Temporal Domain Multiplexing

Temporal domain multiplexing of two red fluorescent proteins(FPs). (A) Acquired photobleaching reference traces of mfRFP and mCardinal in life HeLa-cell, n=10 cells from 2 briefed movies, 41 frames long, from 1 cell culture batch. Data are shown as mean standard deviation ± standard deviation SD. Illumination(594nm at 100% power, 500ms Exposure time). (B) Demultiplexing of co-expressed Keratine-targeted mCardinal and H2B-targeted mfRFP in HeLa cell. Using an identical microscope and image condition as in A, The GT (Ground-truth) image was first refined via background subtraction (ImageJ) followed by denoising (Nikon Element AR software) to produce the input image, the input image was supplied to MATLAB script for linear unmixing of each reference at each pixel. (the GT and input images are brief movies obtained from a single channel imaging(red), containing two different but spectrally similar (red) fluorescent proteins. The output image mfRFP-H2B and mCardinal-Keratine are products of linear un-mixing, the Merge image is a composite of mfRFP-H2B and mCardinal-Keratine. (Scale-Bar 100um). (C). Line profile of a selected ROI (Region of Interest) of the ground-truth (black), input (cyan), and output (orange) and corresponding half violin plot with data on the left comparing the mean said line profile.* signify, significant at the level of 0.05, and n.s. (not significant at the level of 0.05)