MesoTIRF: A prism-based Total Internal Reflection Fluorescence illuminator for high resolution, high contrast imaging of large cell populations

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. However, standard TIRF imaging through the objective requires a high numerical aperture (NA) to generate the evanescent wave. Available lenses have a high magnification with a correspondingly small field of view—ranging from ∼50 μm to 1 mm in diameter. Switching to the older prism-TIRF configuration introduced by Axelrod in the 1980s might seem to remove the requirement for high objective NA and allow the use of existing large-field objectives. Unfortunately, these lenses are unsuitable because their throughput of light is too low for TIRF imaging. As such, high sensitivity TIRF imaging over a much larger mesoscopic field has yet to be demonstrated. We have developed a prism-based TIRF illuminator for the Mesolens—a highly corrected objective lens with an unparalleled ratio of NA to magnification. The imaging field of the Mesolens is 204 times larger than that of the TIRF objectives previously described, increasing the optical throughput of the optical system by a factor of 25 compared to an off-the-shelf microscope objective of the same magnification. We demonstrate MesoTIRF imaging of cell specimens and show the multi-wavelength capability of the modality across more than 700 cells in a single image.