Absorbance Corrected Fluorescence - ACF

Introduction to ACF
_{Supplementary program to NINFE}

Friganović, Šakić, Weitner, 2020-2021

Corrector function to eliminate inner filter effect for fluorescence measurements in microplates using equation:

F_A= F₁∙10^{[(A_ex+A_em)/2]}

Data should be formatted as:

Output data (from above calibration) is formatted as:

Example test file can be downloaded here.

For details about data formatting, export and updates about the method please contact davor.sakic@pharma.unizg.hr.

Help from Mario Gabričević (discussion), Tino Šeba, Valentina Borko, and Robert Kerep (testing) is greatly appreciated.

NOTES

r²: the coefficient of determination representing the proportion of the variance of the dependent variable that is explained by an independent variable(s) in a regression model.

rmsre: root mean squared relative error.

steyx: the standard error of the estimated dependent variable y in a simple linear regression model.

LOD: limit of detection corresponding to the lowest quantity of a substance that can be differentiated from the blank sample (absence of the substance) at a given confidence level.

mErr%: the percentage error of the slope of the line of corrected fluorescence, as compared to the slope of the ideal dependence derived from Beer-Lambert law.

Calibration range: range of concentrations used for calibration (lowest-highest).

Correction range: range of concentrations applicable for correction (LOD-highest).

Absorbance corrected fluorescence (ACF): Corrected fluorescence were obtained via equation F_A= F₁∙10^{[(A_ex+A_em)/2]}. Reference: J. R. Lakowicz, Ed., in Principles of Fluorescence Spectroscopy, Springer US, Boston, MA, 2006, pp. 27–61.

Black-box model (BBm) optimization of ACF: Performed by evaluation of the r² value of a linear regression for a set of equally distributed coefficients in a chosen starting range. Distribution of coefficients within the range is defined by stepsize; larger stepsize equals rougher grid. After each cycle, the coefficient with highest r² value was taken as a starting point for the next cycle with finer grid (smaller stepsize) and equal number of steps. Optimization was stopped if difference in r² between two subsequent cycles is less than 10^-6, or after 20 cycles.

CREDITS

Idea: Tin Weitner, 2020.-2021.

Created by: Tin Weitner, Tomislav Friganović, and Davor Šakić, 2021.

Python version: Tomislav Friganović, 2021.

Web adaptation by: Davor Šakić, 2021.

Funding: HrZZ, GlyMech, LIGHT-N-RING, 2021.