Spline curve measuring SP-D dodecamer arm length with the original AFM, with that image saved to grayscale as “averaged” “lightness” “luminance” and “contrast enhanced in photoshop” shows no significant difference in the length of an arm (One chosen by chance from dozens of images and measured at least five times for each color, and grayscale conversion algoriithm, and also manually contrast enhanced in photoshop) does not cause significant difference in that length .  This is good news since many programs don’t register (or make available) the algorithms used to convert RGB to grayscale. Bottom line…. using discretion… it does not seem to do any much difference:  Left: contrast enhanced, right: original RGB (saved from arroyo et al).

Scores Calculations
Treatment 1 (color image five tracings of vertical dodecamer)
N1: 5
df1 = N – 1 = 5 – 1 = 4
M1: 147.64
SS1: 31.07
s21 = SS1/(N – 1) = 31.07/(5-1) = 7.77
Treatment 2
N2: 5
df2 = N – 1 = 5 – 1 = 4
M2: 147.67
SS2: 136.47
s22 = SS2/(N – 1) = 136.47/(5-1) = 34.12
T-value Calculation
s2p = ((df1/(df1 + df2)) * s21) + ((df2/(df2 + df2)) * s22) = ((4/8) * 7.77) + ((4/8) * 34.12) = 20.94
s2M1 = s2p/N1 = 20.94/5 = 4.19
s2M2 = s2p/N2 = 20.94/5 = 4.19
t = (M1 – M2)/√(s2M1 + s2M2) = -0.03/√8.38 = -0.01 (except reduce the level of noise).