Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part I. Identifying Sources of Nonevanescent Excitation Light

Abstract

Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2).

Calibrating Evanescent-Wave Penetration Depths for Biological TIRF Microscopy

“…(iii), background comes from stray excitation from inside the microscope objective and different optical surfaces [17]. In the critical illumination scheme used in multi-angle TIRF, any scatter on the scanning mirrors (or in any conjugate sample plane) is imaged into the sample plane, Figs.…”

“…2C. 'Spinning' TIRF (spTIRF) or incoherent ring illumination [26] reduces interference fringes and illumination non-uniformities but it does not change the problem of the presence of nonevanescent excitation light [17] as it only redistributes and equalizes intensities, Fig. 2C.…”

“…and k is a constant (°/V) characteristic for the scanner used, and Uθ is the voltage applied to the polar axis of the scanner. Alternatively, for the beam not to suffer TIR, the glass/air surface can be made transmissive by an oil-coupled external prism [25; 35] or a solid-immersion lens (SIL) [17], Alternatively, point emitters can be fixed to the surface of an oblique microscope slide, Fig. 5B, [34], a large convex [70] or concave lens [67].…”

“…All the above test samples have in common the requirement for an evenly lit field of view (see [17] for the limits of this approximation) and they all need z-scans to locate the fluorophores with respect to the reflecting interface. This adds a complication for objectivetype TIRF as the reflected beam displaces laterally upon focusing (see above), and the zdependence of the PSF (detection volume) and z-dependent spherical aberrations require for a correction, see [71] and our calibration protocol, below.…”

Star Light, Star Bright, First Molecule I See Tonight

“…In a set of companion articles in this issue of Biophysical Journal, Brunstein et al (4,5) describe their recent efforts to identify and then address these key challenges. Their motivation was the following: In laser-based objective TIRF systems, the evanescent wave generated through a high-NA objective yields an anisotropic illumination profile across the field of view, an effect that can be obviated through beam scanning (6).…”