Table 1. Effect of red filter bandpass on signal-to-noise ratio.
|
Emission Filter Wavelength Range (nm) |
Near Red: 565–605 |
Far Red: 590–680 |
|||||
|---|---|---|---|---|---|---|---|
| Excitation Wavelength (nm) | 800 | 850 | 900 | 800 | 850 | 900 | |
|
-Red Dye |
AF Bleed-Through |
6774 |
4290 |
2904 |
3972 |
3096 |
2398 |
|
+Red Dye |
Alexa-568 in Red Channel |
18,348 |
4064 |
871 |
27,776 |
8736 |
3626 |
| Ratio of Alexa-568-to- AF Bleed-Through | 2.71 | 0.95 | 0.30 | 6.99 | 2.82 | 1.51 | |
Pixel intensity was calculated in five regions of interest (ROI) positioned in each image frame (see Figure 6). In the absence of Alexa-568 (-Red Dye), fluorescence in the near-red (565–605 nm) and far-red (590–680 nm) channels was attributed to Hoechst and autofluorescence (AF) bleed-through (AF Bleed-Through) and considered background signal or “noise.” In the presence of Alexa-568 dye (+Red Dye); intensity measurements in the same ROIs represented a mix of Alexa-568 signal and background noise. The signal-to-noise ratio (Ratio of Alexa-568-to-AF Bleed Through) decreased with increasing excitation wavelength (from 800 to 900 nm) and use of a far-red filter (590–680 nm).