Three dimensional multi-molecule tracking in thick samples with extended depth-of-field

Area of Science:

• Biophysics
• Optical Microscopy
• Nanotechnology

Background:

• Accurate three-dimensional (3D) molecular localization is crucial for understanding biological processes.
• Conventional microscopy techniques often face limitations in resolution and depth of field (DOF).

Purpose of the Study:

• To develop and validate a non-z-scanning multi-molecule tracking system with enhanced 3D resolution and extended DOF.
• To demonstrate the capability of the system for precise localization and tracking of molecules in biological samples.

Main Methods:

• Development of a custom composite phase mask (PM) integrating distorted grating (DG) and double-helix point spread function (DH-PSF) functionalities.
• Implementation of a distorted grating and double-helix point spread function combination microscopy (DDCM) system.
• Experimental evaluation of 3D localization precision and effective DOF using the DDCM system.

Main Results:

• Achieved 3D localization precisions of (6.5 nm, 9.2 nm, 23.4 nm), (3.7 nm, 2.8 nm, 10.3 nm), and (5.8 nm, 6.9 nm, 18.4 nm) for different diffraction orders.
• Extended the total effective depth of field (DOF) to 14 μm.
• Successfully localized and tracked beads separated over 12 μm axially.

Conclusions:

• The developed DDCM system offers superior 3D localization precision and an extended DOF for multi-molecule tracking.
• The custom-designed phase mask is key to achieving high performance in the DDCM system.
• This advanced microscopy technique holds significant potential for biological imaging and research.