A new UV-visible confocal laser scanning microspectrofluorometer designed for spectral cellular imaging

With the aim to perform spectroscopic studies and spectral images inside living cells, a microspectrofluorometer has been designed for two-dimensional spectral imaging in the visible and in the near-UV region. The main advantage of the device relies on its ability to scan the laser beam along one direction of the sample. This scanning is optically coupled with one direction of a bidimensional detector, allowing an instantaneous recording of a one-dimensional spectral image. The overall scanning of the sample is achieved by means of submicrometric displacements of the stage in the perpendicular direction. The main characteristics and performances of the microspectrofluorometer in terms of sensitivity (detection of a few molecules), spatial resolution (0.5 x 0.5 x 1 microm), and spectral resolution (1 nm) are presented. Finally, applications of this new apparatus concerning in situ localization and spectral characterization of two dyes are shown with Drosophila salivary glands (ethidium bromide) and T47D tumor cells (Hoechst 33342).     

Performance validation of an improved Xenon-arc lamp-based CCD camera system for multispectral imaging in proteomics

Advances in gel-based nonradioactive protein expression and PTM detection using fluorophores has served as the impetus for developing analytical instrumentation with improved imaging capabilities. We describe a CCD camera-based imaging instrument, equipped with both a high-pressure Xenon arc lamp and a UV transilluminator, which provides broad-band wavelength coverage (380-700 nm and UV). With six-position filter wheels, both excitation and emission wavelengths may be selected, providing optimal measurement and quantitation of virtually any dye and allowing excellent spectral resolution among different fluorophores. While spatial resolution of conventional fixed CCD camera imaging systems is typically inferior to laser scanners, this problem is circumvented with the new instrument by mechanically scanning the CCD camera over the sample and collecting multiple images that are subsequently automatically reconstructed into a complete high-resolution image. By acquiring images in succession, as many as four different fluorophores may be evaluated from a gel. The imaging platform is suitable for analysis of the wide range of dyes and tags commonly encountered in proteomics investigations. The instrument is unique in its capabilities of scanning large areas at high resolution and providing accurate selectable illumination over the UV/visible spectral range, thus maximizing the efficiency of dye multiplexing protocols.     

Concepts in imaging and microscopy. Exploring biological structure and function with confocal microscopy

Confocal microscopy is providing new and exciting opportunities for imaging cell structure and physiology in thick biological specimens, in three dimensions, and in time. The utility of confocal microscopy relies on its fundamental capacity to reject out-of-focus light, thus providing sharp, high-contrast images of cells and subcellular structures within thick samples. Computer controlled focusing and image-capturing features allow for the collection of through-focus series of optical sections that may be used to reconstruct a volume of tissue, yielding information on the 3-D structure and relationships of cells. Tissues and cells may also be imaged in two or three spatial dimensions over time. The resultant digital data, which encode the image, are highly amenable to processing, manipulation and quantitative analyses. In conjunction with a growing variety of vital fluorescent probes, confocal microscopy is yielding new information about the spatiotemporal dynamics of cell morphology and physiology in living tissues and organisms. Here we use mammalian brain tissue to illustrate some of the ways in which multidimensional confocal fluorescence imaging can enhance studies of biological structure and function.     

VBA开发动物行为实时记录系统及实例

对动物行为进行实时记录是行为学研究的一项重要工作。传统的行为观察方法常常无法满足实时精确记录行为的要求 ,而一些现代的行为记录系统虽功能强大但却难以推广。作者应用VBA开发了一个简捷方便的行为实时记录系统 ,可以减少记录动物行为观察的工作量 ,降低记录动物行为的观察误差 ,保证了实验数据的可靠性。同时 ,该系统具有开放性和灵活性的特点 ,可以在不同动物行为研究中应用。     

A general computational framework for modeling cellular structure and function.

The "Virtual Cell" provides a general system for testing cell biological mechanisms and creates a framework for encapsulating the burgeoning knowledge base comprising the distribution and dynamics of intracellular biochemical processes. It approaches the problem by associating biochemical and electrophysiological data describing individual reactions with experimental microscopic image data describing their subcellular localizations. Individual processes are collected within a physical and computational infrastructure that accommodates any molecular mechanism expressible as rate equations or membrane fluxes. An illustration of the method is provided by a dynamic simulation of IP3-mediated Ca2+ release from endoplasmic reticulum in a neuronal cell. The results can be directly compared to experimental observations and provide insight into the role of experimentally inaccessible components of the overall mechanism.     

New red-fluorescent calcium indicators for optogenetics,photoactivation and multi-color imaging

Most chemical and, with only a few exceptions, all genetically encoded fluorimetric calcium (Ca²⁺) indicators (GECIs) emit green fluorescence. Many of these probes are compatible with red-emitting cell- or organelle markers. But the bulk of available fluorescent-protein constructs and transgenic animals incorporate green or yellow fluorescent protein (GFP and YFP respectively). This is, in part, not only heritage from the tendency to aggregate of early-generation red-emitting FPs, and due to their complicated photochemistry, but also resulting from the compatibility of green-fluorescent probes with standard instrumentation readily available in most laboratories and core imaging facilities. Photochemical constraints like limited water solubility and low quantum yield have contributed to the relative paucity of red-emitting Ca²⁺ probes compared to their green counterparts, too. The increasing use of GFP and GFP-based functional reporters, together with recent developments in optogenetics, photostimulation and super-resolution microscopies, has intensified the quest for red-emitting Ca²⁺ probes. In response to this demand more red-emitting chemical and FP-based Ca²⁺-sensitive indicators have been developed since 2009 than in the thirty years before.     

Contact x-ray microscopy. A new technique for imaging cellular fine structure.

Contact x-ray microscopy potentially allows living, wet cells to be visualized at a resolution of up to 100 A. Furthermore, differential absorption by specific elements permits the study of the distribution of those elements in biological specimens. In contact x-ray microscopy, soft x-rays (10 A to 100 A) pass through a biological sample and expose an underlying x-ray sensitive polymer (resist), producing an image that reflects the photon absorbance within the specimen. The high penetrating power of soft x-ray enables images to be obtained from specimens up to several microns thick. In this paper, the technique is described, some of the areas currently under study are considered, and biological examples of the use of contact x-ray microscopy are given.     

A comparison of mercury arc lamp and laser illumination for flow cytometers.

Optical differences between a mercury arc lamp and a laser-illuminated flow cytometer are compared. The distributions of spectral intensities of the two light sources are shown in relation to the excitation characteristics of the fluorescent dyes acriflavine, chromomycin A3, mithramycin, ethidium bromide, Hoechst 33258, and 4,6-diamidino-2-phenylindole (DAPI). Fluorescence intensities of microspheres and Hoechst 33258-stained mouse sperm are compared in the two cytometers. The optical efficiencies are similar and depend on the match of the excitation characteristics of the stain with the emission spectra of the light source.     

A novel multisite confocal system for rapid Ca2+ imaging from submicron structures in brain slices

In brain slices, resolving fast Ca²⁺ fluorescence signals from submicron structures is typically achieved using 2‐photon or confocal scanning microscopy, an approach that limits the number of scanned points. The novel multiplexing confocal system presented here overcomes this limitation. This system is based on a fast spinning disk, a multimode diode laser and a novel high‐resolution CMOS camera. The spinning disk, running at 20 000 rpm, has custom‐designed spiral pattern that maximises light collection, while rejecting out‐of‐focus fluorescence to resolve signals from small neuronal compartments. Using a 60× objective, the camera permits acquisitions of tens of thousands of pixels at resolutions of ~250 nm per pixel in the kHz range with 14 bits of digital depth. The system can resolve physiological Ca²⁺ transients from submicron structures at 20 to 40 μm below the slice surface, using the low‐affinity Ca²⁺ indicator Oregon Green BAPTA‐5N. In particular, signals at 0.25 to 1.25 kHz were resolved in single trials, or through averages of a few recordings, from dendritic spines and small parent dendrites in cerebellar Purkinje neurons. Thanks to an unprecedented combination of temporal and spatial resolution with relatively simple implementation, it is expected that this system will be widely adopted for multisite monitoring of Ca²⁺ signals.      

Subcellular heterogeneity of mitochondrial function and dysfunction: Evidence obtained by confocal imaging

Beyond their fundamental role in energy metabolism, mitochondria perform a great variety of other important functions (e.g. in Ca2+ homeostasis, apoptosis, thermogenesis, etc.), thus suggesting their region-specific specializations and intracellular heterogeneity. Although mitochondrial functional heterogeneity has been demonstrated for several cell types, its origin and role under physiological and, in particular, pathophysiological conditions, where the extent of heterogeneity may significantly increase, remain to be elucidated. The present work thus investigated the static and dynamic heterogeneity of mitochondria and mitochondrial function in various cell types in which mitochondria may cope with specific functions including cardiomyocytes, hepatocytes and some cultured carcinoma cells. Modern confocal and two-photon fluorescent microscopy was used for the investigation and direct imaging of region-specific mitochondrial function and heterogeneity. Analysis of the autofluorescence of mitochondrial flavoproteins in hepatocytes and carcinoma cells permitted significant intracellular heterogeneity of mitochondrial redox state to be demonstrated. Comparative homogeneity and clear colocalization of mitochondrial flavoproteins, membrane potential and calcium-sensitive probes were observed in both isolated cardiomyocytes and permeabilized myocardial fibers. After ischemia reperfusion, however, or under conditions of substrate deprivation, significant heterogeneity of all these parameters was detected. Some methodological issues, mechanistic aspects, possible metabolic consequences of mitochondrial functional heterogeneity and its impact under pathological conditions are discussed.     

Nestin structure and predicted function in cellular cytoskeletal organisation

Nestin is an intermediate filament protein expressed in dividing cells during the early stages of development in the CNS, PNS and in myogenic and other tissues. Upon differentiation, nestin becomes downregulated and is replaced by tissue-specific intermediate filament proteins. Interestingly, nestin expression is reinduced in the adult during pathological situations, such as the formation of the glial scar after CNS injury and during regeneration of injured muscle tissue. Although it is utilised as a marker of proliferating and migrating cells very little is known about its functions or regulation. In depth studies on the distribution and expression of nestin in mitotically active cells indicate a complex role in regulation of the assembly and disassembly of intermediate filaments which together with other structural proteins, participate in remodeling of the cell. The role of nestin in dynamic cells, particularly structural organisation of the cell, appears strictly regulated by phosphorylation, especially its integration into heterogeneous intermediate filaments together with vimentin or alpha-internexin.     

A real time TV to computer image input system using run coding.

Run coding applied to the digitized video signal from a TV scan of cell preparations can effect a substantial reduction in the total amount of data, sufficient to permit a moderate size of store to be loaded within one frame time with a representation of the field adequate for computer analysis. This paper describes the design of a run coding interface between a TV scanner and a computer store which also allows control of scan domain, spatial resolution and density resolution. Results are presented showing its efficiency when dealing with cervical smear preparations.     

A handheld real time thermal cycler for bacterial pathogen detection

The handheld advanced nucleic acid analyzer (HANAA) is a portable real time thermal cycler unit that weighs under 1 kg and uses silicon and platinum-based thermalcycler units to conduct rapid heating and cooling of plastic reaction tubes. Two light emitting diodes (LED) provide greater than 1 mW of electrical power at wavelengths of 490 nm (blue) and 525 nm (green), allowing detection of the dyes FAM and JOE/TAMRA. Results are displayed in real time as bar graphs, and up to three, 4-sample assays can be run on the charge of the 12 V portable battery pack. The HANAA was evaluated for detection of defined Escherichia coli strains, and wild-type colonies isolated from stream water, using PCR for the lac Z and Tir genes. PCR reactions using SYBR Green dye allowed detection of E. coli ATCC 11775 and E. coli O157:H7 cells in under 30 min of assay time; however, background fluorescence associated with dye binding to nonspecific PCR products was present. DNA extracted from three isolates of Bacillus anthracis Ames, linked to a bioterrorism incident in Washington DC in October 2001, were also successfully tested on the HANAA using primers for the vrrA and capA genes. Positive results were observed at 32 and 22 min of assay time, respectively. A TaqMan probe specific to the aroQ gene of Erwinia herbicola was tested on the HANAA and when 500 cells were used as template, positive results were observed after only 7 min of assay time. Background fluorescence associated with the use of the probe was negligible. The HANAA is unique in offering real time PCR in a handheld format suitable for field use; a commercial version of the instrument, offering six reaction chambers, is available as of Fall 2002.     

A digital microscope for real time detection of moving microorganisms

A digital microscope which can store time sequences in order to analyse moving objects, without the aid of a videorecorder, is described. This digital microscope can also be used to highlight moving microorganisms, such as protozoa, by suppressing the images of any stationary object, such as particles of dirt, dead or immobile cells. This microscope which operates in real time, is equipped with such standard devices as a TV-camera and a digital frame grabber with hardware facilities (Zoom, Pan, Scroll).     

A real time learning algorithm for recurrent analog neural networks

A new learning algorithm is described for a general class of recurrent analog neural networks which ultimately settle down to a steady state. Recently, Pineda (Pineda 1987; Almeida 1987; Ikeda et al. 1988) has introduced a learning rule for the recurrent net in which the connection weights are adjusted so that the distance between the stable outputs of the current system and the desired outputs will be maximally decreased. In this method, many cycles are needed in order to get a target system. In each cycle, the recurrent net is run until it reaches a stable state. After that, the weight change is calculated by using a linearized recurrent net which receives the current error of the system as a bias input. In the new algorithm the weights are changed so that the total error of neuron outputs over the entire trajectory is minimized. The weights are adjusted in real time when the network is running. In this method, the trajectory to the target system can be controlled, whereas Pineda's algorithm only controls the position of the fixed point. The relation to the back propagation method (Hinton et al. 1986) is also discussed.