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

Three-dimensional (3D) spatial information can be encoded in two-dimensional images of fluorescent nanoparticles by astigmatic imaging. We combined this method with light sheet microscopy for high contrast single particle imaging up to 200 µm deep within living tissue and real-time image analysis to determine 3D particle localizations with nanometer precision and millisecond temporal resolution. Axial information was instantly directed to the sample stage to keep a moving particle within the focal plane in an active feedback loop. We demonstrated 3D tracking of nanoparticles at an unprecedented depth throughout large cell nuclei over several thousand frames and a range of more than 10 µm in each spatial dimension, while simultaneously acquiring optically sectioned wide field images. We conclude that this 3D particle tracking technique employing light sheet microscopy presents a valuable extension to the nanoscopy toolbox.