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

Area of Science:

• Biophysics
• Optical Microscopy
• Cell Biology

Background:

• Total internal reflection fluorescence microscopy (TIRF-M) is crucial for high-contrast surface imaging.
• Generating a uniform excitation field in TIRF-M is challenging with conventional Gaussian beams.
• Existing TIRF-M improvements address artifacts but can be complex.

Purpose of the Study:

• To develop a cost-effective, low-complexity TIRF-M setup.
• To achieve a homogenous, flattop-like excitation profile for quantitative microscopy.
• To demonstrate the benefits of the new setup for live-cell imaging.

Main Methods:

• Utilized a tunable laser ring zoom focus system for 360° TIRF illumination.
• Employed two axicon lenses and one focus lens to control the ring diameter and excitation angle.
• Combined 360° illumination with a radial polarizer to generate an evanescent Bessel-beam excitation field.

Main Results:

• The novel setup generates a flattop intensity profile over an extended field of view.
• The system exhibits a low degree of complexity and no moving parts.
• Demonstrated successful application in live-cell imaging.

Conclusions:

• The axicon-based Bessel beam illumination provides a superior excitation field for TIRF-M.
• This cost-effective setup enhances quantitative microscopy and live-cell imaging capabilities.
• The approach offers a practical advancement for surface imaging techniques.