Two-Photon Excitation Fluorescence Imaging With a Photon-Counting Camera: Improved Resolution for Biomedical Applications

Two-photon excitation fluorescence (TPEF) is a multiphoton imaging technique to visualize non-stained biological tissues. This technique is limited by both the use of a scanning unit and the data sequential acquisition by a single-point detector. The use of alternative detectors based on spatially resolved technologies might enhance TPEF images, overcoming these limitations. In this work, a photon-counting camera was coupled to a custom TPEF imaging microscope. Using first a non-biological sample, the performance of this camera detection mode was compared to that of a photomultiplier tube in terms of signal, contrast, and resolution for different scanning protocols. Results show that the sub-micron resolution of the TPEF images acquired with the camera exhibited more than a 5-fold increase. This enhancement was independent of the scanning protocol, in contrast with that reached with the standard detection mode. Moreover, the evolution of the TPEF signal in areas either with highly visible structural details or with a noticeable presence of noise showed a linear trend with the number of the scanning points. Finally, the capability of the camera detection mode was also demonstrated in a biological sample, revealing morphological details at different depth locations. This study opens the door to the implementation of photon-resolving cameras into TPEF microscopy for biomedical applications, where the increase in resolution and the improved visualization of certain features will benefit characterization and diagnosis of tissues.