Fig. 3: Multiplexed photoconversion strategies for green-to-red and NIR-to-far-red PCFPs. | Nature Communications

Fig. 3: Multiplexed photoconversion strategies for green-to-red and NIR-to-far-red PCFPs.

From: Blue-shift photoconversion of near-infrared fluorescent proteins for labeling and tracking in living cells and organisms

Fig. 3

a Emission spectra for Dendra2 and miRFP713 before (solid) and after (dashed) photoconversion. b, c Efficiency of photoconversion upon various illumination for miRFP713 (dark gray) and Dendra2 (blue). b The fold of conversion for the two proteins is compared over the same energy range of 405 nm illumination (CW). c The energy dependence of the conversion yield for miRFP with 775 nm (700 ps, 40 MHz) illumination is compared to the primed conversion for Dendra2 with fixed 488 nm (CW, 0.13 kW/cm2) and increasing 775 nm (700 ps, 40 MHz) illumination. The comparisons in (b, c) have been performed on a layer of E. coli expressing miRFP713 or Dendra2. Each data point is the mean and SD for bacteria enclosed in a field of view of ~ 15 × 15 µm2 (=50–100 bacteria). Representative example of 3 replica for spatially uncoupled (d) and coupled (e) photoconversion. Scale bars, 5 µm. d Using photoconversion wavelengths and illumination conditions unique for the PCFPs, like pulsed 775 nm for miRFP and low energy of 405 nm light for the green-to-red PCFPs, two different spatial locations (within the dotted square) can be selectively photoconverted in the field of view. e The spatially localized illumination with a combination of high 775 nm and 488 nm light (within the squared dotted area) photoconverts both PCFPs in the same area. Scale bar, 10 µm. f E. coli expressing miRFP720 photoactivated with 775 nm light at saturation level in a region of around 25 µm (dotted square) and observed in the blue-shifted channel (650–690 nm). g Brightfield images of four-time points in the 100 min observation window for the inset in orange. The red square marks the photoconverted area. Scale bars, 10 µm. h Ratio of growth between the photoconverted and non-photoconverted area illuminated with 775 nm (with the energy required to saturate the miRFP720 photoconversion) or 405 nm (with the energy required to saturate Dendra2 photoconversion). The box plot reports 4 independent repetitions (box encloses the 25th to 75th percentile, whiskers include all values and line shows the mean, 0.95 and 0.90, respectively) of which the images in f and g are examples. Source data are provided as a Source Data file.

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