Nanowire-Aperture Probe: Local Enhanced Fluorescence Detection for the Investigation of Live Cells at the Nanoscale

Fluorescence microscopy has tackled many of the burning questions in cellular biology. Probing low-affinity cellular interactions remains one of the major challenges in the field to better understand cellular signaling. We introduce a novel approach the nanowire-aperture probe (NAP) to resolve biological signatures with a nanoscale resolution and a boost in light detection. The NAP takes advantage of the photonic properties of semiconductor nanowires and provides a highly localized excitation volume close to the nanowire surface. The probing region extends less than 20 nm into the solution, which can be exploited as a local light probe in fluorescence microscopy. This confined detection volume is especially advantageous in the study of cellular signaling at the cell membrane, as it wraps tightly around the nanowire. The nanowire acts as a local nanoaperture, both focusing the incoming excitation light and guiding photons emitted by the fluorophore. We demonstrate a 20-fold boost in signal-to-background sensitivity for single fluorophores and membrane-localized proteins in live cells. This work opens a completely new avenue for next-generation studies of live cells

Begonia - a two-photon imaging analysis pipeline for astrocytic Ca2+ signals

Imaging the intact brain of awake behaving mice without the dampening effects of anesthesia, has revealed an exceedingly rich repertoire of astrocytic Ca 2+ signals. Analyzing and interpreting such complex signals pose many challenges. Traditional analyses of fluorescent changes typically rely on manually outlined static region-of-interests, but such analyses fail to capture the intricate spatiotemporal patterns of astrocytic Ca 2+ dynamics. Moreover, all astrocytic Ca 2+ imaging data obtained from awake behaving mice need to be interpreted in light of the complex behavioral patterns of the animal. Hence processing multimodal data, including animal behavior metrics, stimulation timings, and electrophysiological signals is needed to interpret astrocytic Ca 2+ signals. Managing and incorporating these data types into a coherent analysis pipeline is challenging and time-consuming, especially if research protocols change or new data types are added. Here, we introduce Begonia, a MATLAB-based data management and analysis toolbox tailored for the analyses of astrocytic Ca 2+ signals in conjunction with behavioral data. The analysis suite includes an automatic, event-based algorithm with few input parameters that can capture a high level of spatiotemporal complexity of astrocytic Ca 2+ signals. The toolbox enables the experimentalist to quantify astrocytic Ca 2+ signals in a precise and unbiased way and combine them with other types of time series data

Astrocytic Ca2+ signaling is reduced during sleep and is involved in the regulation of slow wave sleep

Astrocytic Ca2+ signaling has been intensively studied in health and disease but has not been quantified during natural sleep. Here, we employ an activity-based algorithm to assess astrocytic Ca2+ signals in the neocortex of awake and naturally sleeping mice while monitoring neuronal Ca2+ activity, brain rhythms and behavior. We show that astrocytic Ca2+ signals exhibit distinct features across the sleep-wake cycle and are reduced during sleep compared to wakefulness. Moreover, an increase in astrocytic Ca2+ signaling precedes transitions from slow wave sleep to wakefulness, with a peak upon awakening exceeding the levels during whisking and locomotion. Finally, genetic ablation of an important astrocytic Ca2+ signaling pathway impairs slow wave sleep and results in an increased number of microarousals, abnormal brain rhythms, and an increased frequency of slow wave sleep state transitions and sleep spindles. Our findings demonstrate an essential role for astrocytic Ca2+ signaling in regulating slow wave sleep

Astrocytic Ca2+ signaling is reduced during sleep and is involved in the regulation of slow wave sleep

Astrocytic Ca2+ signaling has been intensively studied in health and disease but has not been quantified during natural sleep. Here, we employ an activity-based algorithm to assess astrocytic Ca2+ signals in the neocortex of awake and naturally sleeping mice while monitoring neuronal Ca2+ activity, brain rhythms and behavior. We show that astrocytic Ca2+ signals exhibit distinct features across the sleep-wake cycle and are reduced during sleep compared to wakefulness. Moreover, an increase in astrocytic Ca2+ signaling precedes transitions from slow wave sleep to wakefulness, with a peak upon awakening exceeding the levels during whisking and locomotion. Finally, genetic ablation of an important astrocytic Ca2+ signaling pathway impairs slow wave sleep and results in an increased number of microarousals, abnormal brain rhythms, and an increased frequency of slow wave sleep state transitions and sleep spindles. Our findings demonstrate an essential role for astrocytic Ca2+ signaling in regulating slow wave sleep