Dynamic three-dimensional tracking of single fluorescent nanoparticles deep inside living tissue

Area of Science:

• Biophysics
• Optical Microscopy
• Nanotechnology

Background:

• Three-dimensional (3D) spatial information is crucial for understanding biological processes at the nanoscale.
• Traditional imaging techniques often struggle with deep tissue penetration and precise 3D localization of nanoparticles.
• Astigmatic imaging offers a way to encode depth information into 2D images.

Purpose of the Study:

• To develop and validate a novel 3D particle tracking technique for deep-tissue imaging.
• To achieve high-precision, real-time localization of fluorescent nanoparticles in living biological samples.
• To extend the capabilities of nanoscopy for studying cellular dynamics.

Main Methods:

• Combining astigmatic imaging with light sheet microscopy for enhanced contrast and depth penetration.
• Implementing real-time image analysis for rapid 3D particle localization.
• Utilizing an active feedback loop with sample stage control to maintain particles in focus.
• Tracking fluorescent nanoparticles within living tissue up to 200 µm deep.

Main Results:

• Achieved nanometer precision and millisecond temporal resolution for 3D particle localization.
• Demonstrated successful tracking of nanoparticles at unprecedented depths within large cell nuclei.
• Tracked particles over thousands of frames with a spatial range exceeding 10 µm in all dimensions.
• Acquired simultaneous optically sectioned wide-field images.

Conclusions:

• The developed 3D particle tracking technique is a valuable addition to the nanoscopy toolkit.
• This method enables high-resolution imaging and tracking of nanoparticles in challenging biological environments.
• It opens new possibilities for studying molecular mechanisms and dynamics in living tissues.