Precise Three-Dimensional Scan-Free Multiple-Particle Tracking over Large Axial Ranges with Tetrapod Point Spread Functions

[Image: see text] We employ a novel framework for information-optimal microscopy to design a family of point spread functions (PSFs), the Tetrapod PSFs, which enable high-precision localization of nanoscale emitters in three dimensions over customizable axial (z) ranges of up to 20 μm with a high numerical aperture objective lens. To illustrate, we perform flow profiling in a microfluidic channel and show scan-free tracking of single quantum-dot-labeled phospholipid molecules on the surface of living, thick mammalian cells.