Accuracy of adaptive optics correction using fluorescence fluctuations

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.