In Total Internal Reflection Fluorescence (TIRF) microscopy, a specimen is illuminated by the evanescent field produced by a beam undergoing total internal reflection, whose characteristic depth is orders of magnitude below the axial diffraction limit. The axial super-resolution and much improved contrast of TIRF gives a substantially improved signalto-background ratio (SBR) than that which can be achieved with widefield illumination, and it is used extensively for imaging of the cell membrane [1] to [3].
Commercial TIRF objectives allow for a simple adaption to existing microscope systems. To attain a super-critical angle at the specimen plane, the illumination must enter the back focal plane of these objectives off-axis, requiring a high numerical aperture. As such, the magnification of these objectives is generally a minimum of 60x, reducing the lateral imaging field to less than 100 µm in diameter. Sub-cellular axial resolution is therefore restricted to a tiny population of cells and statistically significant data sampling may be difficult to achieve.
To address this, we have developed a TIRF illuminator for the Mesolens, a custom giant objective lens with a 4x/0.47NA specification. The Mesolens provides an imaging field of 4.4 mm x 3.0 mm, and in combination with our new TIRF illuminator which we call MesoTIRF we have performed imaging of large cell populations with sub-micron resolution in three-dimensions. With MesoTIRF we demonstrate more than a five-fold improvement in SBR and significantly reduced photobleaching rate compared to widefield epifluorescence illumination. We will present details of the MesoTIRF system together with current and emerging applications in cell imaging.
