ABSTRACT
Two-photon microscopy together with fluorescent proteins and fluorescent protein-based biosensors are commonly used tools in neuroscience. To enhance their experimental scope, it is important to optimize fluorescent proteins for two-photon excitation. Directed evolution of fluorescent proteins under one-photon excitation is common, but many one-photon properties do not correlate with two-photon properties. A simple system for expressing fluorescent protein mutants is E. coli colonies on an agar plate. The small focal volume of two-photon excitation makes creating a high throughput screen in this system a challenge for a conventional point-scanning approach. We present an instrument and accompanying software that solves this challenge by selectively scanning each colony based on a colony map captured under one-photon excitation. This instrument, called the GIZMO, can measure the two-photon excited fluorescence of 10,000 E. coli colonies in 7 hours. We show that the GIZMO can be used to evolve a fluorescent protein under two-photon excitation.
ABSTRACT
The active properties of dendrites can support local nonlinear operations, but previous imaging and electrophysiological measurements have produced conflicting views regarding the prevalence and selectivity of local nonlinearities in vivo. We imaged calcium signals in pyramidal cell dendrites in the motor cortex of mice performing a tactile decision task. A custom microscope allowed us to image the soma and up to 300 µm of contiguous dendrite at 15 Hz, while resolving individual spines. New analysis methods were used to estimate the frequency and spatial scales of activity in dendritic branches and spines. The majority of dendritic calcium transients were coincident with global events. However, task-associated calcium signals in dendrites and spines were compartmentalized by dendritic branching and clustered within branches over approximately 10 µm. Diverse behavior-related signals were intermingled and distributed throughout the dendritic arbor, potentially supporting a large learning capacity in individual neurons.
Subject(s)
Decision Making , Motor Cortex/physiology , Nerve Net/physiology , Pyramidal Cells/physiology , Animals , Calcium Signaling , Mice , Microscopy , Touch Perception , Vibrissae/physiologyABSTRACT
Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits its speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and computationally recovers high-resolution images, attaining voxel rates of over 1 billion Hz in structured samples. Using a static image as a prior for recording neural activity, we imaged visually evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 µm and frame rates over 1 kHz. Dendritic glutamate transients in anesthetized mice are synchronized within spatially contiguous domains spanning tens of micrometers at frequencies ranging from 1-100 Hz. We demonstrate millisecond-resolved recordings of acetylcholine and voltage indicators, three-dimensional single-particle tracking and imaging in densely labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.
Subject(s)
Cerebral Cortex/physiology , Glutamic Acid/metabolism , Neurons/physiology , Tomography/methods , Animals , Calcium/metabolism , Cerebral Cortex/cytology , Female , Mice , Mice, Inbred C57BL , Neurons/cytology , Photons , RatsABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Imaging is used to map activity across populations of neurons. Microscopes with cellular resolution have small (.
Subject(s)
Brain/physiology , Intravital Microscopy/methods , Neurons/physiology , Optical Imaging/methods , Animals , Female , Male , MiceABSTRACT
We present a bichromatic prism pair interferometer (BPPI) for controlling the delay between laser pulses of two different frequencies propagating collinearly in a single beam. The BPPI is especially useful when working with ultrafast laser pulses because it intrinsically allows for independent control over the second-order dispersion experienced by the differently colored pulses. We use this control to demonstrate successful precompensation for blue (lambda approximately 390 nm) and UV (lambda approximately 260 nm) pulses that pass through 2.2 cm of dispersive material after the interferometer. The BPPI is extremely flexible and works with all frequencies from the UV to the near-infrared. We demonstrate this by describing measurements made with BPPIs configured for three different combinations of central frequencies.
