Two-photon imaging and optogenetic stimulation rely on high illumination powers, particularly for state-of-the-art applications that target deeper structures, achieve faster measurements, or probe ...larger brain areas. However, little information is available on heating and resulting damage induced by high-power illumination in the brain. In the current study we used thermocouple probes and quantum dot nanothermometers to measure temperature changes induced by two-photon microscopy in the neocortex of awake and anaesthetized mice. We characterized heating as a function of wavelength, exposure time, and distance from the center of illumination. Although total power is highest near the surface of the brain, heating was most severe hundreds of micrometers below the focal plane, due to heat dissipation through the cranial window. Continuous illumination of a 1-mm(2) area produced a peak temperature increase of ∼1.8°C/100 mW. Continuous illumination with powers above 250 mW induced lasting damage, detected with immunohistochemistry against Iba1, glial fibrillary acidic protein, heat shock proteins, and activated caspase-3. Higher powers were usable in experiments with limited duty ratios, suggesting an approach to mitigate damage in high-power microscopy experiments.
L-Lactate, traditionally considered a metabolic waste product, is increasingly recognized as an important intercellular energy currency in mammals. To enable investigations of the emerging roles of ...intercellular shuttling of L-lactate, we now report an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. This biosensor, designated eLACCO1.1, enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.
Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing ...fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.
Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the ...brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.
Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are ...needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.
Abstract
Imaging membrane voltage from genetically defined cells offers the unique ability to report spatial and temporal dynamics of electrical signaling at cellular and circuit levels. Here, we ...present a general approach to engineer electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with positive-going fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transform the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further apply this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters.
We examined how correlated firing controls axon remodeling, using in vivo time-lapse imaging and electrophysiological analysis of individual retinal ganglion cell (RGC) axons that were visually ...stimulated either synchronously or asynchronously relative to neighboring inputs in the Xenopus laevis optic tectum. RGCs stimulated out of synchrony rapidly lost the ability to drive tectal postsynaptic partners while their axons grew and added many new branches. In contrast, synchronously activated RGCs produced fewer new branches, but these were more stable. The effects of synchronous activation were prevented by the inhibition of neurotransmitter release and N-methyl-D-aspartate receptor (NMDAR) blockade, which is consistent with a role for synaptic NMDAR activation in the stabilization of axonal branches and suppression of further exploratory branch addition.
Voltage imaging enables monitoring neural activity at sub-millisecond and sub-cellular scale, unlocking the study of subthreshold activity, synchrony, and network dynamics with unprecedented ...spatio-temporal resolution. However, high data rates (>800MB/s) and low signal-to-noise ratios create bottlenecks for analyzing such datasets. Here we present VolPy, an automated and scalable pipeline to pre-process voltage imaging datasets. VolPy features motion correction, memory mapping, automated segmentation, denoising and spike extraction, all built on a highly parallelizable, modular, and extensible framework optimized for memory and speed. To aid automated segmentation, we introduce a corpus of 24 manually annotated datasets from different preparations, brain areas and voltage indicators. We benchmark VolPy against ground truth segmentation, simulations and electrophysiology recordings, and we compare its performance with existing algorithms in detecting spikes. Our results indicate that VolPy's performance in spike extraction and scalability are state-of-the-art.
L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of L-lactate are currently hampered by ...the limited selection and performance of L-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular L-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular L-lactate biosensor, designated R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization and robust fluorescence response. To the best of our knowledge, R-iLACCO1 and its affinity variants exhibit larger fluorescence responses than any previously reported intracellular L-lactate biosensor. We demonstrate spectrally and spatially multiplexed imaging of L-lactate dynamics by coexpression of eLACCO2.1 and R-iLACCO1 in cultured cells, and in vivo imaging of extracellular and intracellular L-lactate dynamics in mice.
Potassium ion (K
+
) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K
+
biosensors are promising tools to further improve our ...understanding of K
+
-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K
+
biosensor, GINKO1, in the K
+
-bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K
+
biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K
+
dynamics in multiple model organisms, including bacteria, plants, and mice.
PDF
