Robert A. A. Campbell

Optogenetic tools are indispensable for understanding the causal neural mechanisms underlying animal behavior, providing millisecond-precision control over genetically defined neural populations. Random-access laser-scanning optogenetics is a powerful and underutilized method for transiently manipulating cortical activity in mice. Despite the utility of this technique, no general-purpose open-source implementation currently exists, and potential users need expertise across optics, real-time hardware control, and programming. This represents a major barrier to adoption, particularly for newly established research groups. Here we present ‘Zapit’, the first open-source general-purpose platform for random-access laser-scanning optogenetic experiments in head-fixed mice. Zapit is fully documented, has a user-friendly GUI, works in stereotaxic coordinates, and comes with easy-to-build hardware options that extend functionality beyond any published system. We validate Zapit’s performance through electrophysiological recordings and cortical photoinhibition in behaving mice. Zapit is a novel and innovative tool with the potential to democratize laser-scanning optogenetics and significantly increase uptake throughout the scientific community.

Volumetric imaging is increasingly important in fields such as neuroscience, where quantifying structure and mapping features are inherently three-dimensional problems. Block-face serial two-photon tomography (STPT) has emerged as a powerful approach for high-resolution, whole-organ volumetric imaging. STPT avoids complex sample preparation and balances image quality, speed, and scalability. However, STPT remains niche, in part because of the absence of flexible, general-purpose control software. Here we present BakingTray, an open-source software platform for STPT that extends ScanImage. BakingTray’s modular framework accommodates diverse hardware configurations and allows users to build systems customised to their needs. Twelve such systems, which we call ‘BrainSaws’, have been constructed worldwide since 2016, with more under construction, and have so far contributed to 35 research papers. The three systems at the Sainsbury Wellcome Centre alone have acquired over 7,000 samples in a busy multi-user facility. We demonstrate the platform across its core neuroscience applications: electrode track localisation, brain-wide cell counting, and bulk projection mapping. Beyond these, we map the cell-type-specific synaptic output of inhibitory neurons in the ventral lateral geniculate nucleus and, with whole-mount staining, amyloid plaques brain-wide in a mouse model of Alzheimer’s disease. BakingTray is a mature platform for groups or facilities needing a customised volumetric imaging solution, whether built new or converted cheaply from an existing two-photon microscope.

Optogenetic tools are indispensable for understanding the causal neural mechanisms underlying animal behavior, providing millisecond-precision control over genetically defined neural populations. Random-access laser-scanning optogenetics is a powerful and underutilized method for transiently manipulating cortical activity in mice. Despite the utility of this technique, no general-purpose open-source implementation currently exists, and potential users need expertise across optics, real-time hardware control, and programming. This represents a major barrier to adoption, particularly for newly established research groups. Here we present ‘Zapit’, the first open-source general-purpose platform for random-access laser-scanning optogenetic experiments in head-fixed mice. Zapit is fully documented, has a user-friendly GUI, works in stereotaxic coordinates, and comes with easy to build hardware options that extend functionality beyond any published system. We validate Zapit’s performance through electrophysiological recordings and cortical photoinhibition in behaving mice. Zapit is a novel and innovative tool with the potential to democratize laser-scanning optogenetics and significantly increase uptake throughout the scientific community.

Optogenetic tools are indispensable for understanding the causal neural mechanisms underlying animal behavior, providing millisecond-precision control over genetically defined neural populations. Random-access laser-scanning optogenetics is a powerful and underutilized method for causally manipulating cortical activity in mice. Despite the utility of this technique, no general-purpose open-source implementation currently exists, and potential users need expertise across optics, real-time hardware control, and programming. This represents a major barrier to adoption, particularly for newly established research groups. Here we present ‘Zapit’, the first open-source general-purpose platform for random-access laser-scanning optogenetic experiments in head-fixed mice. Zapit is fully documented, has a user-friendly GUI, works in stereotaxic coordinates, and comes with easy to build hardware options that extend functionality beyond any published system. We validate Zapit’s performance through electrophysiological recordings and cortical photoinhibition in behaving mice. Zapit is a novel and innovative tool with the potential to democratize laser-scanning optogenetics and significantly increase uptake throughout the scientific community.

The goal of this protocol is to improve the characterization and performance standardization of multiphoton microscopy hardware across a large user base. We purposefully focus on hardware and only briefly touch on software and data analysis routines where relevant. Here we cover the measurement and quantification of laser power, pulse width optimization, field of view, resolution and photomultiplier tube performance. The intended audience is scientists with little expertise in optics who either build or use multiphoton microscopes in their laboratories. They can use our procedures to test whether their multiphoton microscope performs well and produces consistent data over the lifetime of their system. Individual procedures are designed to take 1–2 h to complete without the use of expensive equipment. The procedures listed here help standardize the microscopes and facilitate the reproducibility of data across setups.

Biological systems are complex, encompassing intertwined spatial, molecular and functional features. However, methodological constraints limit the completeness of information that can be extracted. Here, we report the development of INSIHGT, a non-destructive, accessible three-dimensional (3D) spatial biology method utilizing superchaotropes and host-guest chemistry to achieve homogeneous, deep penetration of macromolecular probes up to centimeter scales, providing reliable semi-quantitative signals throughout the tissue volume. Diverse antigens, mRNAs, neurotransmitters, and post-translational modifications are well-preserved and simultaneously visualized. INSIHGT also allows multi-round, highly multiplexed 3D molecular probing and is compatible with downstream traditional histology and nucleic acid sequencing. With INSIHGT, we map undescribed podocyte-to-parietal epithelial cell microfilaments in mouse glomeruli and neurofilament-intensive inclusion bodies in the human cerebellum, and identify NPY-proximal cell types defined by spatial morpho-proteomics in mouse hypothalamus. We anticipate that INSIHGT can form the foundations for 3D spatial multi-omics technology development and holistic systems biology studies.

Optogenetic tools are indispensable for understanding the causal neural mechanisms underlying animal behavior, providing millisecond-precision control over genetically defined neural populations. Random-access laser-scanning optogenetics is a powerful and underutilized method for causally manipulating cortical activity in mice. Despite the utility of this technique, no general-purpose open-source implementation currently exists, and potential users need expertise across optics, real-time hardware control, and programming. This represents a major barrier to adoption, particularly for newly established research groups. Here we present ‘Zapit’, the first open-source general-purpose platform for random-access laser-scanning optogenetic experiments in head-fixed mice. Zapit is fully documented, has a user-friendly GUI, works in stereotaxic coordinates, and comes with easy to build hardware options that extend functionality beyond any published system. We validate Zapit’s performance through electrophysiological recordings and cortical photoinhibition in behaving mice. Zapit is a novel and innovative tool with the potential to democratize laser-scanning optogenetics and significantly increase uptake throughout the scientific community.

The goal of this protocol is to enable better characterisation of multiphoton microscopy hardware across a large user base. The scope of this protocol is purposefully limited to focus on hardware, touching on software and data analysis routines only where relevant. The intended audiences are scientists using and building multiphoton microscopes in their laboratories. The goal is that any scientist, not only those with optical expertise, can test whether their multiphoton microscope is performing well and producing consistent data over the lifetime of their system.

Significance: Some infections are exceedingly difficult to cure, even with adequate antimicrobial chemotherapy. How pathogens can survive exposure to antimicrobials in tissues remains poorly understood. Using three-dimensional whole-organ tomography, we show thatSalmonellacolonization of the mouse spleen is uneven. LowSalmonelladensity in the white pulp triggers only limited local infiltration by inflammatory cells that are crucial for supporting antimicrobialSalmonellaclearance. Inflammatory cell density declines further during treatment in response to recedingSalmonellaloads, resulting in insufficient support for clearance and eradication failure. However, sustaining inflammation during antimicrobial chemotherapy enables effective clearance. Our findings identify heterogeneous host–pathogen interactions in compartmentalized tissues as a main mechanism underlying the antibiotic persistence ofSalmonella.

Mitochondrial dysfunction is recognized as a critical component in the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease (AD). Deficits in oxidative capacity and, specifically, cytochrome c oxidase (CO) activity have been repo