Recent technological advances have seen Total Internal Reflection Fluorescence Microscopy (TIRF-M) join the ranks of the most established tools for imaging surfaces such as plasma cell membranes. Such desirable functionality can be credited to the low-invasive high-contrast surface imaging capabilities of the TIRF-M with optical sectioning ranging 100-200 nanometers. Moreover, improvements on the TIRF-M non-uniform scattering fringes and other artefacts have been made. For quantitative microscopy with TIRF-M, a homogenous evanescent field of excitation over the whole field of view is generally desired, unfortunately, with Gaussian beams it becomes quite challenging to generate this field. This shortcoming can be overcome through the combination of single and multimode fibers or micro-lens arrays. By doing this, a flat-field homogenous epi-fluorescence wide-field illumination with great potential to improve single molecule imaging is generated. Unfortunately, these flat-field approaches have not been applied to TIRF-M.
Professor Katrin Heinze and her PhD student Benjamin Schreiber at University of Würzburg in Germany in collaboration with Dr. Kareem Elsayad at Vienna Biocenter Core Facilities developed a novel objective-based homogeneous TIRF illumination using Bessel beam side lobes. Their aim was to introduce a cost-effective TIRF setup with a very low degree of complexity and no moving parts which would in turn yield a flattop-like excitation profile. They hoped to successfully apply the well-known Bessel-beam illumination, which has previously mainly been implemented in structural illumination and light sheet microscopy. Their work is currently published in the research journal, Optics Letters.
Their experiments involved the formation of a radial symmetric evanescent field at the microscopy coverslip-sample interface by 360° overcritical p-polarized illuminations. To ensure p-polarization was uniform, the researchers focused a radially polarized laser ring at the TIRF objective back focal plane. Two axicon lenses and one focus lens were then employed to allow for generation and control of the ring diameter so as to tune the TIRF excitation angle.
The authors observed that the laser ring diameter could easily be adjusted by moving one lens along the optical axis, which allowed for switching between wide-field and TIRF mode. Additionally, the research team noted that the field of view for AxiTIRF-M became remarkably big when compared to standard single-spot Gaussian illumination where just a few percent of the central area was homogenously illuminated.
The study successfully introduced an axicon-based laser ring focused on a TIRF objective back focal plane which can be used for TIRF-M with added value. It has been seen that the resulting Bessel beam effectively enlarges the field of view and provides a homogeneous TIRF illumination in a cell-friendly objective-based setup. With some additional tweaks, the setup described by Schreiber, Elsayad, and Heinze is potentially suitable for 360° incoherent illuminations, which would further suppress TIRF fringes and would allow for even shadow-less TIRF-M.
Benjamin Schreiber, Kareem Elsayad, Katrin G. Heinze. Axicon-based Bessel beams for flat-field illumination in total internal reflection fluorescence microscopy. Vol. 42, No. 19 / October 1 2017 / Optics Letters
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