant reflection is scanned and a localized reduction in reflection efficiency is induced among strong nanoparticle absorbers at the resonant wavelength allowing them to be detected by changing the resonant wavelength. Finite Difference Time Domain computer simulations were used to confirm experimental results.

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