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.     

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.     

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.     

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+].     

A poly(dimethylsiloxane)-based device enabling time-lapse imaging with high spatial resolution

We have developed a regulator-free device that enables long-term incubation of mammalian cells for epi-fluorescence imaging, based on a concept that the size of sample to be gassed and heated is reduced to observation scale. A poly(dimethylsiloxane) block stamped on a coverslip works as a long-lasting supplier of CO₂-rich gas to adjust bicarbonate-containing medium in a tiny chamber at physiological pH, and an oil-immersion objective warms cells across the coverslip. A time-lapse imaging experiment using HeLa cells stably expressing fluorescent cell-cycle indicators showed that the cells in the chamber proliferated with normal cell-cycle period over 2 days.     

A simple photoactivation and image analysis module for visualizing and analyzing axonal transport with high temporal resolution

We describe a strategy for analyzing axonal transport of cytosolic proteins (CPs) using photoactivatable GFP-PAGFP-with modifications of standard imaging components that can be retroactively fitted to a conventional epifluorescence microscope. The photoactivation and visualization are nearly simultaneous, allowing studies of proteins with rapidly mobile fractions. Cultured hippocampal neurons are transfected with PAGFP-tagged constructs, a discrete protein population within axons is photoactivated, and then the activated population is tracked by live imaging. We show the utility of this method in analyzing axonal transport of CPs that have inherent diffusible pools and distinguish this transport modality from passive diffusion and vesicle transport. The analytical tools used to quantify the motion are also described. Aside from the time needed for preparation of neuronal cultures/transfection, the experiment takes 2-3 h, during which time several axons can be imaged and analyzed. These methods should be easy to adopt by most laboratories and may also be useful for monitoring CP movement in other cell types.     

Quantitative analysis of the local rates of growth of dicot leaves at a high temporal and spatial resolution,using image sequence analysis

A new technique is presented for quantitative mapping of dicot leaf growth at high spatial and temporal resolution, at a speed making online-mapping feasible. Time lapse video sequences of growing leaves are captured by a personal computer (PC) with a frame-grabber board and a standard CCD camera, and evaluated using algorithms that have been recently developed to analyse motion in dynamic image sequences. Growth can be detected at under 1% per hour, with a time resolution of minutes and a spatial resolution of a few millimeters. The new technique has been verified by comparing it with classical approaches to map integrated growth. Diurnal courses of leaf growth of Ricinus communis and tobacco are presented to demonstrate the localised character of growth in leaves. Expansion growth is restricted to the base of the leaf and is restricted to a few hours at the end of the night and the start of the day. The high resolution of the method is illustrated by mapping the responses to step-changes in leaf turgor. A 3 bar turgor jump led to a rapid transient expansion over the entire length of the leaf that was partially reversed when the turgor was relaxed.     

Real time, high resolution video imaging of apoptosis in single cells with a polymeric nanoprobe

We report a new apoptosis nanoprobe (Apo-NP) designed on the basis of a polymer nanoparticle platform. This simple one-step technique is capable of boosting fluorescence signals upon apoptosis in living cells, enabling real-time imaging of apoptosis in single cells and in vivo. The Apo-NP efficiently delivers chemically labeled, dual-quenched caspase-3-sensitive fluorogenic peptides into cells, allowing caspase-3-dependent strong fluorescence amplification to be imaged in apoptotic cells in real-time and at high resolution. The design platform of the Apo-NP is flexible and can be fine-tuned for a wide array of applications such as identification of caspase-related apoptosis in pathologies and for monitoring therapeutic efficacy of apoptotic drugs in cancer treatment.     

Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope.

Fluorescence photobleaching methods have been widely used to study diffusion processes in the plasma membrane of single living cells and other membrane systems. Here we describe the application of a new photobleaching technique, scanning microphotolysis. Employing a recently developed extension module to a commercial confocal microscope, an intensive laser beam was switched on and off during scanning according to a user definable image mask. Thereby the location, geometry, and number of photolysed spots could be chosen arbitrarily, their size ranging from tens of micrometers down to the diffraction limit. Therewith we bleached circular areas on the surface of single living 3T3 cells labeled with the fluorescent lipid analog NBD-HPC. Subsequently, the fluorescence recovery process was observed using the attenuated laser beam for excitation. This yielded image stacks representing snapshots of the spatial distribution of fluorescent molecules. From these we computed the radial distribution functions of the photobleached dye molecules. The variance of these distributions is linearly related to the diffusion constant, time, and the mobile fraction of the diffusing species. Furthermore, we compared directly the theoretically expected and measured distribution functions, and could thus determine the diffusion coefficient from each single image. The results of these two new evaluation methods (D = 0.3 +/- 0.1 micron 2/s) agreed well with the outcome of conventional fluorescence recovery measurements. We show that by scanning microphotolysis information on dynamical processes such as diffusion of lipids or proteins can be acquired at the superior spatial resolution of a confocal laser scanning microscope.     

Construction of a thermotaxis chamber providing spatial or temporal thermal gradients monitored by an infrared video camera system.

A thermotaxis chamber was constructed to quantitatively study thermotaxis in eukaryotic amoeboid cells. The apparatus provided either spatial or temporal temperature gradients in an observation chamber set in an inverted microscope. With an infrared video camera system, spatial thermal gradients were monitored directly and the temperature at the actual location of the cells could be estimated accurately. This enabled a precise determination of the strength of thermal stimuli. With this apparatus, we were able to simultaneously measure temperature and observe cellular behavior directly. This feature permits quantitative studies on stimulus-response relationships. The utility of the apparatus was demonstrated by thermotaxis assay under a spatial thermal gradient in polymorphonuclear leukocytes. Since this apparatus can also provide temporal thermal gradients, it may have several applications in studies of temperature-dependent phenomena in cell biology.