Elliptical mirror-based TIRF microscopy with shadowless illumination and adjustable penetration depth.

ABSTRACT

We propose an elliptical mirror-based total-internal-reflection fluorescence (e-TIRF) microscopy with shadowless illumination and adjustable penetration depth. The elliptical mirror is used to produce a hollow-cone illumination with all azimuthal directions and a large range of incident angle, so as to attenuate the potential shadow effects when utilizing a single-direction illumination, such as asymmetries and low contrast. The experiment demonstrates that the e-TIRF method can realize shadowless imaging with symmetric intensity distribution. Meanwhile, the penetration depth of e-TIRF can be theoretically adjusted from 58 nm to 250 nm by adjusting the size of the aperture or the position of an opaque mask. This method extends the minimum penetration depth, which is useful for high axial resolution.