Exploiting the multiplicative nature of fluoroscopic image stochastic noise to enhance calcium imaging recording quality

One of the main problems that affect fluoroscopic imaging is the difficulty in coupling the recorded activity with the morphological information. The comprehension of fluorescence events in relationship with the internal structure of the cell can be very difficult. At this purpose, we developed a new method able to maximize the fluoroscopic movie quality. The method (maximum intensity enhancement, MIE) works as follow: considering all the frames that compose the fluoroscopic movie, the algorithm extracts, for each pixel of the matrix, the maximal brightness value assumed along all the frames. Such values are collected in a maximum intensity matrix. Then, the method provides the projection of the target molecule oscillations which are present in the DeltaF/F₀ movie onto the maximum intensity matrix. This is done by creating a RGB movie and by assigning to the normalized (DeltaF/F₀) activity a single channel and by reproducing the maximum intensity matrix on all the frames by using the remaining color channels. The application of such a method to fluoroscopic calcium imaging of astrocyte cultures demonstrated a meaningful enhancement in the possibility to discern the internal and external structure of cells.