Application limits and data correction in number of molecules and brightness analysis
Antonio Trullo1, Valeria Corti, Elvira Arza
1Experimental Imaging Centre, San Raffaele Scientific Institute, Milan, Italy; Microscopy and Dynamic Imaging Unit, Vascular Biology and Inflammation Department, Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain.
Microscopy Research and Technique
|August 13, 2013
Summary
This study introduces a corrective method to accurately measure molecular brightness and fluorophore counts in live cells using Number of molecules and Brightness (N&B). The approach corrects for cell movement and photobleaching, enabling precise analysis of protein oligomers.
Area of Science:
- Biophysics
- Cell Biology
- Fluorescence Microscopy
Background:
- Number of molecules and Brightness (N&B) is a technique for quantifying fluorophores in live-cell imaging.
- Oligomer stoichiometry can be inferred from fluorescence brightness changes due to protein clustering.
- Cellular movement and photobleaching can introduce significant errors in N&B analysis.
Purpose of the Study:
- To investigate and correct for extra-fluctuation effects in N&B analysis.
- To develop a robust method for accurate molecular brightness and oligomer stoichiometry determination.
- To establish criteria and assess limitations for N&B analysis under challenging conditions.
Main Methods:
- Developed an offline corrective approach involving frame re-alignment and boxcar filtering.
- Utilized simulations to derive general criteria for N&B analysis.
- Tested the corrective procedure on simulated and experimental data, including GPI-mEGFP constructs.
Main Results:
- Identified and quantified overestimates in cluster size and subunit counts caused by cell shifts and photobleaching.
- Demonstrated that the corrective approach effectively mitigates perturbing effects.
- Successfully recovered accurate brightness measurements for GPI-mEGFP monomers and dimers, even in challenging conditions.
Conclusions:
- The proposed corrective method significantly improves the precision of N&B analysis in live-cell imaging.
- The study provides guidelines for applying N&B analysis, especially in scenarios with limited pixels or high noise.
- Accurate determination of molecular brightness and protein oligomerization is achievable with the developed correction strategy.
