The high sensitivity and spatial resolution enabled by two-photon excitation fluorescence lifetime imaging microscopy/fluorescence resonance energy transfer (2PE-FLIM/FRET) provide an effective approach that reveals protein-protein interactions in a single cell during stimulated exocytosis. Enhanced green fluorescence protein (EGFP)-labeled synaptosomal associated protein of 25 kDa (SNAP25A) and red fluorescence protein (mRFP)-labeled Rabphillin 3A (RPH3A) were co-expressed in PC12 cells as the FRET donor and acceptor, respectively. The FLIM images of EGFP-SNAP25A suggested that SNAP25A/RPH3A interaction was increased during exocytosis. In addition, the multidimensional (three-dimensional with time) nature of the 2PE-FLIM image datasets can also resolve the protein interactions in the z direction, and we have compared several image analysis methods to extract more accurate and detailed information from the FLIM images. Fluorescence lifetime was fitted by using one and two component analysis. The lifetime FRET efficiency was calculated by the peak lifetime (taupeak) and the left side of the half-peak width (tau1/2), respectively. The results show that FRET efficiency increased at cell surface, which suggests that SNAP25A/RPH3A interactions take place at cell surface during stimulated exocytosis. In summary, we have demonstrated that the 2PE-FLIM/FRET technique is a powerful tool to reveal dynamic SNAP25A/RPH3A interactions in single neuroendocrine cells.

In this study, we demonstrate subdiffraction-limited fluorescence lifetime imaging microscopy (FLIM) by engineering the point spread function (PSF) with stimulated emission depletion (STED). The enhanced spatial resolution allows the number of fluorophores in the PSF to reduce in turn the associated heterogeneity in lifetime analysis. Moreover, time gating can be performed using time-correlated single photon counting (TCSPC) to carefully select detected fluorescence photons so as to optimize the spatial resolution and the signal-to-noise ratio in STED imaging. This flexibility also supports the removal of the unintended effects of lifetime reduction that is caused by STED pulses.

The cerebellum has long been known to play an important role in motor and balance control, and accumulating evidence has revealed that it is also involved in multiple cognitive functions. However, the evidence from neuroimaging studies and clinical observations is not well-integrated at the anatomical or molecular level. The goal of this review is to summarize and link different aspects of the cerebellum, including molecular patterning, functional topography images, and clinical cerebellar disorders. More specifically, we explored the potential relationships between the cerebrocerebellar connections and the expression of particular molecules and, in particular, zebrin stripe (a Purkinje cell-specific antibody molecular marker, which is a glycolytic enzyme expressed in cerebellar Purkinje cells). We hypothesized that the zebrin patterns contribute to cerebellar functional maps-especially when cerebrocerebellar circuit changes exist in cerebellar-related diseases. The zebrin stripe receives input from climbing fibers and project to different parts of the cerebral cortex through its cerebrocerebellar connection. Since zebrin-positive cerebellar Purkinje cells are resistant to excitotoxicity and cell injury while zebrin-negative zones are more prone to damage, we suggest that motor control dysfunction symptoms such as ataxia and dysmetria present earlier and are easier to observe than non-ataxia symptoms due to zebrin-negative cell damage by cerebrocerebellar connections. In summary, we emphasize that the molecular zebrin patterns provide the basis for a new viewpoint from which to investigate cerebellar functions and clinico-neuroanatomic correlations.