Predicting future climate at high spatial and temporal resolution

Most studies on the biological effects of future climatic changes rely on seasonally aggregated, coarse‐resolution data. Such data mask spatial and temporal variability in microclimate driven by terrain, wind and vegetation, and ultimately bear little resemblance to the conditions that organisms experience in the wild. Here, I present the methods for providing fine‐grained, hourly and daily estimates of current and future temperature and soil moisture over decadal timescales. Observed climate data and spatially coherent probabilistic projections of daily future weather were disaggregated to hourly and used to drive empirically calibrated physical models of thermal and hydrological microclimates. Mesoclimatic effects (cold‐air drainage, coastal exposure and elevation) were determined from the coarse‐resolution climate surfaces using thin‐plate spline models with coastal exposure and elevation as predictors. Differences between micro and mesoclimate temperatures were determined from terrain, vegetation and ground properties using energy balance equations. Soil moisture was computed in a thin upper layer and an underlying deeper layer, and the exchange of water between these layers was calculated using the van Genuchten equation. Code for processing the data and running the models is provided as a series of R packages. The methods were applied to the Lizard Peninsula, United Kingdom, to provide hourly estimates of temperature (100 m grid resolution over entire area, 1 m for a selected area) for the periods 1983–2017 and 2041–2049. Results indicated that there is a fine‐resolution variability in climatic changes, driven primarily by interactions between landscape features and decadal trends in weather conditions. High‐temporal resolution extremes in conditions under future climate change were predicted to be considerably less novel than the extremes estimated using seasonally aggregated variables. The study highlights the need to more accurately estimate the future climatic conditions experienced by organisms and equips biologists with the means to do so.     

Imaging cortical dynamics at high spatial and temporal resolution with novel blue voltage-sensitive dyes

Conventional imaging techniques have provided high-resolution imaging either in the spatial domain or in the temporal domain. Optical imaging utilizing voltage-sensitive dyes has long had the unrealized potential to achieve high resolution in both domains simultaneously, providing subcolumnar spatial detail with millisecond precision. Here, we present a series of developments in voltage-sensitive dyes and instrumentation that make functional imaging of cortical dynamics practical, in both anesthetized and awake behaving preparations, greatly facilitating exploration of the cortex. We illustrate this advance by analyzing the millisecond-by-millisecond emergence of orientation maps in cat visual cortex.     

Solid phase gene extraction isolates mRNA at high spatial and temporal resolution

Rapid, localized changes in gene expression require mRNA extraction at high temporal and spatial resolution. Current small-scale mRNA extractions depend on the removal of the cells/tissue from an organism or preserved specimens. What these methods have in common is that they are destructive and do not distinguish between genomic DNA and RNA. Therefore, extracted (m)RNA is typically contaminated by extracted cytoplasm, nuclear DNA, or other compounds, and the required purification leads to loss of especially low-abundant mRNA. The need to repeatedly remove mRNA from living material has led to the development of solid phase gene extraction (SPGE). SPGE sampling can be achieved using gene-specific or generic sequences and is not species-specific. Here we demonstrate the versatility and validity of this novel RNA extraction by simultaneously profiling nanos and bicoid mRNA in individual Drosophila eggs. The SPGE technique detects previously described distribution profiles of nanos and bicoid. Its low impact is underscored by the normal development of repeatedly sampled eggs. In our study, quantification of actin mRNA in germinating flax seeds linked gene expression to distinct developmental processes. These data demonstrate the universality of SPGE as a simple generic, analytical, and diagnostic procedure.     

Electron cryo-microscopy of vitrified biological specimens: towards high spatial and temporal resolution

A decade after the development of electron cryo-microscopy for vitrified specimens, its advantages and limitations are analysed. Indeed, recent work carried out by different laboratories strengthens the idea that electron cryo-microscopy might soon be an alternative method to X-ray crystallography and NMR techniques for determining the structure of biological assemblies with both high spatial and temporal resolutions. High pressure freezing allows vitrification of larger volumes of biological suspensions. Thick vitrified objects can be cryosectioned. Electron cryo-microscopy of the sections gives images having a resolution better than 2 nm. Although the high resolution imaging mode under low dose conditions is not yet fully understood, microscopes are being developed to provide better and better images. Image averaging is being facilitated by the development of both crystallization and computer methods. Thus, we can expect that electron microscopy will soon become a potential technique for structural determination at atomic resolution. Finally, much effort is being devoted to improving the temporal resolution of electron cryo-microscopy. Soon, we may be able to observe molecules during their biological activity.     

Epi-illumination dark-field microscopy enables direct visualization of unlabeled small organisms with high spatial and temporal resolution

Dark-field microscopy is known to offer both high resolution and direct visualization of thin samples. However, its performance and optimization on thick samples is under-explored and so far, only meso-scale information from whole organisms has been demonstrated. In this work, we carefully investigate the difference between trans- and epi-illumination configurations. Our findings suggest that the epi-illumination configuration is superior in both contrast and fidelity compared to trans-illumination, while having the added advantage of experimental simplicity and an “open top” for experimental intervention. Guided by the theoretical analysis, we constructed an epi-illumination dark-field microscope with measured lateral and axial resolutions of 260 nm and 520 nm, respectively. Subcellular structures in whole organisms were directly visualized without the need for image reconstruction, and further confirmed via simultaneous fluorescence imaging. With an imaging speed of 20 to 50 fps, we visualize fast dynamic processes such as cell division and pharyngeal pumping in Caenorhabditis elegans.     

Probing DNA conformational changes with high temporal resolution by tethered particle motion

The tethered particle motion (TPM) technique informs about conformational changes of DNA molecules, e.g. upon looping or interaction with proteins, by tracking the Brownian motion of a particle probe tethered to a surface by a single DNA molecule and detecting changes of its amplitude of movement. We discuss in this context the time resolution of TPM, which strongly depends on the particle-DNA complex relaxation time, i.e. the characteristic time it takes to explore its configuration space by diffusion. By comparing theory, simulations and experiments, we propose a calibration of TPM at the dynamical level: we analyze how the relaxation time grows with both DNA contour length (from 401 to 2080 base pairs) and particle radius (from 20 to 150 nm). Notably we demonstrate that, for a particle of radius 20 nm or less, the hydrodynamic friction induced by the particle and the surface does not significantly slow down the DNA. This enables us to determine the optimal time resolution of TPM in distinct experimental contexts which can be as short as 20 ms.     

Electro-deformation and poration of giant vesicles viewed with high temporal resolution

Fast digital imaging was used to study the deformation and poration of giant unilamellar vesicles subjected to electric pulses. For the first time the dynamics of response and relaxation of the membrane at micron-scale level is revealed at a time resolution of 30 micros. Above a critical transmembrane potential the lipid bilayer ruptures. Formation of macropores (diameter approximately 2 microm) with pore lifetime of approximately 10 ms has been detected. The pore lifetime has been interpreted as interplay between the pore edge tension and the membrane viscosity. The reported data, covering six decades of time, show the following regimes in the relaxation dynamics of the membrane. Tensed vesicles first relax to release the acquired stress due to stretching, approximately 100 micros. In the case of poration, membrane resealing occurs with a characteristic time of approximately 10 ms. Finally, for vesicles with excess area an additional slow regime was observed, approximately 1 s, which we associate with relaxation of membrane curvature. Dimensional analysis can reasonably well explain the corresponding characteristic timescales. Being performed on cell-sized giant unilamellar vesicles, this study brings insight to cell electroporation. The latter is widely used for gene transfection and drug transport across the membrane where processes occurring at different timescales may influence the efficiency.     

Enhanced temporal and spatial resolution in super-resolution covariance imaging algorithm with deconvolution optimization

Based on the numerical analysis that covariance exhibits superior statistical precision than cumulant and variance, a new SOFI algorithm by calculating the n orders covariance for each pixel is presented with an almost -fold resolution improvement, which can be enhanced to 2ⁿ via deconvolution. An optimized deconvolution is also proposed by calculating the (n + 1) order SD associated with each n order covariance pixel, and introducing the results into the deconvolution as a damping factor to suppress noise generation. Moreover, a re-deconvolution of the covariance image with the covariance-equivalent point spread function is used to further increase the final resolution by above 2-fold. Simulated and experimental results show that this algorithm can significantly increase the temporal–spatial resolution of SOFI, meanwhile, preserve the sample's structure. Thus, a resolution of 58 nm is achieved for 20 experimental images, and the corresponding acquisition time is 0.8 seconds.     

Evaluating ecosystem effects of climate change on tropical island streams using high spatial and temporal resolution sampling regimes

Climate change is expected to alter precipitation patterns worldwide, which will affect streamflow in riverine ecosystems. It is vital to understand the impacts of projected flow variations, especially in tropical regions where the effects of climate change are expected to be one of the earliest to emerge. Space‐for‐time substitutions have been successful at predicting effects of climate change in terrestrial systems by using a spatial gradient to mimic the projected temporal change. However, concerns have been raised that the spatial variability in these models might not reflect the temporal variability. We utilized a well‐constrained rainfall gradient on Hawaii Island to determine (a) how predicted decreases in flow and increases in flow variability affect stream food resources and consumers and (b) if using a high temporal (monthly, four streams) or a high spatial (annual, eight streams) resolution sampling scheme would alter the results of a space‐for‐time substitution. Declines in benthic and suspended resource quantity (10‐ to 40‐fold) and quality (shift from macrophyte to leaf litter dominated) contributed to 35‐fold decreases in macroinvertebrate biomass with predicted changes in the magnitude and variability in the flow. Invertebrate composition switched from caddisflies and damselflies to taxa with faster turnover rates (mosquitoes, copepods). Changes in resource and consumer composition patterns were stronger with high temporal resolution sampling. However, trends and ranges of results did not differ between the two sampling regimes, indicating that a suitable, well‐constrained spatial gradient is an appropriate tool for examining temporal change. Our study is the first to investigate resource to community wide effects of climate change on tropical streams on a spatial and temporal scale. We determined that predicted flow alterations would decrease stream resource and consumer quantity and quality, which can alter stream function, as well as biomass and habitat for freshwater, marine, and terrestrial consumers dependent on these resources.     

Proteohistography--direct analysis of tissue with high sensitivity and high spatial resolution using ProteinChip technology.

On the proteomic level, all tissues, tissue constituents, or even single cells are heterogeneous, but the biological relevance of this cannot be adequately investigated with any currently available technique. The analysis of proteins of small tissue areas by any proteomic approach is limited by the number of required cells. Increasing the number of cells only serves to lower the spatial resolution of expressed proteins. To enhance sensitivity and spatial resolution we developed Proteohistography. Laser microdissection was used to mark special areas of interest on tissue sections attached to glass slides. These areas were positioned under microscopic control directly on an affinity chromatographic ProteinChip Array so that cells were lysed and their released proteins bound on a spatially defined point. The ProteinChip System, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), allows the laser to be steered to up to 215 distinct positions across the surface of the spot, enabling a high spatial resolution of measured protein profiles for the analyzed tissue area. Protein profiles of the single positions were visually plotted over the used tissue section to visualize distribution proteohistologically. Results show that the spatial distribution of detectable proteins could be used as a Proteohistogram for a given tissue area. Consequently, this procedure can provide additional information to both a matrix-assisted laser desorption/ionization (MALDI)-based approach and immunohistochemistry, as it is more sensitive, highly quantitative, and no specific antibody is needed. Hence, proteomic heterogeneity can be visualized even if proteins are not known or identified.     

Imaging in vivo redox status in high spatial resolution with OMRI

Redox-reactions are playing a significant role in regulation of homeostasis of organism. Disorder of the redox-status is related with the onset and/or propagation of oxidative diseases such as lifestyle-related diseases, including cancers and cardiac diseases, etc. In vivo imaging of redox-status is thereby important in the analysis of mechanisms of oxidative diseases and developments of new medicines for the diseases. Aminoxyl radicals are redox-sensitive reporter molecules, which lose their paramagnetic moiety by reactions of free radicals or reducing compounds. Electron spin resonance (ESR) technique has been used to measure the molecules in vivo. In vivo spatial resolution in ESR imaging is in the range of a few millimeters and is not sufficient for the detailed diagnosis of disease models. Overhauser enhanced MRI (OMRI) is an emerging free radical imaging technique, which utilised electron-proton coupling to image the distribution of free radicals. In vivo imaging of redox-status is applicable with OMRI/aminoxyl radical technique. The detailed imaging analysis was demonstrated in oxidative diseases, such as tumour-bearing, neurodegeneration or gastric ulcer models. The OMRI/aminoxyl radical technique has a large potential as a diagnostic system for biomedical applications in the future.     

Imaging in vivo redox status in high spatial resolution with OMRI

Abstract

Redox-reactions are playing a significant role in regulation of homeostasis of organism. Disorder of the redox-status is related with the onset and/or propagation of oxidative diseases such as lifestyle-related diseases, including cancers and cardiac diseases, etc. In vivo imaging of redox-status is thereby important in the analysis of mechanisms of oxidative diseases and developments of new medicines for the diseases. Aminoxyl radicals are redox-sensitive reporter molecules, which lose their paramagnetic moiety by reactions of free radicals or reducing compounds. Electron spin resonance (ESR) technique has been used to measure the molecules in vivo. In vivo spatial resolution in ESR imaging is in the range of a few millimeters and is not sufficient for the detailed diagnosis of disease models. Overhauser enhanced MRI (OMRI) is an emerging free radical imaging technique, which utilised electron–proton coupling to image the distribution of free radicals. In vivo imaging of redox-status is applicable with OMRI/aminoxyl radical technique. The detailed imaging analysis was demonstrated in oxidative diseases, such as tumour-bearing, neurodegeneration or gastric ulcer models. The OMRI/aminoxyl radical technique has a large potential as a diagnostic system for biomedical applications in the future.     

Trace metal imaging with high spatial resolution: applications in biomedicine

New generations of analytical techniques for imaging of metals are pushing hitherto boundaries of spatial resolution and quantitative analysis in biology. Because of this, the application of these imaging techniques described herein to the study of the organization and dynamics of metal cations and metal-containing biomolecules in biological cell and tissue is becoming an important issue in biomedical research. In the current review, three common metal imaging techniques in biomedical research are introduced, including synchrotron X-ray fluorescence (SXRF) microscopy, secondary ion mass spectrometry (SIMS), and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). These are exemplified by a demonstration of the dopamine-Fe complexes, by assessment of boron distribution in a boron neutron capture therapy cell model, by mapping Cu and Zn in human brain cancer and a rat brain tumor model, and by the analysis of metal topography within neuromelanin. These studies have provided solid evidence that demonstrates that the sensitivity, spatial resolution, specificity, and quantification ability of metal imaging techniques is suitable and highly desirable for biomedical research. Moreover, these novel studies on the nanometre scale (e.g., of individual single cells or cell organelles) will lead to a better understanding of metal processes in cells and tissues.     

Improving spatial and temporal resolution in evoked EEG responses using surface Laplacians

Spline generated surface Laplacian temporal wave forms are presented as a method to improve both spatial and temporal resolution of evoked EEG responses. Middle latency and the N1 components of the auditory evoked response were used to compare potential-based methods with surface Laplacian methods in the time domain. Results indicate that surface Laplacians provide better estimates of underlying cortical activity than do potential wave forms. Spatial discrimination among electrode sites was markedly better with surface Laplacian than with potential wave forms. Differences in the number and latencies of peaks, and their topographic distributions, were observed for surface Laplacian, particularly during the time period encompassing the middle latency responses. Focal activities were observed in surface Laplacian wave forms and topographic maps which were in agreement with previous findings from auditory evoked response studies. Methodological issues surrounding the application of spline methods to the time domain are also discussed. Surface Laplacian methods in the time domain appear to provide an improved way for studying evoked EEG responses by increasing temporal and spatial resolution of component characteristics.     

Real-time confocal microscopy and calcium measurements in heart muscle cells: towards the development of a fluorescence microscope with high temporal and spatial resolution

Preliminary experiments and characterization of a modified confocal fluorescence microscope have been carried out and are presented in this article. We have made use of commercially available hardware and software (having modified and extended the system) and are continuing the process of making modifications to this system to enable us to carry out investigations in living and mobile cells. We report on identified problems in measuring intracellular calcium in myocardial cells, important lessons that have been learned and new findings regarding myocardial cells. Specifically, we have found that myocardial cellular organelles (e.g. nuclei and mitochondria) are sharply defined unlike images obtained with standard epifluorescence microscopes using intracellular indicators. Furthermore, we show that the organelles can accumulate or largely exclude certain indicators relative to the concentration in the cytosol. Additionally, we have examined the use of the new calcium indicator fluo-3 in contracting heart cells and have more clearly defined certain limitations in its use in this preparation. We are working on modifications of the present equipment to enable the system to work with an ultraviolet laser as a light source. The confocal microscope offers the prospect of extraordinarily good spatial and temporal resolution under specific conditions in heart cells.     

New double-tracer digital radiography for analysis of spatial and temporal myocardial flow heterogeneity

A new high-resolution digital radiographic technique based on the deposition of (125)I- and (3)H-labeled desmethylimipramine (IDMI and HDMI, respectively) was developed for the assessment of spatial and temporal myocardial flow heterogeneity at a microvascular level. The density distributions of two tracers, or relative flow distributions, were determined by subtraction digital radiography using two imaging plates of different sensitivity. The regions resolved are comparable in size to vascular regulatory units (400 x 400 microm(2)). This method was applied to the measurement of within-layer myocardial flow distributions in Langendorff-perfused rabbit hearts. The validity of this method was confirmed by the strong correlation between regional densities of two tracers injected simultaneously (r = 0.89 +/- 0.03, n = 8). The temporal flow stability was evaluated by a 90-s continuous IDMI injection and subsequent bolus HDMI injection (n = 8). Regional densities of the two tracers were fairly correlated (r = 0.86 +/- 0.03), indicating that the spatial pattern of flow distribution was stable even at a microvascular level over a 90-s period. The effect of microsphere embolization on the flow distribution was also investigated by the sequential injections of IDMI, 15-microm microspheres, and HDMI at 20-s intervals (n = 8). Microembolization increased the coefficient of variation of tracer density from 19 to 25% (P < 0.05), whereas the regional densities of two tracers were still correlated substantially, as in the case of no embolization (r = 0.84 +/- 0.06). Thus the microsphere embolization enhanced flow heterogeneity with increasing flow differences between control high-flow and control low-flow regions but rather maintained the pattern of flow distribution. In conclusion, double-tracer digital radiography will be a promising method for the spatial and temporal myocardial flow analysis at microvascular levels.     

A simple method for high temporal resolution calcium imaging with dual excitation dyes.

Calcium-sensitive dual excitation dyes, such as fura-2, are now widely used to measure the free calcium concentration ([Ca2+]) in living cells. Preferentially, [Ca2+] is calculated in a ratiometric manner, but if calcium images need to be acquired at high temporal resolution, a potential drawback of ratiometry is that it requires equally fast switching of the excitation light between two wavelengths. To circumvent continuous excitation switching, some investigators have devised methods for calculating [Ca2+] from single-wavelength measurements combined with the acquisition of a single ratiometric pair of fluorescence images at the start of the recording. These methods, however, are based on the assumption that the concentration of the dye does not change during the experiment, a condition that is often not fulfilled. We describe here a method of single-wavelength calcium imaging, in which the dye concentration is estimated from ratiometric fluorescence image pairs acquired at regular intervals during the recording period, that furthermore includes a correction for the changing dye concentration in the calculation of [Ca2+].