A comparative study of different instrumental concepts for spectrally and lifetime-resolved multiphoton intravital tomography (5D-IVT) in dermatological applications

Multiphoton optical tomography or intravital tomography (IVT) provides non-invasive optical sectioning of biological specimens, e.g. skin, with subcellular spatial resolution without any need of contrast agents. It can be used to distinguish between normal and diseased tissue due to the differences in morphological appearance. Additional information beyond morphology can be obtained by analyzing the collected fluorescence light spectroscopically and by means of its fluorescence decay time. This is frequently termed spectral fluorescence lifetime imaging (SFLIM) or 5D-intravital tomography (5D-IVT). Spectral and temporal resolution scales with the number of detection increments (i.e. spectral channels and time bins). 5D-IVT enables us to detect new physiological parameters, however accompanied by a decrease in intensity per channel. Moreover, the increase of data requests a higher need of software skills. In this study we investigate and evaluate different technical modes of 5D-IVT with respect to their clinical relevance: (1) a multichannel photomultiplier tube (PMT) array coupled to a diffraction grating, each channel being analyzed by timecorrelated single photon counting (TCSPC), (2) three separate PMTs in spectral separation path using dichroic mirrors, each channel being analyzed by TCSPC and (3) a single PMT TCSPC setup in combination with a high-resolution CCDspectrograph for pointwise microspectroscopy.