Table 2.
Optimal parameters that were determined for detecting green fluorescent protein (GFP) emission in mixed samples with high autofluorescence.
| Experiment/Parameter | Widefield Microscope | Confocal Microscope |
|---|---|---|
| Confocal pinhole | NA | 4.9 Airy disk units |
| Illumination power | Arc lamp–100% | 405 laser–60% 57 laser–2% |
| Detector gain | EMCCD–3900 | PMT–138 |
| Photobleaching | Similar bleaching rates in widefield and confocal for AF. Hoechst and GFP-Less | Hoechst and GFP comparatively high |
| SNR | Low SNR due to high background signal (SNR:2–3) | Medium SNR (SNR: 3–10) |
| RMS error | High | Low |
| Sensitivity | Linear: 0.929 slope (GFP detected/GFP added) | Linear: 0.96 slope (GFP detected/ GFP added) |
| Specificity | 0.06% Type I errors | 0.16 % Type I errors |