Recent evidence suggests that capillary pericytes are contractile and play a crucial role in the regulation of microcirculation. However, failure to detect components of the contractile apparatus in capillary pericytes, most notably α-smooth muscle actin (α-SMA), has questioned these findings. Using strategies that allow rapid filamentous-actin (F-actin) fixation (i.e. snap freeze fixation with methanol at −20°C) or prevent F-actin depolymerization (i.e. with F-actin stabilizing agents), we demonstrate that pericytes on mouse retinal capillaries, including those in intermediate and deeper plexus, express α-SMA. Junctional pericytes were more frequently α-SMA-positive relative to pericytes on linear capillary segments. Intravitreal administration of short interfering RNA (α-SMA-siRNA) suppressed α-SMA expression preferentially in high order branch capillary pericytes, confirming the existence of a smaller pool of α-SMA in distal capillary pericytes that is quickly lost by depolymerization. We conclude that capillary pericytes do express α-SMA, which rapidly depolymerizes during tissue fixation thus evading detection by immunolabeling.

The Neuron Doctrine formulated by Ramón y Cajal at the turn of the 19th century is an embodiment of neuroscience discovery empowered by optics. Cajal used Camillo Golgi’s “black reaction” to sparsely impregnate neurons with light-absorbing silver nitrate in histological brain sections. Inspecting these sections with a light microscope, Cajal concluded that information was flowing in one specific direction—from dendrites to axons—within a network of discrete nerve cells connected by synapses. This conceptual advance, enabled through utilization of a light-absorbing probe in combination with widefield microscopy, laid the foundation for modern neuroscience. Fast-forward to the present day, and optical tools for measurement and manipulation of neuronal activity are so ubiquitous and versatile that “neurophotonics” has become a common word in the scientific vocabulary: we talk about neurophotonic technologies and research centers, we have the Neurophotonics journal, and recognize neurophotonics as its own field of research.

Chronic cranial windows allow for longitudinal brain imaging experiments in awake, behaving mice. Different imaging technologies have their unique advantages and combining multiple imaging modalities offers measurements of a wide spectrum of neuronal, glial, vascular, and metabolic parameters needed for comprehensive investigation of physiological and pathophysiological mechanisms. Here, we detail a suite of surgical techniques for installation of different cranial windows targeted for specific imaging technologies and their combination. Following these techniques and practices will yield higher experimental success and reproducibility of results.

Stroke is the leading cause of long-term disability and the second leading cause of death worldwide.¹ Strokes are either ischemic, which account for over 85% of all strokes and occur due to the interruption of blood flow caused by thrombosis, or hemorrhagic, which occur due to bleeding.^2,3 During ischemic stroke, a reduction or complete loss in blood supply causes a starved oxygen environment and leads to neuronal damage within minutes and ultimately to sensorimotor and cognitive impairment.^2,3

Optical coherence tomography (OCT) is a volumetric imaging technique that has been widely used to image the microstructure of biological samples.¹ The OCT signal originates from back-scattered photons and provides image contrast that reflects intrinsic differences in tissue properties within the sample. The depth profile of the OCT signal is determined by five factors: the refraction index of the sample, the optical attenuation coefficient, the back-scattering coefficient, the numerical aperture (NA) of focusing optics, and the depth of the focus in the sample.² The attenuation coefficient, i.e., the sum of the tissue scattering and absorption coefficients, is a spatially varying intrinsic property of the sample and is independent of the optics and the incident power of the system. For low-NA OCT systems, where the lateral resolution is typically larger than $10\mu \mathrm{m}$ and the confocal parameter (i.e., two times the Rayleigh range) is comparable to or larger than the imaging depth, a single-scattering exponential model is appropriate to describe OCT signal attenuation versus depth.^3,4 Previous studies have shown that extracting the attenuation coefficient from low-NA OCT measurements based on Beer’s law is useful in quantifying the optical properties of the sample for applications, including detecting cancerous tissue in skin, bladder, and brain,^{5 – 7} monitoring blood glucose concentration,⁸ characterizing atherosclerosis plaques^3,9 and correlating collagen content with histological staining.¹⁰ However, for high-NA OCT systems where the lateral resolution reaches a few microns or less, the shape of the axial point spread function (PSF) plays an important role in modulating the OCT signal depth profile, and thus the effect of the confocal parameter and focus depth cannot be neglected when estimating the optical properties of the sample.^4,11 We have previously shown that OCT with a $3.5\text{-}\mu \mathrm{m}$ lateral resolution is able to differentiate laminar structures in the neocortex of human brain samples using the average intensity projection (AIP) over the effective Rayleigh range.^2,12 Moreover, we showed that estimating the intrinsic optical properties of the sample could provide additional information to differentiate brain structures, albeit with high inter-parameter correlations that resulted in large variance.²

SignificanceFluorescence head-mounted microscopes, i.e., miniscopes, have emerged as powerful tools to analyze in-vivo neural populations but exhibit a limited depth-of-field (DoF) due to the use of high numerical aperture (NA) gradient refractive index (GRIN) objective lenses.AimWe present extended depth-of-field (EDoF) miniscope, which integrates an optimized thin and lightweight binary diffractive optical element (DOE) onto the GRIN lens of a miniscope to extend the DoF by 2.8 × between twin foci in fixed scattering samples.ApproachWe use a genetic algorithm that considers the GRIN lens’ aberration and intensity loss from scattering in a Fourier optics-forward model to optimize a DOE and manufacture the DOE through single-step photolithography. We integrate the DOE into EDoF-Miniscope with a lateral accuracy of 70 μm to produce high-contrast signals without compromising the speed, spatial resolution, size, or weight.ResultsWe characterize the performance of EDoF-Miniscope across 5- and 10-μm fluorescent beads embedded in scattering phantoms and demonstrate that EDoF-Miniscope facilitates deeper interrogations of neuronal populations in a 100-μm-thick mouse brain sample and vessels in a whole mouse brain sample.ConclusionsBuilt from off-the-shelf components and augmented by a customizable DOE, we expect that this low-cost EDoF-Miniscope may find utility in a wide range of neural recording applications.