Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM ...techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.
We presented a simple and efficient method to eliminate redundant vertices produced by standard Marching Tetrahedra approach. More than 60% of time cost in quantitative analysis of molecular surface ...of electrostatic potential can be saved without evident deterioration of accuracy of the results. Display omitted
► A new surface refinement method is applied to quantitative molecular surface analysis. ► More than 60% of time cost in surface analysis can be saved by eliminating unreasonably distributed vertices. ► Bisection algorithm improves interpolation accuracy in Marching Tetrahedra significantly. ► Optimal grid spacing for surface analysis was explored.
Quantitative analysis of molecular surface is a valuable technique for analyzing non-covalent interaction, studying molecular recognition mode, predicting reactive site and reactivity. An efficient way to realize the analysis was first proposed by Bulat et al. (J. Mol. Model., 16, 1679), in which Marching Tetrahedra (MT) approach commonly used in computer graphics is employed to generate vertices on molecular surface. However, it has been found that the computations of the electrostatic potential in the MT vertices are very expensive and some artificial surface extremes will be presented due to the uneven distribution of MT vertices. In this article, we propose a simple and reliable method to eliminate these unreasonably distributed surface vertices generated in the original MT. This treatment can save more than 60% of total analysis time of electrostatic potential, yet the loss in accuracy is almost negligible. The artificial surface extremes are also largely avoided as a byproduct of this algorithm. In addition, the bisection iteration procedure has been exploited to improve accuracy of linear interpolation in MT. The most appropriate grid spacing for surface analysis has also been investigated. 0.25 and 0.20bohr are recommended to be used for surface analysis of electrostatic potential and average local ionization energy, respectively.
Body condition scoring (BCS) is a farm-management tool for estimating dairy cows’ energy reserves. Today, BCS is performed manually by experts. This paper presents a 3-dimensional algorithm that ...provides a topographical understanding of the cow’s body to estimate BCS. An automatic BCS system consisting of a Kinect camera (Microsoft Corp., Redmond, WA) triggered by a passive infrared motion detector was designed and implemented. Image processing and regression algorithms were developed and included the following steps: (1) image restoration, the removal of noise; (2) object recognition and separation, identification and separation of the cows; (3) movie and image selection, selection of movies and frames that include the relevant data; (4) image rotation, alignment of the cow parallel to the x-axis; and (5) image cropping and normalization, removal of irrelevant data, setting the image size to 150×200 pixels, and normalizing image values. All steps were performed automatically, including image selection and classification. Fourteen individual features per cow, derived from the cows’ topography, were automatically extracted from the movies and from the farm’s herd-management records. These features appear to be measurable in a commercial farm. Manual BCS was performed by a trained expert and compared with the output of the training set. A regression model was developed, correlating the features with the manual BCS references. Data were acquired for 4 d, resulting in a database of 422 movies of 101 cows. Movies containing cows’ back ends were automatically selected (389 movies). The data were divided into a training set of 81 cows and a test set of 20 cows; both sets included the identical full range of BCS classes. Accuracy tests gave a mean absolute error of 0.26, median absolute error of 0.19, and coefficient of determination of 0.75, with 100% correct classification within 1 step and 91% correct classification within a half step for BCS classes. Results indicated good repeatability, with all standard deviations under 0.33. The algorithm is independent of the background and requires 10 cows for training with approximately 30 movies of 4 s each.
Cryogenic electron microscopy (cryo-EM) enables structure determination of macromolecular objects and their assemblies. Although the techniques have been developing for nearly four decades, they have ...gained widespread attention in recent years due to technical advances on numerous fronts, enabling traditional microscopists to break into the world of molecular structural biology. Many samples can now be routinely analyzed at near-atomic resolution using standard imaging and image analysis techniques. However, numerous challenges to conventional workflows remain, and continued technical advances open entirely novel opportunities for discovery and exploration. Here, I will review some of the main methods surrounding cryo-EM with an emphasis specifically on single-particle analysis, and I will highlight challenges, open questions, and opportunities for methodology development.
Optical tissue transparency permits scalable cellular and molecular investigation of complex tissues in 3D. Adult human organs are particularly challenging to render transparent because of the ...accumulation of dense and sturdy molecules in decades-aged tissues. To overcome these challenges, we developed SHANEL, a method based on a new tissue permeabilization approach to clear and label stiff human organs. We used SHANEL to render the intact adult human brain and kidney transparent and perform 3D histology with antibodies and dyes in centimeters-depth. Thereby, we revealed structural details of the intact human eye, human thyroid, human kidney, and transgenic pig pancreas at the cellular resolution. Furthermore, we developed a deep learning pipeline to analyze millions of cells in cleared human brain tissues within hours with standard lab computers. Overall, SHANEL is a robust and unbiased technology to chart the cellular and molecular architecture of large intact mammalian organs.
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•CHAPS forms smaller micelles allowing full permeabilization of aged human organs•SHANEL enables centimeters deep molecular labeling and clearing of whole human organs•SHANEL renders intact adult human brain and kidney transparent•Deep learning and light-sheet microscopy with SHANEL allows human organ mapping
Zhao et al. present an approach for intact human organ mapping that uses a new tissue permeabilization method to clear and deeply label whole organs followed by light-sheet microscopy imaging and a deep learning-based pipeline for 3D reconstruction and data analysis.
The past quarter century has witnessed rapid developments of fluorescence microscopy techniques that enable structural and functional imaging of biological specimens at unprecedented depth and ...resolution. The performance of these methods in multicellular organisms, however, is degraded by sample-induced optical aberrations. Here I review recent work on incorporating adaptive optics, a technology originally applied in astronomical telescopes to combat atmospheric aberrations, to improve image quality of fluorescence microscopy for biological imaging.
Mapping Sub-Second Structure in Mouse Behavior Wiltschko, Alexander B.; Johnson, Matthew J.; Iurilli, Giuliano ...
Neuron (Cambridge, Mass.),
12/2015, Letnik:
88, Številka:
6
Journal Article
Recenzirano
Odprti dostop
Complex animal behaviors are likely built from simpler modules, but their systematic identification in mammals remains a significant challenge. Here we use depth imaging to show that 3D mouse pose ...dynamics are structured at the sub-second timescale. Computational modeling of these fast dynamics effectively describes mouse behavior as a series of reused and stereotyped modules with defined transition probabilities. We demonstrate this combined 3D imaging and machine learning method can be used to unmask potential strategies employed by the brain to adapt to the environment, to capture both predicted and previously hidden phenotypes caused by genetic or neural manipulations, and to systematically expose the global structure of behavior within an experiment. This work reveals that mouse body language is built from identifiable components and is organized in a predictable fashion; deciphering this language establishes an objective framework for characterizing the influence of environmental cues, genes and neural activity on behavior.
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•Computational modeling reveals structure in mouse behavior without observer bias•Mouse behavior appears to be composed of stereotyped, sub-second modules•From this perspective, new behaviors result from altering both modules and transitions•Unsupervised analysis reveals how genes and neural activity impact behavior
Mouse behavior appears inherently divided into brief modules of 3D motion. This sub-second structure reveals the influence of the environment, genes and neural activity on action.
The visualization of molecularly labeled structures within large intact tissues in three dimensions is an area of intense focus. We describe a simple, rapid, and inexpensive method, iDISCO, that ...permits whole-mount immunolabeling with volume imaging of large cleared samples ranging from perinatal mouse embryos to adult organs, such as brains or kidneys. iDISCO is modeled on classical histology techniques, facilitating translation of section staining assays to intact tissues, as evidenced by compatibility with 28 antibodies to both endogenous antigens and transgenic reporters like GFP. When applied to degenerating neurons, iDISCO revealed unexpected variability in number of apoptotic neurons within individual sensory ganglia despite tight control of total number in all ganglia. It also permitted imaging of single degenerating axons in adult brain and the first visualization of cleaved Caspase-3 in degenerating embryonic sensory axons in vivo, even single axons. iDISCO enables facile volume imaging of immunolabeled structures in complex tissues. PAPERCLIP:
To identify sex-related differences and age-related changes in individual retinal layer thicknesses in a population of healthy eyes across the lifespan, using spectral domain optical coherence ...tomography (SD-OCT).
In seven institutes in Japan, mean thicknesses of the retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptor inner segment (IS), and photoreceptor outer segment (OS) were measured using SD-OCT with a new automated segmentation protocol in 256 healthy subjects.
Interoperator coefficients of variability for measurements of each layer ranged from 0.012 to 0.038. The RNFL, GCL, IPL, and INL were thinnest in the foveal area, whereas the OPL+ONL and OS were thickest in this area. Mean thicknesses of the INL and the OPL+ONL were significantly greater in men (P = 0.002 and 0.001, respectively). However, mean RNFL thickness was greater in women (P = 0.006). Thicknesses of the RNFL, GCL, IPL, INL, and IS correlated negatively with age. Thickness of the OPL+ONL was not correlated with age, and thickness of the OS correlated positively with age. Inner retinal (RNFL+GCL+IPL) thickness over the whole macula correlated negatively with age (P < 0.001), but outer retinal (OPL+ONL+IS+OS) thickness did not. Thicknesses of layers did not correlate with axial length.
Macular layer thicknesses measured on SD-OCT images in healthy eyes showed significant variations by sex and age. These findings should inform macular layer thickness analyses in SD-OCT studies of retinal diseases and glaucoma.
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