Fluorescence fluctuation methods, such as fluorescence correlation spectroscopy, are very sensitive to optical
aberrations. That is why it is possible to use a fluctuations-based metric, the molecular brightness, to correct aberrations
using a sensorless modal adaptive optics approach. We have investigated the performance of this method by correcting
known aberrations under various experimental conditions. The signal-to-noise ratio of the brightness measurement was
examined theoretically and experimentally and found to be directly related to the accuracy of aberration correction, so
that the latter can be predicted for a given sample brightness and measurement duration. We have also shown that the
initial measurement conditions play a key role in the correction dynamics and we provide guidelines to optimize the
corrections accuracy and speed. The molecular brightness, used as a metric, has the advantage that it depends on
aberrations as the square of the Strehl ratio, regardless of the nature of the sample. Therefore, it is straightforward to
predict the achievable correction accuracy and the same performance can be obtained in samples with different structure
and contrast, which would not be possible with image-based optimization metrics.
Micro-fabrication and surface functionalization imply to know the equilibrium surface concentration of various kinds of molecules. Paradoxically, this crucial parameter is often poorly controlled and even less quantified. We have used a technique belonging to the family of fluorescence fluctuation microscopy, namely Image Correlation Spectroscopy (ICS), to measure the absolute surface concentration of fibrinogen molecules adsorbed on glass substrates. As these molecules are immobile, the width of the autocorrelation of the confocal image obtained by scanning the sample only reflects that of the confocal Point Spread Function. Conversely, the amplitude of the autocorrelation is directly related to the average number of proteins simultaneously illuminated by the laser beam and therefore to their surface concentration. We have studied the surface concentration of fibrinogen proteins versus the initial concentration of these molecules, solubilized in the solution which has been deposited on the surface. The estimation of this relation can be biased for several reasons: the concentration of fibrinogen molecules in solution is difficult to control; the measurement of the surface concentration of adsorbed molecules can be strongly underestimated if the surface coverage or the molecular brightness is not uniform. We suggest methods to detect these artifacts and estimate the actual surface concentration, together with control parameters. Globally, fluorescence fluctuation microscopy is a powerful set of techniques when one wants to quantify the surface concentration of molecules at the micrometer scale.