isoSTED microscopy with water-immersion lenses and background reduction

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Fluorescence microscopy is crucial for studying cellular structures and processes.
• Stimulated emission depletion (STED) microscopy offers nanometer resolution but often lacks isotropic capabilities.
• Isotropic resolution is essential for accurately imaging cellular structures in any orientation.

Purpose of the Study:

• To present an isoSTED microscope enabling isotropic super-resolution imaging.
• To demonstrate its application in imaging living cellular structures with high fidelity.
• To introduce a method for background signal removal in super-resolution images.

Main Methods:

• Utilized water-immersion objective lenses in the STED microscope setup.
• Achieved isotropic resolution better than 60 nm in living samples at room temperature.
• Developed a method to identify and remove unspecific background fluorescence signals.

Main Results:

• The isoSTED microscope provides isotropic resolution of <60 nm in living cells.
• Imaging was performed without CO2 supply or pH control, at room temperature.
• Imaging speed reached 0.8 s/μm³, enabling rapid acquisition.
• A novel method effectively removed background noise from the super-resolution images.

Conclusions:

• The isoSTED microscope significantly enhances the visualization of cellular structures with isotropic resolution.
• This technique offers a powerful tool for studying dynamic biological processes in living cells.
• The background removal method improves image quality and data interpretation in super-resolution microscopy.