Multiphoton fluorescence microscopy (MPM), using near infrared excitation light, provides increased penetration depth, decreased detection background, and reduced phototoxicity. Using stimulated emission depletion (STED) approach, MPM can bypass the diffraction limitation, but it requires both spatial alignment and temporal synchronization of high power (femtosecond) lasers, which is limited by the inefficiency of the probes. Here, we report that upconversion nanoparticles (UCNPs) can unlock a new mode of near-infrared emission saturation (NIRES) nanoscopy for deep tissue super-resolution imaging with excitation intensity several orders of magnitude lower than that required by conventional MPM dyes. Using a doughnut beam excitation from a 980 nm diode laser and detecting at 800 nm, we achieve a resolution of sub 50 nm, 1/20th of the excitation wavelength, in imaging of single UCNP through 93 μm thick liver tissue. This method offers a simple solution for deep tissue super resolution imaging and single molecule tracking.

Gut ischemia is common in critically ill patients, promoting thrombosis and inflammation in distant organs. The mechanisms linking hemodynamic changes in the gut to remote organ thrombosis remain ill-defined. We demonstrate that gut ischemia in the mouse induces a distinct pulmonary thrombotic disorder triggered by neutrophil macroaggregates. These neutrophil aggregates lead to widespread occlusion of pulmonary arteries, veins, and the microvasculature. A similar pulmonary neutrophil-rich thrombotic response occurred in humans with the acute respiratory distress syndrome. Intravital microscopy during gut ischemia-reperfusion injury revealed that rolling neutrophils extract large membrane fragments from remnant dying platelets in multiple organs. These platelet fragments bridge adjacent neutrophils to facilitate macroaggregation. Platelet-specific deletion of cyclophilin D, a mitochondrial regulator of cell necrosis, prevented neutrophil macroaggregation and pulmonary thrombosis. Our studies demonstrate the existence of a distinct pulmonary thrombotic disorder triggered by dying platelets and neutrophil macroaggregates. Therapeutic targeting of platelet death pathways may reduce pulmonary thrombosis in critically ill patients.