An Open-Source Whole Slide Image Registration Workflow at Cellular Precision Using Fiji, QuPath and Elastix

Image analysis workflows for Histology increasingly require the correlation and combination of measurements across several whole slide images. Indeed, for multiplexing, as well as multimodal imaging, it is indispensable that the same sample is imaged multiple times, either through various systems for multimodal imaging, or using the same system but throughout rounds of sample manipulation (e.g. multiple staining sessions). In both cases slight deformations from one image to another are unavoidable, leading to an imperfect superimposition Redundant and thus a loss of accuracy making it difficult to link measurements, in particular at the cellular level. Using pre-existing software components and developing missing ones, we propose a user-friendly workflow which facilitates the nonlinear registration of whole slide images in order to reach sub-cellular resolution level. The set of whole slide images to register and analyze is at first defined as a QuPath project. Fiji is then used to open the QuPath project and perform the registrations. Each registration is automated by using an elastix backend, or semi-automated by using BigWarp in order to interactively correct the results of the automated registration. These transformations can then be retrieved in QuPath to transfer any regions of interest from an image to the corresponding registered images. In addition, the transformations can be applied in QuPath to produce on-the-fly transformed images that can be displayed on top of the reference image. Thus, relevant data can be combined and analyzed throughout all registered slides, facilitating the analysis of correlative results for multiplexed and multimodal imaging.

All-optical imaging and patterned stimulation with a one-photon endoscope

While in vivo calcium imaging makes it possible to record activity in defined neuronal populations with cellular resolution, optogenetics allows selective manipulation of neural activity. Recently, these two tools have been combined to stimulate and record neural activity at the same time, but current approaches often rely on two-photon microscopes that are difficult to use in freely moving animals. To address these limitations, we have developed a new integrated system combining a one-photon endoscope and a digital micromirror device for simultaneous calcium imaging and precise optogenetic photo-stimulation with near cellular resolution (Miniscope with All-optical Patterned Stimulation and Imaging, MAPSI). Using this highly portable system in freely moving mice, we were able to image striatal neurons from either the direct pathway or the indirect pathway while simultaneously activating any neuron of choice in the field of view, or to synthesize arbitrary spatiotemporal patterns of photo-stimulation. We could also select neurons based on their relationship with behavior and recreate the behavior by mimicking the natural neural activity with photo-stimulation. MAPSI thus provides a powerful tool for interrogation of neural circuit function in freely moving animals.

Striatum-projecting prefrontal cortex neurons support working memory maintenance

ABSTRACTThe medial prefrontal cortex (mPFC) and the dorsomedial striatum (dmStr) are linked to working memory (WM) but how striatum-projecting mPFC neurons contribute to WM encoding, maintenance, or retrieval remains unclear. Here, we probed mPFC→dmStr pathway function in freely-moving mice during a T-maze alternation test of spatial WM. Fiber photometry of GCaMP6m-labeled mPFC→dmStr projection neurons revealed strongest activity during the delay period that requires WM maintenance. Demonstrating causality, optogenetic inhibition of mPFC→dmStr neurons only during the delay period impaired performance. Conversely, enhancing mPFC→dmStr pathway activity—via pharmacological suppression of HCN1 or by optogenetic activation during the delay— alleviated WM impairment induced by NMDA receptor blockade. Consistently, cellular-resolution miniscope imaging resolved preferred activation of >50% mPFC→dmStr neurons during WM maintenance. This subpopulation was distinct from neurons showing preference for encoding and retrieval. In all periods, including the delay, neuronal sequences were evident. Striatum-projecting mPFC neurons thus critically contribute to spatial WM maintenance.

Simultaneous two-photon imaging of action potentials and subthreshold inputs in vivo

To better understand the input-output computations of neuronal populations, we developed ArcLight-ST, a genetically-encoded voltage indicator, to specifically measure subthreshold membrane potentials. We combined two-photon imaging of voltage and calcium, and successfully discriminated subthreshold inputs and spikes with cellular resolution in vivo. We demonstrate the utility of the method by mapping epileptic seizures progression through cortical circuits, revealing divergent sub- and suprathreshold dynamics within compartmentalized epileptic micronetworks. Two-photon, two-color imaging of calcium and voltage enables mapping of inputs and outputs in neuronal populations in living animals.

A genetically encoded fluorescent biosensor for extracellular l-lactate

Abstractl-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.