Axicon-based Bessel beams for flat-field illumination in total internal reflection fluorescence microscopy


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.

Axicon-based Bessel beams for flat-field illumination in total internal reflection fluorescence microscopy. Advances in Engineering

About the author

Benjamin Schreiber is a PhD student with Prof. Dr. Katrin G. Heinze at the Rudolf-Virchow-Centre for Experimental Biomedicine at the University Würzburg, Germany. He holds a BSc and MSc degree in physics from The Technische Universität Dresden (TUD), Germany. He performed his undergraduate studies in the field of nanotechnology at the Helmholtz-Zentrum Dresden-Rossendorf, Germany, and was involved in DNA origami research, plasmonics as well as ion-induced nanostructures. Afterwards, Benjamin Schreiber moved to Würzburg for an interdisciplinary PhD project where he is currently developing new surface imaging methods for biomedical applications. Therefore, Benjamin Schreiber combines his expertise in nanotechnology and optics to reach his goals. Within the framework of his PhD project, Benjamin Schreiber designs nanocoatings to enhance the fluorescence signal readout of cell receptor activation processes.

Particularly optical instrumentation became his passion. Benjamin Schreiber could further enhance his skills in optical engineering during a half year research stay at the Advanced Microscopy facility of the Vienna Biocenter with Dr. Kareem Elsayad, Austria, where the AxiTIRF setup presented in this feature was brought to life.

About the author

Katrin Heinze is a Professor at the Rudolf-Virchow-Centre for Experimental Biomedicine at the University Würzburg, Germany. She is a physicist by training, and got first in touch with biophysics as a PhD student with Petra Schwille in Göttingen and Dresden when “taming and tweaking” Fluorescence Correlation methods for intracellular analysis of biomolecular interactions. Katrin Heinze received her PhD degree in 2002, and was honored by the May-Planck Society with the Otto-Hahn-Medal. With the Max-Planck fellowship, she joined Paul Wiseman’s group at McGill University in Montreal as a Postdoc. Afterwards she accepted a position at the Research Institute of Molecular Pathology in Vienna where she started her first own research group. In 2011, she returned to her home country Germany, and joined the Rudolf-Virchow-Center at the University of Würzburg as a research group leader, and since May 2017 as an associate professor for Molecular Microscopy.

Her research interest ranges from single molecules to whole organ imaging and spectroscopy. Bridging physical, biological and medical research is the most fun part, and the main strength of her work.


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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