EZ Clear for simple, rapid, and robust mouse whole organ clearing

Tissue clearing for whole organ cell profiling has revolutionized biology and imaging for exploration of organs in three-dimensional space without compromising tissue architecture. But complicated, laborious procedures, or expensive equipment, as well as the use of hazardous, organic solvents prevents the widespread adoption of these methods. Here we report a simple and rapid tissue clearing method, EZ Clear, that can clear whole adult mouse organs in 48 hours in just three simple steps. Samples stay at room temperature and remain hydrated throughout the clearing process, preserving endogenous and synthetic fluorescence, without altering sample size. After wholemount clearing and imaging, EZ Cleared samples can be further processed for downstream embedding and cryosectioning followed by standard histology or immunostaining, without loss of endogenous or synthetic fluorescence signal. Overall, the simplicity, speed, and flexibility of EZ Clear make it easy to adopt and apply to diverse approaches in biomedical research.

Improved clearing method contributes to deep imaging of plant organs

Tissue clearing methods are increasingly essential for the microscopic observation of internal tissues of thick biological organs. We previously developed TOMEI, a clearing method for plant tissues; however, it could not entirely remove chlorophylls nor reduce the fluorescent signal of fluorescent proteins. Here, we developed an improved TOMEI method (iTOMEI) to overcome these limitations. First, a caprylyl sulfobetaine was determined to efficiently remove chlorophylls from Arabidopsis thaliana seedlings without GFP quenching. Next, a weak alkaline solution restored GFP fluorescence, which was mainly lost during fixation, and an iohexol solution with a high refractive index increased sample transparency. These procedures were integrated to form iTOMEI. iTOMEI enables the detection of much brighter fluorescence than previous methods in tissues of A. thaliana, Oryza sativa, and Marchantia polymorpha. Moreover, a mouse brain was also efficiently cleared by the iTOMEI-Brain method within 48 h, and strong fluorescent signals were detected in the cleared brain.

Optimizing the Experimental Method for Stomata-Profiling Automation of Soybean Leaves Based on Deep Learning

Stomatal observation and automatic stomatal detection are useful analyses of stomata for taxonomic, biological, physiological, and eco-physiological studies. We present a new clearing method for improved microscopic imaging of stomata in soybean followed by automated stomatal detection by deep learning. We tested eight clearing agent formulations based upon different ethanol and sodium hypochlorite (NaOCl) concentrations in order to improve the transparency in leaves. An optimal formulation—a 1:1 (v/v) mixture of 95% ethanol and NaOCl (6–14%)—produced better quality images of soybean stomata. Additionally, we evaluated fixatives and dehydrating agents and selected absolute ethanol for both fixation and dehydration. This is a good substitute for formaldehyde, which is more toxic to handle. Using imaging data from this clearing method, we developed an automatic stomatal detector using deep learning and improved a deep-learning algorithm that automatically analyzes stomata through an object detection model using YOLO. The YOLO deep-learning model successfully recognized stomata with high mAP (~0.99). A web-based interface is provided to apply the model of stomatal detection for any soybean data that makes use of the new clearing protocol.

Modelling the disappearance of coarse woody debris, following a land clearing event

Background Land clearing generates coarse woody debris (CWD), much of which ultimately becomes atmospheric CO2. Schemes for greenhouse gas accounting must consider the contribution from land clearing, but the timing of the contribution will have large uncertainty, due to a paucity of knowledge about the rate of CWD disappearance. To better understand above-ground CWD disappearance following a land clearing event—through the actions of microorganisms, invertebrates, wildfire, or deliberate burning—we combined statistical modelling with an archive of semi-quantitative observations (units of CWD %), made within Queensland, Australia. Results Using a generalised additive mixed-effects model (median absolute error = 14.7%), we found that CWD disappearance was strongly influenced by the: (i) number of years elapsed since clearing; (ii) clearing method; (iii) bioregion (effectively a climate-by-tree species interaction); and (iv) the number of times burned. Years-since-clearing had a strongly non-linear effect on the rate of CWD disappearance. The data suggested that disappearance was reverse-sigmoidal, with little change in CWD apparent for the first three years after clearing. In typical conditions for Queensland, the model predicted that it will take 38 years for 95% of CWD to disappear, following a land clearing event; however, accounting for uncertainty in the data and model, this value could be as few as 5 years, or > 100 years. In contrast, due to an assumption about the propensity of land managers to burn CWD, the official method used to assess Australia’s greenhouse gas emissions predicted that 95% of CWD will disappear in < 1 year. Conclusions In Queensland, the CWD generated by land clearing typically takes 38 years to disappear. This ultimately implies that a key assumption of Australia’s official greenhouse gas reporting—i.e. that 98% of CWD is burned soon after a clearing event—does not adequately account for delayed CO2 emissions.

Immunolabelling and clearing of intact spinal cord for visualization of lower urinary tract afferents v1

The whole-mount immunolabeling and clearing method (iDISCO) was used to visualize cholera toxin subunit B-labelled lower urinary tract afferents in the lumbosacral spinal cord of the rat. Imaging of spinal cord was performed on a light sheet microscope with a 12x lens. Concurrently, choline acetyltransferase identified preganglionic autonomic neurons and motoneurons within the spinal cord, which were used to confirm the rostrocaudal location of afferents.