Wide-field two-photon random illumination microscopy (2P-RIM)

Area of Science:

• Biophotonics
• Microscopy
• Optical Imaging

Background:

• Focused two-photon (2P) microscopy offers axial sectioning but causes sample damage and has lower resolution than one-photon (1P) microscopy.
• Wide-field 2P microscopy promises reduced phototoxicity and high acquisition rates but struggles with axial sectioning over large fields of view (FOVs).

Purpose of the Study:

• Introduce 2P random illumination microscopy (2P-RIM) for low photo-damage, high-resolution, wide-field 2P imaging.
• Achieve effective axial sectioning and enhanced lateral resolution for large FOVs using an easy-to-implement technique.

Main Methods:

• Implemented wide-field 2P microscopy using random, speckled illuminations.
• Employed an image standard deviation matching algorithm for image reconstruction.
• Utilized parallel detection across millions of pixels with extended integration times.

Main Results:

• Demonstrated multicolor imaging over FOVs exceeding 200 μm.
• Achieved a lateral resolution of 220 nm and axial sectioning of 2 μm.
• Reduced peak excitation powers by approximately 10 times compared to focused laser scanning microscopy.

Conclusions:

• 2P-RIM is an effective wide-field technique for low photo-damage biological imaging.
• The method enhances lateral resolution and provides micrometric axial sectioning for large FOVs.
• 2P-RIM offers a promising alternative to conventional 2P microscopy for various applications.