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Fig. 1 | Molecular Brain

Fig. 1

From: Accelerated super-resolution imaging with FRET-PAINT

Fig. 1

Principle and characterization of FRET-PAINT. a Docking (black), donor (blue), and acceptor (red) strands used to characterize FRET-PAINT. The docking strand contains biotin (B) at the 5’-end for surface immobilization. The donor strand is labeled with either Alexa488 or Cy3 at the 3’-end. The acceptor strand is labeled with Cy5 at one of the underlined sites. b Scheme of FRET-PAINT. Donor fluorophores are excited but only acceptor fluorophores excited via FRET are detected. c Representative Cy5 fluorescence intensity time traces with 1000 nM Donor_P1_Alexa488 and 100 nM Acceptor_P11_Cy5. d Normalized FRET efficiency as a function of the donor-acceptor distance for Cy3-Cy5 (black) and Alexa488-Cy5 (red) pairs. (e) DNA-PAINT images of surface immobilized docking strand (Docking_P0) at indicated concentrations of Acceptor_P11_Cy3. f FRET-PAINT images of Docking_P0 at indicated concentrations of Donor_P1_Cy3 with Acceptor_P6_Cy5 fixed at 10 nM. g FRET-PAINT images of Docking_P0 at indicated concentrations of Acceptor_P6_Cy5 with Donor_P1_Cy3 fixed at 10 nM. h FRET-PAINT images of Docking_P0 at the indicated concentration of Donor_P1_Alexa488 with Acceptor_P2_Cy5 fixed at 10 nM. i FRET-PAINT images of Docking_P0 at the indicated concentration of Acceptor_P2_Cy5 with Donor_P1_Alexa488 fixed at 10 nM. Scale bars: 1 μm. j Comparison of SNRs of DNA-PAINT at varying Cy3 imager strand concentration (black) and FRET-PAINT at varying Cy3 donor strand (red solid), and Cy5 acceptor strand (red open) concentration. k Comparison of SNRs of DNA-PAINT at varying Cy3 imager strand concentration (black) and FRET-PAINT at varying Alexa488 donor strand (red solid), and Cy5 acceptor strand (red open) concentrations. SNR was defined as the ratio of spot brightness (amplitude of two-dimensional Gaussian fit of spot) to the background fluctuation (FWHM of Gaussian fit of background signal). The data were fitted to an inverse square root of imager concentration. Green dashed lines are added to help find the data points with SNR = 3.3

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