Resin embedding is a well-established technique to prepare biological specimens for microscopic imaging. However, it is not compatible with modern green-fluorescent protein (GFP) fluorescent-labelling technique because it significantly quenches the fluorescence of GFP and its variants. Previous empirical optimization efforts are good for thin tissue but not successful on macroscopic tissue blocks as the quenching mechanism remains uncertain. Here we show most of the quenched GFP molecules are structurally preserved and not denatured after routine embedding in resin, and can be chemically reactivated to a fluorescent state by alkaline buffer during imaging. We observe up to 98% preservation in yellow-fluorescent protein case, and improve the fluorescence intensity 11.8-fold compared with unprocessed samples. We demonstrate fluorescence microimaging of resin-embedded EGFP/EYFP-labelled tissue block without noticeable loss of labelled structures. This work provides a turning point for the imaging of fluorescent protein-labelled specimens after resin embedding.

Neural circuits are fundamental for brain functions. However, obtaining long range continuous projections of neurons in the entire brain is still challenging. Here a two-photon fluorescence micro-optical sectioning tomography (2p-fMOST) method is developed for high-throughput, high-resolution visualization of the brain circuits. Two-photon imaging technology is used to obtain high resolution, and acoustical optical deflector (AOD), an inertia-free beam scanner is used to realize fast and prolonged stable imaging. The combination of these techniques with imaging and then sectioning method of a plastic-embedded mouse brain facilitated the acquisition of a three-dimensional data set of a fluorescent mouse brain with a resolution adequate to resolve the spines. In addition, the brain circuit tracing ability is showed by several neurons projecting across different brain regions. Besides brain imaging, 2p-fMOST could be used in many studies that requires sub-micro resolution or micro resolution imaging of a large sample.

The sensory responses of cortical neuronal populations following training have been extensively studied. However, the spike firing properties of individual cortical neurons following training remain unknown. Here, we have combined two-photon Ca 2+ imaging and single-cell electrophysiology in awake behaving mice following auditory associative training. We find a sparse set (~5%) of layer 2/3 neurons in the primary auditory cortex, each of which reliably exhibits high-rate prolonged burst firing responses to the trained sound. Such bursts are largely absent in the auditory cortex of untrained mice. Strikingly, in mice trained with different multitone chords, we discover distinct subsets of neurons that exhibit bursting responses specifically to a chord but neither to any constituent tone nor to the other chord. Thus, our results demonstrate an integrated representation of learned complex sounds in a small subset of cortical neurons.

Two-photon laser scanning microscopy has been extensively applied to study in vivo neuronal activity at cellular and subcellular resolutions in mammalian brains. However, the extent of such studies is typically confined to a single functional region of the brain. Here, we demonstrate a novel technique, termed the multiarea two-photon real-time in vivo explorer (MATRIEX), that allows the user to target multiple functional brain regions distributed within a zone of up to 12 mm in diameter, each with a field of view (FOV) of ~200 μm in diameter, thus performing two-photon Ca2+ imaging with single-cell resolution in all of the regions simultaneously. For example, we demonstrate real-time functional imaging of single-neuron activities in the primary visual cortex, primary motor cortex and hippocampal CA1 region of mice in both anesthetized and awake states. A unique advantage of the MATRIEX technique is the configuration of multiple microscopic FOVs that are distributed in three-dimensional space over macroscopic distances (>1 mm) both laterally and axially but that are imaged by a single conventional laser scanning device. In particular, the MATRIEX technique can be effectively implemented as an add-on optical module for an existing conventional single-beam-scanning two-photon microscope without requiring any modification to the microscope itself. Thus, the MATRIEX technique can be readily applied to substantially facilitate the exploration of multiarea neuronal activity in vivo for studies of brain-wide neural circuit function with single-cell resolution.