bluuebunny/crossref_metadata_2025_split huggingface dataset:
An automatic procedure for evaluation of single cell motility
Authors: Dmytriyev Anatoliy , Tkach Vadim , Rudenko Olga , Bock Elisabeth , Berezin Vladimir 
Published: September 2006

AbstractBackground:Cell motility is vital in many physiological and pathological processes, such as embryogenesis, inflammation, wound healing, and metastasis. However, the time‐consuming step in the evaluation of individual cell motility is the analysis of hundreds of recorded images of cell cultures in general consisting of retrieving images, one at a time, and marking the positions of individual cells by a computer mouse. Therefore, the aim of the present study was to develop a novel automatic procedure for the evaluation of cell motility.Materials and Methods:The procedure was tested on fibroblasts and glioma and adenocarcinoma cells engineered to express the green fluorescent protein by either transient transfection or adenovirus transduction, allowing automatic recognition of cell coordinates on retrieved images.Results:The effects of serum growth factors, teratogenic compounds, and overexpression of transcription factors on the motile behavior of cultured cells were determined. Cell motility was estimated by both manual and automatic marking of cell position and subsequently motility parameters were computed. The results obtained by the two procedures were found to correlate significantly.Conclusions:We developed a procedure allowing automatic video recording of sparsely seeded cells transfected with a plasmid or tranduced with a recombinant virus expressing the enhanced green fluorescent protein (EGFP). © 2006 International Society for Analytical Cytology
Spectral imaging microscopy web sites and data
Authors: McNamara George , Gupta Amit , Reynaert James , Coates Thomas D. , Boswell Carl 
Published: September 2006

AbstractThe Internet is enabling greater access to spectral imaging publications, spectral graphs, and data than that was available a generation ago. The spectral imaging systems discussed in this issue of Cytometry work because reagent and hardware spectra are reproducible, reusable, and provide input to spectral unmixing and spectral components recognition algorithms. These spectra need to be readily available in order to determine what to purchase, how to use it, and what the output means. We refer to several commercially sponsored and academic spectral web sites and discuss our spectral graphing and data sites. Sites include fluorescent dye graph servers from Invitrogen/Molecular Probes, BD Biosciences, Zeiss/Bio‐Rad Cell Sciences, and filter set servers from Chroma Technology and Omega Optical. Several of these sites include data download capabilities. Recently, two microscope manufacturers have published on their web sites transmission curves for select objective lenses—crucial data for anyone doing multiphoton excitation microscopy. Notable among the academic sites, PhotoChemCAD 2.0 has over 200 dyes and a downloadable database/graphing program, and the USC‐A Chemistry UV–vis Database displays absorption spectra of many dyes and indicators used in clinical histology and pathology. Our Fluorescent Spectra graphing/calculator site presents dyes, filters, and illumination data from many of these and additional sources. PubSpectra is our free download site which uses Microsoft Excel files as standardized human/machine readable format with over 2,000 biomedical spectra. The principle that data is not subject to copyright provides a framework in which all scientific data should be made freely accessible. © 2006 International Society for Analytical Cytology
The Nikon C1si combines high spectral resolution, high sensitivity, and high acquisition speed
Authors: Larson Jeffrey M. 
Published: September 2006

AbstractSpectral imaging is a natural extension of the capabilities of confocal microscopes. The first confocal spectral imaging (CSI) instruments were able to acquire spectral data that allowed the emissions of overlapping fluorescent probes to be assigned to data channels representing a spectrum rather than a range of emission wavelengths. This marked a significant improvement over what could be done by channel series with standard confocal microscopes. However the performance of these earlier designs can fall short in one or more of the following areas; sensitivity, spectral resolution and reproducibility, acquisition speed, and unmixing accuracy. Nikon has recently introduced a new CSI instrument, C1si, that overcomes some of the more serious performance deficiencies of earlier designs through unique optical, electronic, and data handling advances. C1si uses a multianode photomultiplier tube (PMT) as the detector and typically acquires spectral data in a single scan. Sensitivity is enhanced over designs diffracting randomly polarized fluorescence by rotating the polarization of all emission photons to the S‐plane, the plane for which the diffraction grating is most efficient. Three diffraction gratings are provided supporting wavelength sampling increments of 2.5, 5, and 10 nm. Improvements have been made in the digitization process to increase detection efficiency as well. C1si is calibrated to a high enough standard that it is possible to share and reproduce data between instruments. The algorithm implemented in the EZ‐C1 software is able to accurately and repeatedly unmix fluorescent probes with emission peaks separated by as little as 5 nm. It is possible to unmix probes with emission peaks separated by 20 nm with a 10–1 brightness difference. Three probes can be unmixed with emission peaks contained within a 20 nm range. Acquisition is fast enough and the sensitivity is sufficient for C1si to acquire more than 100 frames of spectral time series data without serious photobleaching. © 2006 International Society for Analytical Cytology
Spectral mapping tools from the earth sciences applied to spectral microscopy data
Authors: Harris A. Thomas 
Published: September 2006

AbstractBackground:Spectral imaging, originating from the field of earth remote sensing, is a powerful tool that is being increasingly used in a wide variety of applications for material identification. Several workers have used techniques like linear spectral unmixing (LSU) to discriminate materials in images derived from spectral microscopy. However, many spectral analysis algorithms rely on assumptions that are often violated in microscopy applications. This study explores algorithms originally developed as improvements on early earth imaging techniques that can be easily translated for use with spectral microscopy.Methods:To best demonstrate the application of earth remote sensing spectral analysis tools to spectral microscopy data, earth imaging software was used to analyze data acquired with a Leica confocal microscope with mechanical spectral scanning. For this study, spectral training signatures (often referred to as endmembers) were selected with the ENVI (ITT Visual Information Solutions, Boulder, CO) “spectral hourglass” processing flow, a series of tools that use the spectrally over‐determined nature of hyperspectral data to find the most spectrally pure (or spectrally unique) pixels within the data set. This set of endmember signatures was then used in the full range of mapping algorithms available in ENVI to determine locations, and in some cases subpixel abundances of endmembers.Results:Mapping and abundance images showed a broad agreement between the spectral analysis algorithms, supported through visual assessment of output classification images and through statistical analysis of the distribution of pixels within each endmember class.Conclusions:The powerful spectral analysis algorithms available in COTS software, the result of decades of research in earth imaging, are easily translated to new sources of spectral data. Although the scale between earth imagery and spectral microscopy is radically different, the problem is the same: mapping material locations and abundances based on unique spectral signatures. © 2006 International Society for Analytical Cytology
Aged mice exhibit distinct peripheral B‐cell phenotypes differing in apoptotic susceptibility: An ex vivo analysis
Authors: Verdier Mireille , Malissein Emilie , Munteanu Eliza , Jayat‐Vignoles Chantal , Ratinaud Marie‐Hélène , Troutaud Danielle 
Published: September 2006

AbstractBackground:Age‐related changes in the antibody response have been classically associated with alterations in T‐cell help, but increasing evidence shows that intrinsic B‐cell defects exist. This article analyzes the apoptotic susceptibility of peripheral B‐cells in aged and young control mice.Materials and Methods:Freshly isolated lymphocytes from spleen and Peyer's patches (PPs) were labeled for B‐cell lineage (B220+ cells) and germinal center B subset (GCs, B220+/PNA+ cells). Alternatively, splenic B‐cells purified by MACS were used. Apoptosis was monitored by the Annexin V binding, incorporation of 3,3′‐dihexyloxacarbocyanine iodide (DiOC6(3)), propidium iodide (PI) staining, and morphological changes. Moreover, intracellular Bcl‐2 expression and Bad phosphorylation status were also analyzed in B‐cells.Results:We showed in aging mice an enhanced Annexin V+/PI− cell percentage in splenic B‐lymphocytes, which was correlated with a lower ΔΨm. By contrast, no change in apoptosis was observed in compartments known to be enriched in activated B‐cells (GCs and PPs). Analysis of Bcl‐2 levels revealed no modification. When using B‐cells purified by MACS, we strongly confirm data obtained on staining cells. Moreover, enhanced spontaneous apoptosis of splenic B‐cells in aged mice was found to be correlated with a reduced phosphorylated Bad expression.Conclusion:Increased apoptosis of resting B‐cells in old mice may be determined by an altered Bad phosphorylation, which in turn contributes to cell death by lowering the mitochondrial threshold for apoptosis. © 2006 International Society for Analytical Cytology
Spectral imaging: Principles and applications
Authors: Garini Yuval , Young Ian T. , McNamara George 
Published: September 2006

AbstractBackground:Spectral imaging extends the capabilities of biological and clinical studies to simultaneously study multiple features such as organelles and proteins qualitatively and quantitatively. Spectral imaging combines two well‐known scientific methodologies, namely spectroscopy and imaging, to provide a new advantageous tool. The need to measure the spectrum at each point of the image requires combining dispersive optics with the more common imaging equipment, and introduces constrains as well.Methods and Results:The principles of spectral imaging and a few representative applications are described. Spectral imaging analysis is necessary because the complex data structure cannot be analyzed visually. A few of the algorithms are discussed with emphasis on the usage for different experimental modes (fluorescence and bright field). Finally, spectral imaging, like any method, should be evaluated in light of its advantages to specific applications, a selection of which is described.Conclusions:Spectral imaging is a relatively new technique and its full potential is yet to be exploited. Nevertheless, several applications have already shown its potential. © 2006 International Society for Analytical Cytology
DNA methylation in stingless bees with low and high heterochromatin contents as assessed by restriction enzyme digestion and image analysis
Authors: Mampumbu André R. , Mello Maria Luiza S. 
Published: September 2006

AbstractBackground:The stingless bee genus Melipona has been divided into two groups, based on their heterochromatin content. Melipona quadrifasciata and Melipona rufiventris have low and high levels of heterochromatin, respectively. Since condensed chromatin may be rich in methylated DNA sequences, M. quadrifasciata and M. rufiventris nuclei may contain different amounts of methylated CpG. These differences could be assessed by comparing Feulgen‐DNA values obtained by image analysis of cells treated with the restriction enzymes Msp I and Hpa II that distinguish between methylated and unmethylated DNA. Msp I and Hpa II cleave the sequence ‐CCGG‐, but there is no cleavage by Hpa II if the cytosine of the central CG dinucleotide is methylated.Methods:Malpighian tubules of M. quadrifasciata and M. rufiventris were treated with Msp I and Hpa II prior to the Feulgen reaction, and analyzed by automatic scanning microspectrophotometry.Results:The Feulgen‐DNA values for the heterochromatin of M. rufiventris and for the small heterochromatin and some euchromatin domains of M. quadrifasciata mostly decreased after treatment with Msp I, but were unchanged after treatment with Hpa II.Conclusion:CpG methylation, although detected in diverse chromatin compartments in different bee species, may induce silencing effects required for the same cell physiology. © 2006 International Society for Analytical Cytology
Confocal laser scanning microscopy of whole mouse ovaries: Excellent morphology, apoptosis detection, and spectroscopy
Authors: Zucker Robert M. , Jeffay Susan C. 
Published: September 2006

AbstractBackground:Ovaries consist of numerous follicles, oocytes, and granulosa cells in different stages of development. Many of these follicles will undergo an apoptotic process during the lifetime of the animal. By using proper tissue preparation methods, the events within the whole ovary can be observed by using 3D confocal microscopy.Methods:Whole ovaries were stained with LysoTracker Red (LT), fixed with 4% paraformaldehyde (PF) and 1% glutaraldehyde (Glut), dehydrated with methanol (MEOH), and cleared with benzyl alcohol and benzyl benzoate (BABB). Using this tissue preparation technique, the ovary becomes relatively transparent, allowing its morphology to be observed with confocal microscopes. A spectral imaging system (PARISS) located on a conventional microscope was used to interpret the LT dye spectra and fixation products in the tissues with different excitation wavelengths.Results:Apoptosis in the follicle was detected as clusters of intensely stained granulosa cells located in close proximity to the oocytes. The fixation with Glut and PF preserved morphological details, increased tissue fluorescence, thus increased the signal to noise of the background image.Conclusions:Thick tissues can be imaged after they are properly stained, aldehyde fixed, and BABB cleared. LT intensely stained single cells or clusters of apoptotic cells in the follicles and the nucleolus. Spectral differences between LT as an indicator of apoptosis and Glut‐PF fixation was used to visualize ovarian morphology and apoptosis. The PARISS spectrophotometer revealed spectral peaks for LT at 609.6 nm and for Glut‐PF at 471.3 nm. The proper use of the spectra from these fluorescence molecules is the foundation for high quality morphological images of apoptosis. By sequentially imaging the two probes with a 488 nm laser and a 543/568 nm laser, there was a reduction in fluorescent cross talk and an increase in image quality. © 2006 International Society for Analytical Cytology
Increasing lanthanide luminescence by use of the RETEL effect
Authors: Leif Robert C. , Vallarino Lidia M. , Becker Margie C. , Yang Sean 
Published: September 2006

AbstractBackground:Luminescent lanthanide complexes produce emissions with the narrowest‐known width at half maximum; however, their significant use in cytometry required an increase in luminescence intensity. The companion review, Leif et al., Cytometry 2006;69A:767–778, described a new technique for the enhancement of lanthanide luminescence, the Resonance Energy Transfer Enhanced Luminescence (RETEL) effect, which increases luminescence and is compatible with standard slide microscopy.Methods:The luminescence of the europium ion macrocyclic complex, EuMac, was increased by employing the RETEL effect. After adding the nonluminescent gadolinium ion complex of the thenoyltrifluoroacetonate (TTFA) ligand or the sodium salt of TTFA in ethanol solution, the EuMac‐labeled sample was allowed to dry. Both a conventional arc lamp and a time‐gated UV LED served as light sources for microscopic imaging. The emission intensity was measured with a CCD camera. Multiple time‐gated images were summed with special software to permit analysis and effective presentation of the final image.Results:With the RETEL effect, the luminescence of the EuMac‐streptavidin conjugate increased at least six‐fold upon drying. Nuclei of apoptotic cells were stained with DAPI and tailed with 5BrdUrd to which a EuMac‐anti‐5BrdU conjugate was subsequently attached. Time‐gated images showed the long‐lived EuMac luminescence but did not show the short‐lived DAPI fluorescence. Imaging of DNA‐synthesizing cells with an arc lamp showed that both S phase and apoptotic cells were labeled, and that their labeling patterns were different. The images of the luminescent EuMac and fluorescent DAPI were combined to produce a color image on a white background. This combination of simple chemistry, instrumentation, and presentation should make possible the inexpensive use of the lanthanide macrocycles, Quantum Dyes®, as molecular diagnostics for cytological and histopathological microscopic imaging. © 2006 International Society for Analytical Cytology
Single particle high resolution spectral analysis flow cytometry
Authors: Goddard Gregory , Martin John C. , Naivar Mark , Goodwin Peter M. , Graves Steven W. , Habbersett Robb , Nolan John P. , Jett James H. 
Published: September 2006

AbstractBackground:While conventional multiparameter flow cytometers have proven highly successful, there are several types of analytical measurements that would benefit from a more comprehensive and flexible approach to spectral analysis including, but certainly not limited to spectral deconvolution of overlapping emission spectra, fluorescence resonance energy transfer measurements, metachromic dye analysis, free versus bound dye resolution, and Raman spectroscopy.Methods:Our system utilizes a diffraction grating to disperse the collected fluorescence and side‐scattered light from cells or microspheres passing through the interrogation region over a rectangular charge‐coupled‐device image sensor. The flow cell and collection optics are taken from a conventional flow cytometer with minimal modifications to assure modularity of the system.Results:Calibration of the prototype spectral analysis flow cytometer included wavelength characterization and calibration of the dispersive optics. Benchmarking of the system demonstrated a single particle/cell intensity sensitivity of 2160 MESF of R‐Phycoerythrin. Single particle spectra taken with our instrument were validated against bulk solution fluorimeter and conventional flow cytometer measurements. Coefficients of variation of integrated spectral fluorescence intensity of several sets of standard fluorescent microspheres ranged from 1.4 to 4.8% on the spectral system. Spectral discrimination of free versus PI bound to cells is also demonstrated.Conclusions:It is demonstrated that the flow spectrometer has sufficient sensitivity and wavelength resolution to detect single cells and microspheres, including multi‐fluorophore labeled microspheres. The capability to use both standard mathematical deconvolution techniques for data analysis, coupled with the feasibility of integration with existing flow cytometers, will improve the accuracy and precision of ratiometric measurements, enable the analysis of more discrete emission bands within a given wavelength range, and allow more precise resolution of the relative contribution of individual fluorophores in multiply‐tagged samples, thereby enabling a range of new applications involving the spectral analysis of single cells and particles. © 2006 International Society for Analytical Cytology
Increasing the luminescence of lanthanide complexes
Authors: Leif Robert C. , Vallarino Lidia M. , Becker Margie C. , Yang Sean 
Published: September 2006

AbstractThis review compares the chemical and physical properties of lanthanide ion complexes and of other narrow‐emitting species that can be used as labels for cytometry. A series of luminescent lanthanide ion macrocyclic complexes, Quantum Dyes®, which do not release or exchange their central lanthanide ion, do accept energy transfer from ligands, and are capable of covalent binding to macromolecules, including proteins and nucleic acids, is described and their properties are discussed.Two methods are described for increasing the luminescence intensity of lanthanide ion complexes, which intrinsically is not as high as that of standard fluorophores or quantum dots. One method consists of adding a complex of a second lanthanide ion in a micellar solution (columinescence); the other method produces dry preparations by evaporation of a homogeneous solution containing an added complex of a second lanthanide ion or an excess of an unbound antenna ligand. Both methods involve the Resonance Energy Transfer Enhanced Luminescence, RETEL, effect as the mechanism for the luminescence enhancement. © 2006 International Society for Analytical Cytology
Factors affecting flow karyotype resolution
Authors: Ng Bee Ling , Carter Nigel P. 
Published: September 2006

AbstractBackground:One of the major factors which influences the chromosome purity achievable particularly during high speed sorting is the analytical resolution of individual chromosome peaks in the flow karyotype, as well as the amount of debris and fragmented chromosomes. We have investigated the factors involved in the preparation of chromosome suspensions that influence karyotype resolution.Methods:Chromosomes were isolated from various human and animal cell types using a series of polyamine buffer isolation protocols modified with respect to pH, salt concentration, and chromosome staining time. Each preparation was analyzed on a MoFlo sorter (DAKO) configured for high speed sorting and the resolution of the flow karyotypes compared.Results:High resolution flow cytometric data was obtained with chromosomes optimally isolated using hypotonic solution buffered at pH 8.0 and polyamine isolation buffer (with NaCl excluded) between pH 7.50 and 8.0. Extending staining time to more than 8 h with chromosome suspensions isolated from cell lines subjected to sufficient metaphase arrest times gave the best result with the lowest percentage of debris generated, tighter chromosome peaks with overall lower coefficients of variation, and a 1‐ to 5‐fold increase in the yield of isolated chromosomes.Conclusions:Optimization of buffer pH and the length of staining improved karyotype resolution particularly for larger chromosomes and reduced the presence of chromosome fragments (debris). However, the most interesting and surprising finding was that the exclusion of NaCl in PAB buffer improved the yield and resolution of larger chromosomes. © 2006 International Society for Analytical Cytology