Publications
Dynamic three-dimensional tracking of single fluorescent nanoparticles deep inside living tissue
JH Spille, T Kaminski, HP Königshoven, U Kubitscheck. Optics Express 20 (18), 19697-19707, (2012).
Abstract:
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.
Light sheet microscopy is great for single particle tracking because you can get confocal-like background suppression - but a camera speed! What's limiting now is the depth of focus of your high NA objective. Single molecule trajectories end because the particle diffuses out of the focal plane and the light sheet. So let's bring them back to focus! We encoded 3D information in the PSF shape using an astigmatic lens and evaluated image data on-the-fly to reposition a piezo stage holding the sample. The heavy detection objective stays in place and we don't need a galvo to displace the light sheet. This extends our axial tracking range from 1um (depth of focus and light sheet thickness) to 100um (piezo range). We demonstrate that we can follow fluorescent beads in buffer and 50nm dextran molecules micro-injected into live cell nuclei for hundreds of frames. Later, we got much better and were able to follow single molecules!
