Comparison of I5M and 4Pi-microscopy

Area of Science:

• Super-resolution fluorescence microscopy
• Optical imaging
• Biophysics

Background:

• 4Pi and I5M microscopy achieve ~100 nm axial resolution by combining spherical wavefronts from opposing high numerical aperture lenses.
• Both techniques generate a point-spread function (PSF) with a central spot and axially shifted sidelobes, causing replication artefacts in raw images.

Purpose of the Study:

• To investigate the capability of 4Pi and I5M fluorescence microscopy for artefact-free imaging of aqueous specimens.
• To compare the performance of 4Pi and I5M microscopes by analyzing their optical transfer function (OTF) and signal-to-noise ratio (SNR).

Main Methods:

• Utilized an interconvertible setup with high numerical aperture (NA=1.2) water-immersion lenses for direct comparison of 4Pi and I5M imaging modes.
• Performed imaging of E. coli bacteria.
• Corroborated experimental results with simulations based on experimental signal strength.

Main Results:

• I5M generated approximately 10 times brighter raw image data compared to two-photon excitation (2PE) 4Pi-microscopy.
• Despite higher signal brightness, I5M data could not be entirely cleared of artefacts.
• 4Pi microscopy exhibited less pronounced sidelobes without significant lateral substructure, facilitating reliable and faster artefact removal.

Conclusions:

• The suppression of sidelobes is crucial for artefact-free imaging in super-resolution microscopy.
• Trading sidelobe suppression for increased image signal (as in I5M) is not advisable with current aperture angles and fluorescence signal strengths.
• 4Pi microscopy remains a more reliable choice for artefact-free imaging despite its lower signal intensity compared to I5M.