Ratiometric 4Pi single molecule localization with optimal resolution and color assignment

4Pi single molecule localization microscopy (4Pi-SMLM) with two opposing objectives achieves sub-10 nm isotropic 3D resolution with as few as 250 photons collected by each objective. Here, we developed a new ratiometric multi-color imaging strategy for 4Pi-SMLM which employed the intrinsic multi-phase interference intensity without increasing the complexity of the system and achieved both optimal 3D resolution and color separation. By partially linking the photon parameters between channels with interference difference of π during global fitting of the multi-channel 4Pi single molecule data, we showed on simulated data that the loss of the localization precision is minimal compared with the theoretical minimum uncertainty, the Cramer-Rao lower bound (CRLB).

Global fitting for high-accuracy multi-channel single-molecule localization

Multi-channel detection in single-molecule localization microscopy (SMLM) greatly increases information content for various biological applications. Here, we present globLoc, a graphics processing unit (GPU) based global fitting algorithm with flexible PSF modeling and parameter sharing, to extract maximum information from multi-channel single molecule data. We show, both in simulations and experiments, that global fitting can substantially improve the 3D localization precision for biplane and 4Pi SMLM and color assignment for ratiometric multicolor imaging.

Numerical Analysis on Color Preference and Visual Comfort from Eye Tracking Technique

Color preferences in engineering are very important, and there exists relationship between color preference and visual comfort. In this study, there are thirty university students who participated in the experiment, supplemented by pre- and posttest questionnaires, which lasted about an hour. The main purpose of this study is to explore the visual effects of different color assignment with subjective color preferences via eye tracking technology. Eye-movement data through a nonlinear analysis detect slight differences in color preferences and visual comfort, suggesting effective physiological indicators as extensive future research discussed. Results found that the average pupil size of eye-movement indicators can effectively reflect the differences of color preferences and visual comfort. This study more confirmed that the subjective feeling will make people have misjudgment.