Sequential immunofluorescence staining and image analysis for detection of large numbers of antigens in individual cell nuclei.

BACKGROUND: Visualization of more than one antigen by multicolor immunostaining is often desirable or even necessary to explore spatial and temporal relationships of functional significance. Previously presented staining protocols have been limited to the visualization of three or four antigens. METHODS: Immunofluorescence staining was performed both on slices of formalin-fixed tissue and on cells in culture. Images of the stained material were recorded using digital imaging fluorescence microscopy. The primary and secondary antibodies, as well as the fluorophores, were thereafter removed using a combination of denaturation and elution techniques. After removal of the fluorescence stain, a new immunofluorescence staining was performed, visualizing a new set of antigens. The procedure was repeated up to three times. A method for image registration combined with segmentation, extraction of data, and cell classification was developed for efficient and objective analysis of the image data. RESULTS: The results show that immunofluorescence stains in many cases can be repeatedly removed without major effects on the antigenicity of the sample. CONCLUSIONS: The concentration of at least six different antigens in each cell can thus be measured semiquantitatively using sequential immunofluorescence staining and the described image analysis techniques. The number of antigens that can be visualized in a single sample is considerably increased by the presented protocol.     

Light element analysis of individual bacteria by x-ray microanalysis

A method based on X-ray microanalysis (XRMA) with the transmission electron microscope for measurement of total amounts of elements in single microbial cells has been developed. All major elements in cells except hydrogen can be measured simultaneously. XRMA provided N/C ratios (means (plusmn) standard errors of the mean) for stationary-phase and growing Escherichia coli of 0.23 (plusmn) 0.01 and 0.30 (plusmn) 0.01, respectively, while CHN analysis gave values of 0.276 and 0.307, respectively, for samples from the same cultures. Analyses of free coccoliths from Emiliana huxleyi provided weight fractions close to those of CaCO(inf3): 0.35 (plusmn) 0.01, 0.15 (plusmn) 0.01, and 0.47 (plusmn) 0.01 for calcium, carbon, and oxygen, respectively. Calibration is based on monodisperse latex beads and on microdrops of defined compounds. Elements in particles in the size range from 5 fg to 500 pg are measured with a relative precision between 500 and 5,000 ppm, depending on size. As a single-cell method, XRMA avoids the shortcomings of commonly used fractionation techniques associated with bulk methods, which are based on centrifugation or filtration. On the basis of morphology and XRMA, particles may be classified more precisely into groups (e.g., biotic versus abiotic) than is possible by bulk methods. Single-cell elemental analysis may provide insight into topics like nutritional and energetic status, macromolecular composition, and (by multivariate statistics) community structure.     

A novel method for measuring lag times in division of individual bacterial cells using image analysis

A method is presented for determining the time to first division of individual bacterial cells growing on agar media. Bacteria were inoculated onto agar-coated slides and viewed by phase-contrast microscopy. Digital images of the growing bacteria were captured at intervals and the time to first division estimated by calculating the "box area ratio". This is the area of the smallest rectangle that can be drawn around an object, divided by the area of the object itself. The box area ratios of cells were found to increase suddenly during growth at a time that correlated with cell division as estimated by visual inspection of the digital images. This was caused by a change in the orientation of the two daughter cells that occurred when sufficient flexibility arose at their point of attachment. This method was used successfully to generate lag time distributions for populations of Escherichia coli, Listeria monocytogenes and Pseudomonas aeruginosa, but did not work with the coccoid organism Staphylococcus aureus. This method provides an objective measure of the time to first cell division, whilst automation of the data processing allows a large number of cells to be examined per experiment.     

Monitoring hairy-root growth by image analysis

Hairy roots, incited byAgrobacterium rhizogenes, form a useful system for analysing the expression and phenotypic effects of foreign genes in plant root tissue. Image analysis offers a non-invasive method of describing their growth in culture. Images of pea (coarse) andBrassica (fine) hairy roots were captured, processed and analysed without difficulty using a commercially available image analysis system. The value of this method in monitoring intermediate changes in growth pattern was illustrated by following the changes in five putatively chlorsulfuron-resistantBrassicc hairy-root lines cultured with and without a selective level of chlorsulfuron. Areas of hairy-root research where this technique will be particularly useful are discussed.     

Location of respiration activity in filamentous bacteria by image analysis

Respiration activity was located in Streptomyces ambofaciens using formazan crystals and image analysis. The technique was validated by comparing it to a global respiration rate measurement method. A uniform distribution of formazan crystals along the hyphae was found in cultivations in flasks and fermenter, throughout the cultures, although respiration is globally decreased when cultures age.     

Quantitation of specific parameters of motility in large numbers of human sperm by digital image processing

The CellSoft computer-assisted digital image analysis system was validated for quantitating specific motility parameters in large numbers of human sperm. Motility patterns ranging from linear head trajectories (Type 1) to nonlinear, asymmetric patterns with overlapping trajectory (Type 5) were subjectively identified in semen and washed samples prepared for in vitro fertilization. A representative of each type was used for optimizing the digital imaging set-up parameters, tracking rate, and frequency. Each cell type was also characterized according to the following motility parameters: curvilinear velocity (Vcl), straight line velocity (Vsl), linearity of forward progression (Lin), maximum and mean lateral head amplitude (maxLHA; mean LHA), and beat cross frequency (BCF). Comparison of all parameters that could be determined both digitally and manually (Vcl, Vsl, Lin, and BCF) indicated no differences (p greater than 0.05) in Vcl, Lin, or BCF and only slight differences (5-6%) in Vsl measurements. After validation of the digital imaging technique, populations of seminal and washed cells were studied. Replicate analysis of the same sample demonstrated no significant intraassay variability. A comparison of semen and washed cells from 10 different donors indicated that all of the motility parameters, with the exception of Lin, were significantly higher (p less than 0.05) in washed cells. It was concluded that the digital imaging system can adequately and rapidly quantitate a large number of cells with heterogeneous motility patterns. This technique may prove to be useful in defining motility characteristics associated with capacitation, the acrosome reaction, and fertility of human sperm.     

The determination of growth rates of individual colonies in agarose using high-resolution automated image analysis

This paper describes the evaluation of a colony formation assay using automated image analysis, which permits the tracking of growth at the individual colony level, such that a growth rate can be estimated for each colony followed. In principle, this will permit quantitative characterization of cellular heterogeneity in growth rate and cellular heterogeneity in response to proliferation-modifying agents. In addition, we have demonstrated the possibility of using correlative microscopy to relate growth rate to other parameters, using metabolic viability as an example. This should be useful for determining cellular characteristics associated with proliferative behavior and response to proliferation-modifying agents.     

Biodegradation of sulfamethoxazole by individual and mixed bacteria

Antibiotic compounds, like sulfamethoxazole (SMX), have become a concern in the aquatic environment due to the potential development of antibacterial resistances. Due to excretion and disposal, SMX has been frequently detected in wastewaters and surface waters. SMX removal in conventional wastewater treatment plants (WWTPs) ranges from 0% to 90%, and there are opposing results regarding its biodegradability at lab scale. The objective of this research was to determine the ability of pure cultures of individual and mixed consortia of bacteria (Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas putida, Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus rhodocrous, and Rhodococcus zopfii) known to exist in WWTP activated sludge to remove SMX. Results showed that R. equi alone had the greatest ability to remove SMX leading to 29% removal (with glucose) and the formation of a metabolite. Degradation pathways and metabolite structures have been proposed based on the potential enzymes produced by R. equi. When R. equi was mixed with other microorganisms, a positive synergistic effect was not observed and the maximum SMX removal achieved was 5%. This indicates that pure culture results cannot be extrapolated to mixed culture conditions, and the methodology developed here to study the biodegradability of compounds under controlled mixed culture conditions offers an alternative to conventional studies using pure bacterial cultures or inocula from activated sludge sources consisting of unknown and variable microbial populations.     

Determination of yeast glycogen content by individual cell spectroscopy using image analysis

A rapid technique has been developed to determine the glycogen content of yeast on an individual cell basis using a combination of image analysis technology and staining of yeast cells with an I(2):KI solution. Changes in mean cellular glycogen content during alcoholic fermentation have been reported using this technique. The glycogen content of stored brewer's yeast is heterogeneous compared to freshly propagated yeast which have a more uniform distribution of glycogen. Analysis of the distribution of yeast glycogen during fermentation indicates that a fraction of yeast cells do not dissimilate glycogen. Therefore, conventional analysis of the mean glycogen content of yeast used to inoculate fermentations is of limited use, unless information regarding the proportion of cells which utilize glycogen is known. Analysis of the distribution of glycogen within a yeast population can serve as a useful indicator of yeast quality.     

A simple and rapid technique for identification of large numbers of individual mosquitoes using DNA hybridization

A general method for obtaining species-specific repetitive DNA sequences is described. The method is based on the detection of recombinant DNA clones containing repetitive sequences using labeled total genomic DNA. These repetitive DNA sequences can be used to identify individual mosquito adults, pupae, and larvae squashed on filter membranes (squash blots). This technique was used to distinguish individuals of the four sibling species of the Anopheles quadrimaculatus complex. Repetitive DNA sequences and squash blots can be of use for rapid identification of other insect species in field collections.     

Yeast peroxisomes multiply by growth and division

Peroxisomes can arise de novo from the endoplasmic reticulum (ER) via a maturation process. Peroxisomes can also multiply by fission. We have investigated how these modes of multiplication contribute to peroxisome numbers in Saccharomyces cerevisiae and the role of the dynamin-related proteins (Drps) in these processes. We have developed pulse-chase and mating assays to follow the fate of existing peroxisomes, de novo-formed peroxisomes, and ER-derived preperoxisomal structures. We find that in wild-type (WT) cells, peroxisomes multiply by fission and do not form de novo. A marker for the maturation pathway, Pex3-GFP, is delivered from the ER to existing peroxisomes. Strikingly, cells lacking peroxisomes as a result of a segregation defect do form peroxisomes de novo. This process is slower than peroxisome multiplication in WT cells and is Drp independent. In contrast, peroxisome fission is Drp dependent. Our results show that peroxisomes multiply by growth and division under our assay conditions. We conclude that the ER to peroxisome pathway functions to supply existing peroxisomes with essential membrane constituents.     

Nanoscale mapping and functional analysis of individual adhesins on living bacteria

Although much progress has been made in the identification and characterization of adhesins borne by pathogenic bacteria, the molecular details underlying their interaction with host receptors remain largely unknown owing to the lack of appropriate probing techniques. Here we report a method, based on atomic force microscopy (AFM) with tips bearing biologically active molecules, for measuring the specific binding forces of individual adhesins and for mapping their distribution on the surface of living bacteria. First, we determined the adhesion forces between the heparin-binding haemagglutinin adhesin (HBHA) produced by Mycobacterium tuberculosis and heparin, used as a model sulphated glycoconjugate receptor. Both the adhesion frequency and adhesion force increased with contact time, indicating that the HBHA-heparin complex is formed via multiple intermolecular bridges. We then mapped the distribution of single HBHA molecules on the surface of living mycobacteria and found that the adhesin is not randomly distributed over the mycobacterial surface, but concentrated into nanodomains.     

Analysis of growth kinetics by division tracking

Cell division tracking using fluorescent dyes, such as carboxyfluorescein diacetate succinimidyl ester, provides a unique opportunity for analysis of cell growth kinetics. The present review article presents new methods for enhancing resolution of division tracking data as well as derivation of quantities that characterize growth from time-series data. These include the average time between successive divisions, the proportion of cells that survive and the proliferation per division. The physical significance of these measured quantities is interpreted by formulation of a two-compartment model of cell cycle transit characterized by stochastic and deterministic cell residence times, respectively. The model confirmed that survival is directly related to the proportion of cells that enter the next cell generation. The proportion of time that cells reside in the stochastic compartment is directly related to the proliferation per generation. This form of analysis provides a starting point for more sophisticated physical and biochemical models of cell cycle regulation.     

Evolution of cell division in bacteria

Molecular evolution in bacteria is examined with an emphasis on cell division. For a bacterial cell to assemble and then divide required an immense amount of integrated cell and molecular biology structures/functions to be present, such as a stable cellular structure, enzyme catalysis, minimal genome, septum formation at mid-cell and mechanisms to take up nutrients and produce and use energy, as well as store it. The first bacterial cell(s) capable of division must have had complex cell and molecular biology functions. At this stage of evolution, they would not have been primitive cells but would have reached a threshold in evolution where cell division occurred in a regulated manner.     

Recombinant DNA techniques: storage and screening of cDNA libraries with large numbers of individual colonies from initial transformations

A typical cDNA library with a large number of initial transformants, plated in soft agarose, can be stored and shipped in 12% glycerol at -70 degrees C. To prepare the library for storage, the soft agarose layer is made into a paste and the agarose is removed by Sephadex G-25 filtration. This method of cDNA library storage does not alter the relative representation of the plasmids carried in the library. To achieve a very uniform distribution of colonies at high colony density, an aliquot of the cDNA library is diluted to 3000 to 10,000 colonies/ml. One milliliter of this suspension is evenly distributed on a nitrocellulose filter on an agar plate and air-dried. Filter copies are made and screened by published methods.