RESUMO
Homeostatic and pathological phenomena often affect multiple organs across the whole organism. Tissue clearing methods, together with recent advances in microscopy, have made holistic examinations of biological samples feasible. Here, we report the detailed protocol for nanobody(VHH)-boosted 3D imaging of solvent-cleared organs (vDISCO), a pressure-driven, nanobody-based whole-body immunolabeling and clearing method that renders whole mice transparent in 3 weeks, consistently enhancing the signal of fluorescent proteins, stabilizing them for years. This allows the reliable detection and quantification of fluorescent signal in intact rodents enabling the analysis of an entire body at cellular resolution. Here, we show the high versatility of vDISCO applied to boost the fluorescence signal of genetically expressed reporters and clear multiple dissected organs and tissues, as well as how to image processed samples using multiple fluorescence microscopy systems. The entire protocol is accessible to laboratories with limited expertise in tissue clearing. In addition to its applications in obtaining a whole-mouse neuronal projection map, detecting single-cell metastases in whole mice and identifying previously undescribed anatomical structures, we further show the visualization of the entire mouse lymphatic system, the application for virus tracing and the visualization of all pericytes in the brain. Taken together, our vDISCO pipeline allows systematic and comprehensive studies of cellular phenomena and connectivity in whole bodies.
Assuntos
Encéfalo , Imageamento Tridimensional , Camundongos , Animais , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Solventes/química , Neuritos , CorantesRESUMO
Histological analysis of biological tissues by mechanical sectioning is significantly time-consuming and error-prone due to loss of important information during sample slicing. In the recent years, the development of tissue clearing methods overcame several of these limitations and allowed exploring intact biological specimens by rendering tissues transparent and subsequently imaging them by laser scanning fluorescence microscopy. In this review, we provide a guide for scientists who would like to perform a clearing protocol from scratch without any prior knowledge, with an emphasis on DISCO clearing protocols, which have been widely used not only due to their robustness, but also owing to their relatively straightforward application. We discuss diverse tissue-clearing options and propose solutions for several possible pitfalls. Moreover, after surveying more than 30 researchers that employ tissue clearing techniques in their laboratories, we compiled the most frequently encountered issues and propose solutions. Overall, this review offers an informative and detailed guide through the growing literature of tissue clearing and can help with finding the easiest way for hands-on implementation.
Assuntos
Óptica e Fotônica/métodos , Especificidade de Órgãos , Animais , Árvores de Decisões , Humanos , Solventes/toxicidade , Coloração e RotulagemRESUMO
Sphingosine-1-phosphate (S1P), is a signaling sphingolipid which acts as a bioactive lipid mediator. We assessed whether S1P had multiplex effects in regulating the large-conductance Ca2+-activated K+ channel (BKCa) in catecholamine-secreting chromaffin cells. Using multiple patch-clamp modes, Ca2+ imaging, and computational modeling, we evaluated the effects of S1P on the Ca2+-activated K+ currents (IK(Ca)) in bovine adrenal chromaffin cells and in a pheochromocytoma cell line (PC12). In outside-out patches, the open probability of BKCa channel was reduced with a mean-closed time increment, but without a conductance change in response to a low-concentration S1P (1 µM). The intracellular Ca2+ concentration (Cai) was elevated in response to a high-dose (10 µM) but not low-dose of S1P. The single-channel activity of BKCa was also enhanced by S1P (10 µM) in the cell-attached recording of chromaffin cells. In the whole-cell voltage-clamp, a low-dose S1P (1 µM) suppressed IK(Ca), whereas a high-dose S1P (10 µM) produced a biphasic response in the amplitude of IK(Ca), i.e., an initial decrease followed by a sustained increase. The S1P-induced IK(Ca) enhancement was abolished by BAPTA. Current-clamp studies showed that S1P (1 µM) increased the action potential (AP) firing. Simulation data revealed that the decreased BKCa conductance leads to increased AP firings in a modeling chromaffin cell. Over a similar dosage range, S1P (1 µM) inhibited IK(Ca) and the permissive role of S1P on the BKCa activity was also effectively observed in the PC12 cell system. The S1P-mediated IK(Ca) stimulation may result from the elevated Cai, whereas the inhibition of BKCa activity by S1P appears to be direct. By the differentiated tailoring BKCa channel function, S1P can modulate stimulus-secretion coupling in chromaffin cells.
Assuntos
Cálcio/metabolismo , Células Cromafins/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Animais , Bovinos , Sistema Livre de Células , Células Cromafins/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eletrofisiologia/métodos , Lisofosfolipídeos/administração & dosagem , Lisofosfolipídeos/farmacologia , Células PC12 , Ratos , Esfingosina/administração & dosagem , Esfingosina/metabolismo , Esfingosina/farmacologiaRESUMO
Wavefront shaping makes it possible to form a focus through opaque scattering materials. In some cases, this focus may be scanned over a small distance using the optical memory effect. However, in many cases of interest, the optical memory effect has a limited range or is even too small to be measured. In such cases, one often resorts to measuring the full transmission matrix (TM) of the sample to completely control the light transmission. However, this process is time-consuming and may not always be possible. We introduce a new method, to the best of our knowledge, for focusing and scanning the focus at any arbitrary position behind the medium by measuring only a subset of the TM, called sparse field focusing (SFF). With SFF, the scan range is not limited to the memory effect, and there is no need to measure the full TM. Our experimental results agree well with our theoretical model. We expect that this method will find applications in imaging through scattering media, especially when the optical memory effect range is small.
RESUMO
Ca2+ influx triggers the release of synaptic vesicles at the presynaptic active zone (AZ). A quantitative characterization of presynaptic Ca2+ signaling is critical for understanding synaptic transmission. However, this has remained challenging to establish at the required resolution. Here, we employ confocal and stimulated emission depletion (STED) microscopy to quantify the number (20-330) and arrangement (mostly linear 70 nm × 100-600 nm clusters) of Ca2+ channels at AZs of mouse cochlear inner hair cells (IHCs). Establishing STED Ca2+ imaging, we analyze presynaptic Ca2+ signals at the nanometer scale and find confined elongated Ca2+ domains at normal IHC AZs, whereas Ca2+ domains are spatially spread out at the AZs of bassoon-deficient IHCs. Performing 2D-STED fluorescence lifetime analysis, we arrive at estimates of the Ca2+ concentrations at stimulated IHC AZs of on average 25 µM. We propose that IHCs form bassoon-dependent presynaptic Ca2+-channel clusters of similar density but scalable length, thereby varying the number of Ca2+ channels amongst individual AZs.
Assuntos
Sinalização do Cálcio/fisiologia , Células Ciliadas Auditivas Internas/fisiologia , Microscopia/métodos , Nanotecnologia/métodos , Algoritmos , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/fisiologia , Células Ciliadas Auditivas Internas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Modelos Neurológicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Sinapses/metabolismo , Sinapses/fisiologia , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologiaRESUMO
For sounds of a given frequency, spiral ganglion neurons (SGNs) with different thresholds and dynamic ranges collectively encode the wide range of audible sound pressures. Heterogeneity of synapses between inner hair cells (IHCs) and SGNs is an attractive candidate mechanism for generating complementary neural codes covering the entire dynamic range. Here, we quantified active zone (AZ) properties as a function of AZ position within mouse IHCs by combining patch clamp and imaging of presynaptic Ca(2+) influx and by immunohistochemistry. We report substantial AZ heterogeneity whereby the voltage of half-maximal activation of Ca(2+) influx ranged over â¼20 mV. Ca(2+) influx at AZs facing away from the ganglion activated at weaker depolarizations. Estimates of AZ size and Ca(2+) channel number were correlated and larger when AZs faced the ganglion. Disruption of the deafness gene GIPC3 in mice shifted the activation of presynaptic Ca(2+) influx to more hyperpolarized potentials and increased the spontaneous SGN discharge. Moreover, Gipc3 disruption enhanced Ca(2+) influx and exocytosis in IHCs, reversed the spatial gradient of maximal Ca(2+) influx in IHCs, and increased the maximal firing rate of SGNs at sound onset. We propose that IHCs diversify Ca(2+) channel properties among AZs and thereby contribute to decomposing auditory information into complementary representations in SGNs.