Removing the effect of blooming from potential energy measurement by employing total internal reflection microscopy integrated with video microscopy.

Total internal reflection microscopy (TIRM) measures the interactions between a colloidal particle and a flat surface in aqueous solution. Recently, TIRM has further integrated with video microscopy (VM) and enabled the simultaneous measurements of multi-particle colloid-surface interactions in the same ensemble. However, there still remain challenges about accurate image acquisition due to blooming. Blooming means the number of photons reaching the detector exceeds its maximum capacity, and the excess photons will either spill to adjacent pixels or not be counted, leading to an obstacle from precise determination of intensity. Our result shows that blooming gives rise to a deviation of the measured potential energy from the classical theory of Derjaguin, Landau, Verway, and Overbeek (DLVO). Therefore, a correction method was developed in this work to deduce the real intensity from the experimental measurement. The relationship between scattered light intensity and exposure time deviates from linearity when blooming occurs. A correction equation was developed to recover the real intensity, which was then confirmed by the accordance between the corresponding potential energy profiles and the DLVO theory. This correction method is suitable for VM systems of colloidal probes illuminated by scattered light, broadening the application of VM imaging to investigate colloidal interactions. [ABSTRACT FROM AUTHOR]