Compressive three-dimensional super-resolution microscopy with speckle-saturated fluorescence excitation.

Area of Science:

• Biophysics
• Optical Microscopy
• Super-resolution Imaging

Background:

• Nonlinear structured illumination microscopy (nSIM) offers high resolution but faces challenges in 3D imaging due to fluorophore photobleaching.
• Extending 2D nSIM to 3D is difficult because of the fading of organic fluorophores under intense illumination conditions.

Purpose of the Study:

• To develop a 3D sub-diffraction imaging method using nSIM that overcomes limitations of photobleaching and acquisition time.
• To present a compressed sensing approach for 3D nSIM imaging of cultured cells.

Main Methods:

• Utilized a compressed sensing approach with saturated fluorescence excitation for 3D nSIM.
• Exploited the natural orthogonality of speckles at different axial planes for 3D sample probing via a single 2D scan.
• Leveraged optical vortices in polarized speckle patterns to ensure high fluorescence contrast under saturated excitation.

Main Results:

• Achieved 3D sub-diffraction imaging of cultured cells using nSIM with saturated excitation.
• Demonstrated that the method allows 3D probing with a single 2D scan by exploiting speckle orthogonality.
• Ensured high fluorescence contrast through the density of intensity minima in polarized speckle patterns.

Conclusions:

• Compressed speckle microscopy is a simple and effective method for 3D super-resolved nSIM imaging.
• The presented approach offers potentially reduced acquisition times and photobleaching compared to conventional methods.
• This technique advances 3D super-resolution microscopy for biological imaging applications.