We describe an adaptive optics method that modulates the intensity or phase of light rays at multiple pupil segments in parallel to determine the sample-induced aberration. Applicable to fluorescent protein-labeled structures of arbitrary complexity, it allowed us to obtain diffraction-limited resolution in various samples in vivo. For the strongly scattering mouse brain, a single aberration correction improved structural and functional imaging of fine neuronal processes over a large imaging volume.

BACKGROUND: The use of genetically-encoded fluorescent reporters is essential for the identification and observation of cells that express transgenic modulatory proteins. Near-infrared (NIR) fluorescent proteins have superior light penetration through biological tissue, but are not yet widely adopted.

NEW METHOD: Using the near-infrared fluorescent protein, iRFP713, improves the imaging resolution in thick tissue sections or the intact brain due to the reduced light-scattering at the longer, NIR wavelengths used to image the protein. Additionally, iRFP713 can be used to identify transgenic cells without photobleaching other fluorescent reporters or affecting opsin function. We have generated a set of adeno-associated vectors in which iRFP713 has been fused to optogenetic channels, and can be expressed constitutively or Cre-dependently.

RESULTS: iRFP713 is detectable when expressed in neurons both in vitro and in vivo without exogenously supplied chromophore biliverdin. Neuronally-expressed iRFP713 has similar properties to GFP-like fluorescent proteins, including the ability to be translationally fused to channelrhodopsin or halorhodopsin, however, it shows superior photostability compared to EYFP. Furthermore, electrophysiological recordings from iRFP713-labeled cells compared to cells labeled with mCherry suggest that iRFP713 cells are healthier and therefore more stable and reliable in an ex vivo preparation. Lastly, we have generated a transgenic rat that expresses iRFP713 in a Cre-dependent manner.

CONCLUSIONS: Overall, we have demonstrated that iRFP713 can be used as a reporter in neurons without the use of exogenous biliverdin, with minimal impact on viability and function thereby making it feasible to extend the capabilities for imaging genetically-tagged neurons in slices and in vivo.

The mouse has become an important model for understanding the neural basis of visual perception. Although it has long been known that mouse lens transmits ultraviolet (UV) light and mouse opsins have absorption in the UV band, little is known about how UV visual information is processed in the mouse brain. Using a custom UV stimulation system and in vivo calcium imaging, we characterized the feature selectivity of layer 2/3 neurons in mouse primary visual cortex (V1). In adult mice, a comparable percentage of the neuronal population responds to UV and visible stimuli, with similar pattern selectivity and receptive field properties. In young mice, the orientation selectivity for UV stimuli increased steadily during development, but not direction selectivity. Our results suggest that, by expanding the spectral window through which the mouse can acquire visual information, UV sensitivity provides an important component for mouse vision.

Major resources are now available to develop tools and technologies aimed at dissecting the circuitry and computations underlying behavior, unraveling the underpinnings of brain disorders, and understanding the neural substrates of cognition. Scientists from around the world shared their views around new tools and technologies to drive advances in neuroscience.

Two-photon excitation fluorescence microscopy has revolutionized our understanding of brain structure and function through the high resolution and large penetration depth it offers. Investigating neural structures in vivo requires gaining optical access to the brain, which is typically achieved by replacing a part of the skull with one or several layers of cover glass windows. To compensate for the spherical aberrations caused by the presence of these layers of glass, collar-correction objectives are typically used. However, the efficiency of this correction has been shown to depend significantly on the tilt angle between the glass window surface and the optical axis of the imaging system. Here, we first expand these observations and characterize the effect of the tilt angle on the collected fluorescence signal with thicker windows (double cover slide) and compare these results with an objective devoid of collar-correction. Second, we present a simple optical alignment device designed to rapidly minimize the tilt angle in vivo and align the optical axis of the microscope perpendicularly to the glass window to an angle below 0.25°, thereby significantly improving the imaging quality. Finally, we describe a tilt-correction procedure for users in an in vivo setting, enabling the accurate alignment with a resolution of <0.2° in only few iterations.

The intrinsic aberrations of high-NA gradient refractive index (GRIN) lenses limit their image quality as well as field of view. Here we used a pupil-segmentation-based adaptive optical approach to correct the inherent aberrations in a two-photon fluorescence endoscope utilizing a 0.8 NA GRIN lens. By correcting the field-dependent aberrations, we recovered diffraction-limited performance across a large imaging field. The consequent improvements in imaging signal and resolution allowed us to detect fine structures that were otherwise invisible inside mouse brain slices.

Optical aberrations deteriorate the performance of microscopes. Adaptive optics can be used to improve imaging performance via wavefront shaping. Here, we demonstrate a pupil-segmentation based adaptive optical approach with full-pupil illumination. When implemented in a two-photon fluorescence microscope, it recovers diffraction-limited performance and improves imaging signal and resolution.

Inhomogeneous optical properties of biological samples make it difficult to obtain diffraction-limited resolution in depth. Correcting the sample-induced optical aberrations needs adaptive optics (AO). However, the direct wavefront-sensing approach commonly used in astronomy is not suitable for most biological samples due to their strong scattering of light. We developed an image-based AO approach that is insensitive to sample scattering. By comparing images of the sample taken with different segments of the pupil illuminated, local tilt in the wavefront is measured from image shift. The aberrated wavefront is then obtained either by measuring the local phase directly using interference or with phase reconstruction algorithms similar to those used in astronomical AO. We implemented this pupil-segmentation-based approach in a two-photon fluorescence microscope and demonstrated that diffraction-limited resolution can be recovered from nonbiological and biological samples.

Neural circuitry has evolved to form distributed networks that act dynamically across large volumes. Conventional microscopy collects data from individual planes and cannot sample circuitry across large volumes at the temporal resolution relevant to neural circuit function and behaviors. Here we review emerging technologies for rapid volume imaging of neural circuitry. We focus on two critical challenges: the inertia of optical systems, which limits image speed, and aberrations, which restrict the image volume. Optical sampling time must be long enough to ensure high-fidelity measurements, but optimized sampling strategies and point-spread function engineering can facilitate rapid volume imaging of neural activity within this constraint. We also discuss new computational strategies for processing and analyzing volume imaging data of increasing size and complexity. Together, optical and computational advances are providing a broader view of neural circuit dynamics and helping elucidate how brain regions work in concert to support behavior.

Understanding the functions of a brain region requires knowing the neural representations of its myriad inputs, local neurons and outputs. Primary visual cortex (V1) has long been thought to compute visual orientation from untuned thalamic inputs, but very few thalamic inputs have been measured in any mammal. We determined the response properties of ~28,000 thalamic boutons and ~4,000 cortical neurons in layers 1–5 of awake mouse V1. Using adaptive optics that allows accurate measurement of bouton activity deep in cortex, we found that around half of the boutons in the main thalamorecipient L4 carried orientation-tuned information and that their orientation and direction biases were also dominant in the L4 neuron population, suggesting that these neurons may inherit their selectivity from tuned thalamic inputs. Cortical neurons in all layers exhibited sharper tuning than thalamic boutons and a greater diversity of preferred orientations. Our results provide data-rich constraints for refining mechanistic models of cortical computation.