MesoTIRF: a Total Internal Reflection Fluorescence illuminator for axial super-resolution membrane imaging at the mesoscale

Abstract

Total Internal Reflection Fluorescence (TIRF) illumination bypasses the axial diffraction limit of light by using an evanescent field to excite fluorophores close to a sample substrate. TIRF illumination significantly improves image contrast, allowing researchers to study membrane structure and dynamics with localized reductions in photobleaching. However, a significant limitation of most TIRF microscopes is the relatively small field of view (FOV). TIRF objectives require a high numerical aperture (NA) to generate the evanescent wave. Such lenses invariably have a high magnification and result in a [~] 50 {micro}m diameter imaging field, requiring many subsequent images for accurate statistical analysis. Waveguide and prism-based TIRF systems are, in principle, compatible with lower magnification lenses to widen the FOV but these have a correspondingly low NA and lateral resolution. To overcome these limitations, we present a prism-based TIRF illuminator for the Mesolens - a specialist objective lens with the unusual combination of low magnification and high NA. This new imaging mode - MesoTIRF - enables TIRF imaging across a 4.4 mm x 3.0 mm FOV. We demonstrate evanescent wave illumination of cell specimens, and show the multi-wavelength capability of the modality across more than 700 cells in a single image. MesoTIRF images have up to a 6-fold improvement in signal-to-background ratio compared to widefield epi-fluorescence illumination, and we illustrate the benefit of this improved contrast for the detection and quantification of focal adhesions in fixed cells. Fluorescence intensities and resolvable structural detail do not vary considerably in homogeneity across the MesoTIRF FOV.