Molecular Vision: Gonzalez, Mol Vis 2016; 22:203-212. Table 1

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).