Unbiased quantitative analysis of macroscopic biological samples demands fast imaging systems capable of maintaining high resolution across large volumes. Here we introduce RAPID (rapid autofocusing via pupil-split image phase detection), a real-time autofocus method applicable in every widefield-based microscope. RAPID-enabled light-sheet microscopy reliably reconstructs intact, cleared mouse brains with subcellular resolution, and allowed us to characterize the three-dimensional (3D) spatial clustering of somatostatin-positive neurons in the whole encephalon, including densely labeled areas. Furthermore, it enabled 3D morphological analysis of microglia across the entire brain. Beyond light-sheet microscopy, we demonstrate that RAPID maintains high image quality in various settings, from in vivo fluorescence imaging to 3D tracking of fast-moving organisms. RAPID thus provides a flexible autofocus solution that is suitable for traditional automated microscopy tasks as well as for quantitative analysis of large biological specimens.
Visualizing neuronal activation on a brain-wide scale yet with cellular resolution is a fundamental technical challenge for neuroscience. This would enable analyzing how different neuronal circuits are disrupted in pathology and how they could be rescued by pharmacological treatments. Although this goal would have appeared visionary a decade ago, recent technological advances make it eventually feasible. Here, we review the latest developments in the fields of genetics, sample preparation, imaging, and image analysis that could be combined to afford whole-brain cell-resolution activation mapping. We show how the different biochemical and optical methods have been coupled to study neuronal circuits at different spatial and temporal scales, and with celltype specificity. The inventory of techniques presented here could be useful to find the tools best suited for a specific experiment. We envision that in the next years, mapping of neuronal activation could become routine in many laboratories, allowing dissecting the neuronal counterpart of behavior.
The status of glaciers is alarming globally with still unknown effects on freshwater ecosystems. Thegeneral aim of this study was to investigate the structural and functional changes in the macroinvertebrate communityin stream networks fed by shrinking glaciers in relation to environmental variables. Feeding glaciers haddifferent surface areas and retreating rates. We selected 10 study sites in the Italian Alps, spanning five kryal, twoglacio-rhithral, two krenal and one proglacial pond, sampled twice in summer 2018. Eight of these sites weresampled previously between 1996 and 2014. In all, in 2018, > 15,000 individuals (73 taxa) were collected, of which82 % were chironomids (Diptera Chironomidae) (33 taxa). Diamesa zernyi gr. (Chironomidae Diamesinae) wasthe most frequent and abundant taxon, followed by Oligochaeta and Chironomidae Orthocladiinae. Taxonomical(Shannon index) and functional (based on functional feeding groups) diversity both increased with decreasingglacial influence (estimated as glacial index, GI, based on distance from the glacier snout of each site and glacierarea), from kryal to glacio-rhithral and krenal habitats. Taxa distribution was explained mainly by GI, maximumwater temperature, substrate stability, silica, epilithic chlorophyll-a, and benthic particulate organic matter. Thesame variables explained temporal differences in the community structure for the eight sites re-sampled in the lasttwo decades. Among the taxa best associated with high GI was the chironomid Diamesa steinboecki, that in 2018was exclusive of the kryal sites with GCC (% glacier cover in the catchment, expressed in a range from 0 %–100 %)> 50 % and maximum temperature < 5 °C. This species was absent only in the kryal site C0 (GCC = 33 %), whereit was dominant in 1996 –1997. This site was still fed by ice melt in 2018, but resembled a glacio-rhithral site inhabitat features (e.g. maximum temperature > 6 °C) and biota (e.g. % Diamesa spp. < 30 %). In C0, it was evidentthat in the last 22 years, the macroinvertebrate community changed remarkably. This change was due to upstreammigration of generalist insect species to sites once exclusive for kryal species with consequent changes in food webstructure and loss of strictly kryal species, first D. steinboecki that we propose as the “flagship” species of the kryalin the Alps. The site C0 represents a “tipping point”, showing us the effects of climate change on alpine biodiversityin a relatively short period; unfortunately, there are many such sites in the Italian Alps.
The combination of biological tissue clearing methods with light-sheet fluorescence microscopy (LSFM) allows acquiring images of specific biological structures of interest at whole organ scale and microscopic resolution. Differently to classical epifluorescence techniques, where the sample is cut into slices, LSFM preserves the whole organ architecture, which is of particular relevance for investigations of long-range neuronal circuits. This imaging modality comes with the need of new protocols for sample mounting. Gel matrix, hooks, tips, glues, and quartz cuvettes have been used to keep whole rodent organs in place during image acquisitions. The last one has the advantage of avoiding sample damage and optical aberrations when using a quartz refractive index (RI) matching solution. However, commercially available quartz cuvettes for such large samples are expensive. We propose the use of polydimethylsiloxane (PDMS) for creating tailor-made cuvettes for sample holding. For validation, we compared PDMS and quartz cuvettes by measuring light transmittance and performing whole mouse-brain imaging with LSFM. Moreover, imaging can be performed using an inexpensive RI matching solution, which further reduces the cost of the imaging process. Worth of note, the RI matching solution used in combination with PDMS leads to a moderate expansion of the sample with respect to its original size, which may represent an advantage when investigating small components, such as neuronal processes. Overall, we found the use of custom-made PDMS cuvettes advantageous in term of cost, image quality, or preservation of sample integrity with respect to other whole-mouse brain mounting strategies adopted for LSFM.
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