A Programmable Light Engine For Quantitative TIRF And HILO Imaging

Abstract

We report on a simple yet powerful implementation of objective type total internal reflection fluorescence (TIRF) and highly inclined laminated optical sheet (HILO) illumination microscopy. Both rely on an asymmetric oblique illumination to confine spatially fluorescence excitation to a dielectric boundary or a thin sheet of light, respectively. Undesired side effects are intensity variations across the field of view, due to scattering and interference. Utilizing an acousto-optical deflector (AOD) based scanner, we restore the symmetry of illumination by generating a hollow cone of light, impinging at the sample at an angle adjustable within μs. Parameters relevant for quantitative image analysis are measured on-line, during fluorescence image acquisition by capturing an objective back-focal plane image on a second small CCD detector. Our device affords background-free variable-angle TIRF/HILO even for the shortest practical exposure times. We expect it to be useful for evanescent-wave imaging involving multiple excitation wavelengths, multiple penetration depths (Variable-angle TIRF) and experiments using objectives with different magnification (and hence different back pupils). We illustrate the system performance by demonstrating ultra-low background TIRF imaging of 200 Hz Qdot blinking, vinculin-EGFP labeled cellular adhesion sites and lysosomal dynamics in cortical astrocytes.