ISM-assisted tomographic STED microscopy.

Area of Science:

• Microscopy
• Optical Imaging
• Biophysics

Background:

• Stimulated emission depletion (STED) microscopy offers high resolution but is limited by photobleaching in biological samples.
• Tomographic STED (tomoSTED) microscopy addresses photobleaching by using 1D depletion patterns from multiple directions.
• Achievable resolution in tomoSTED depends on the number of orientations, impacting light dose and bleaching.

Purpose of the Study:

• To introduce and demonstrate a novel approach combining tomographic STED (tomoSTED) microscopy with image scanning microscopy (ISM).
• To reduce the number of depletion pattern orientations required in tomoSTED microscopy while maintaining high resolution and image quality.
• To mitigate photobleaching in high-resolution imaging of biological samples.

Main Methods:

• Development and implementation of an integrated ISM-tomoSTED microscope.
• Acquisition of 2D images using successively applied 1D depletion patterns from reduced orientations.
• Comparison of image quality and resolution achieved with the ISM-tomoSTED versus conventional tomoSTED methods.

Main Results:

• The novel ISM-tomoSTED microscope successfully reduces the number of required depletion pattern orientations by approximately a factor of two.
• The achieved resolution and image quality are comparable to those obtained with conventional tomoSTED microscopy.
• This reduction in orientations leads to a lower overall light dose, thereby minimizing photobleaching.

Conclusions:

• Combining ISM with tomoSTED microscopy is a viable strategy to enhance imaging efficiency and reduce photobleaching.
• The ISM-tomoSTED approach offers a significant advantage for high-resolution biological imaging where photobleaching is a critical concern.
• This technique provides a pathway to achieve superior resolution with reduced light exposure, preserving sample integrity.