Design and simulation of a snapshot multi-focal interferometric microscope.

Area of Science:

• Biophysics
• Optical Microscopy
• Nanotechnology

Background:

• Achieving high temporal and spatial resolution in 3D fluorescent imaging remains a significant challenge.
• Existing methods often struggle to provide isotropic resolution across large volumes.

Purpose of the Study:

• To introduce and validate an interferometric multifocus microscopy (iMFM) system.
• To achieve simultaneous multifocal plane interferometry for axial super-resolution and isotropic 3D resolution in a single exposure.

Main Methods:

• Designed and simulated an iMFM system combining multifocus microscopy with opposing objectives and diffractive optical elements.
• Employed Richardson-Lucy deconvolution with total variation regularization for 3D object recovery.
• Utilized a maximum likelihood estimator (MLE) for single molecule tracking, with a novel method for initial axial position determination.

Main Results:

• Demonstrated theoretically and numerically that iMFM achieves isotropic 3D nanoscopic localization accuracy within a 2um axial range.
• Showcased a 3-4 times improvement in diffraction-limited axial resolution for single-shot, wide-field 3D extended object recovery.
• Validated the capability for simultaneous multifocal plane interferometry.

Conclusions:

• The iMFM system offers a powerful solution for 3D fluorescent imaging challenges.
• iMFM provides isotropic 3D resolution and enhanced axial resolution, crucial for dynamic biological processes.
• This technique is poised to become a valuable tool for imaging fast 3D events with high precision.