Table 8. Theoretical bleed through for Qdot 565 in Cy3 filter, using semrocksearchlight.com.
| Theoretical data | single wave length LED | LED white light | ||||
| Name | Qdot565 in Qdot filter | Cy3 in Cy3 filter | Qdot in Cy3 filter | Qdot565 in Qdot filter | Cy3 in Cy3 filter | Qdot in Cy3 filter |
| Filter Set | specific | specific | bleed through | specific | specific | bleed through |
| Exciter | FF02-435/40 | FF01-543/22 | FF01-543/22 | FF02-435/40 | FF01-543/22 | FF01-543/22 |
| Emitter | FF01-496/LP | FF01-593/40 | FF01-593/40 | FF01-496/LP | FF01-593/40 | FF01-593/40 |
| Dichroic | FF482-Di01 | FF562-Di03 | FF562-Di03 | FF482-Di01 | FF562-Di03 | FF562-Di03 |
| Fluorophore Emission | Qdot 565 | Cy3 | Qdot 565 | Qdot 565 | Cy3 | Qdot 565 |
| Light Source | CoolLED pE4000 | Lumencor SOLA light engine | ||||
| 435 nm | 525 nm | 525 nm | ||||
| Fluorescence Signal (mW) | 2.71E-06 | 3.54E-06 | 1.86E-07 | 7.60E-07 | 8.39E-07 | 4.22E-08 |
| Excitation Light Noise (mW) | 1.29E-08 | 2.72E-09 | 2.72E-09 | 2.15E-08 | 1.49E-09 | 1.49E-09 |
| Autofluorescence Noise (mW) | 0.0000325 | 0.0000034 | 0.0000034 | 0.00000937 | 0.000000747 | 0.000000747 |
| Signal-to-Noise Ratio | 0.083442 | 1.039 | 0.054611 | 0.080983 | 1.1212 | 0.056396 |
| Ratio single wave length /LED white light | Qdot565 in Qdot filter | Cy3 in Cy3 filter | Qdot in Cy3 filter | |||
| specific | specific | bleed through | ||||
| Fluorescence Signal (mW) | 3.565789474 | 4.219308701 | 4.407582938 | (1) | ||
| Excitation Light Noise (mW) | 0.6 | 1.825503356 | 1.825503356 | (2) | ||
| Autofluorescence Noise (mW) | 3.468516542 | 4.551539491 | 4.551539491 | (3) | ||
| Signal-to-Noise Ratio | 1.030364397 | 0.926685694 | 0.968348819 | (4) | ||
| Single wave length | LED white light | Ratio single wave length/white light LED (7) | ||||
| Bleed through signal Qdot/specific signal Qdot (5) | 0.06863 | 0.05553 | 1.23607 | |||
| Bleed though into Cy3 / specific Cy3 (6) | 0.05254 | 0.0503 | 1.04462 | |||
From the theoretical data, ratio between the data given for single wave length and the white light illumination were calculated. They suggest that:
(1) The fluorescence signal would be stronger with single wave length LED but this would lead to more bleed through too
(2) The light noise for Qdot would be better with single wave length LED but worse than the white light illumination for Cy3
(3) The autofluorescence for Qdot would be worse with single wave length LED for Cy3 and Qdot
(4) The signal to noise ratio would slightly better for Qdot with single wave length LED but worse for Cy3
(5) The equivalent of 6.86% of Qdot specific fluorescence would be observed through Cy3 filter with single wave length LED & 5.55% with LED white light
(6) The equivalent of 5.25% of Qdot specific would go through Cy3 filter with LED white light & 5.03% with LED white light
(7) There would be more bleed through with single wave length LED, about 1.2 time more than with white light LED