Dual-mode phase and fluorescence imaging with a confocal laser scanning microscope

Area of Science:

• Biomedical Optics
• Microscopy Techniques
• Cellular Imaging

Background:

• Confocal laser scanning microscopy (CLSM) is a powerful tool for biological imaging.
• Phase imaging offers quantitative structural information but often suffers from limited depth of field.
• Fluorescence imaging provides functional and molecular details but can be limited in structural resolution.

Purpose of the Study:

• To develop a dual-mode imaging system integrating phase and fluorescence capabilities within a CLSM.
• To enhance the phase imaging capability by extending the depth of field.
• To enable simultaneous acquisition of complementary structural and functional information from live cells.

Main Methods:

• Utilized a tunable acoustic gradient index of refraction lens for fast axial scanning to extend CLSM depth of field.
• Employed transillumination and recorded intensity images at varying defocusing distances for phase reconstruction.
• Reconstructed phase images using the transport-of-intensity equation.
• Acquired 3D fluorescence images via standard confocal scanning.
• Ensured pixel-to-pixel correspondence between phase and fluorescence images.

Main Results:

• Successfully implemented dual-mode phase and fluorescence imaging in a CLSM system.
• Achieved extended depth of field for phase imaging.
• Generated phase and fluorescence images with precise spatial correlation.
• Demonstrated the complementary nature of the obtained structural and functional data.
• Enabled standalone determination of the refractive index of live cells.

Conclusions:

• The developed dual-mode imaging system provides comprehensive, quantitative information from live cells.
• The combination of phase and fluorescence imaging overcomes limitations of individual modalities.
• This technique offers a novel approach for label-free refractive index measurement in biological samples.