NIR fluorescence imaging setup

An Olympus BX53 microscope equipped with 20× (MPlanFL N 20×/0.45, Olympus) and 100× (UPlanSApo 100×/1.35 Sil, Olympus) objectives was used. A Xeva-1.7-320 NIR camera (Xenics®) and a Zyla 5.5 sCMOS camera (Oxford Instruments) were used to observe the EB-NS fluorescence excited by a 561 nm laser (Cobolt Jive^TM 561 nm). Typically, a droplet of an EB-NS dispersion was put on glass slides, dried and imaged at 10–50 mW excitation power. To assess bleaching, a solution of Rhodamin B (Sigma-Aldrich) in isopropanol was prepared and a drop of this solution was put on a glass slide for imaging. Dried EB-NS were put on a separate glass slide. At a continuous excitation of 100 mW at 561 nm, images with an integration time of 100 ms were recorded 120 times every 0.5 min in the case of Rhodamin B and every 1 min with an integration time of 1 s in the case of EB-NS. For Rhodamin B the Zyla camera was used and for EB the Xenics NIR camera was used. The specified fluorescence intensity corresponds to the mean gray value of the images.

For video-rate imaging and size-fluorescence correlation experiments, a modified setup equipped with a Cheetah 640TE3 camera (Xenics NV, Belgium) was used for NIR detection. Here, the light was passed through a dichroic mirror (HC BS R785, AHF, Germany) and a long-pass filter (FELH0900, Thorlabs, Inc., USA) before reaching the camera’s sensor. Images were captured with the 100× objective.