Preservation of comet assay slides: comparison with fresh slides.

The single cell gel electrophoresis assay (comet assay) is an inexpensive, rapid and highly sensitive method for the determination of DNA damage, crosslinks, and alkaline-labile lesions in individual cells. A limitation of the procedure is that the microelectrophoretic gels must be scored rapidly as the comet configuration deteriorates on storage due to dehydration of the agarose and diffusion of DNA. The objectives of this study were firstly to evaluate drying regimes as rapid and simple methods of preservation of the microgels as close to their original fresh state as possible, and secondly to examine the effects of storage of the slides. Human hepatoma (HepG2) cells challenged for 30 min with hydrogen peroxide (H(2)O(2)) were used in the study. Microgel slides were prepared and evaluated immediately, or after drying with or without a methanol fixation step. Microgels that were dried at a variety of temperatures (22-50 degrees C) and re-hydrated did not differ in the values obtained for H(2)O(2)-induced DNA damage when compared to fresh samples. Samples could also be continually dried and re-hydrated over a period of up to 3 months with no obvious loss of information. In conclusion, drying of microgels represents a simple and inexpensive method of preserving comet assay slides.     

Restoration of broken cytology slides and creation of multiple slides from a single smear preparation.

A technique was developed for restoring broken cytology slides so that they are close to their original condition and for making multiple slides from a single smear preparation. The method is applicable to both cytologic preparations and histologic sections. In this study the fragmented smear preparation was treated with Pro-Texx, which penetrated, impregnated and solidified the full thickness of the pieces of the smear, enabling them to be lifted from the pieces of the broken slide. The removed pieces of the smear preparation were reassembled onto a new slide, which was then restained and coverslipped. In preparing multiple teaching slides, the treated smear preparation was divided as planned, with each portion mounted onto a separate slide, which was then restained and coverslipped. Ten other fine needle aspiration cases with broken slides have been restored, and more teaching slides were prepared from a single smear preparation using the same technique. All were equally successful. This technique provides an excellent method of smear transfer in cases of broken slides and creation of multiple slides from a single smear preparation for cytology teaching. This is particularly useful for unusual cases.     

Protein immobilization on plasma-polymerized ethylenediamine-coated glass slides

For protein chip construction, protein immobilization on the surface of the glass slide is essential. It was previously reported that glass slides are embedded with chemicals that contain primary amines and aldehydes for protein immobilization. We fabricated a plasma-polymerized ethylenediamine (PPEDA)-coated slide that exposed primary amines. For the plasma polymer deposition on the glass slide, the inductively coupled plasma (ICP) power was found to be a critical factor in sustaining a high density of amine on the surface of the PPEDA films. We prepared PPEDA-coated slides at three different ICP powers (3, 30, or 70 W). In the slide that was prepared at a low ICP power (3 W), we detected a high density of primary amine. Therefore, the fluorescein isothiocyanate-conjugated immunoglobulin G (IgG) was highly immobilized to the PPEDA-coated slide that was prepared at the low ICP power. For protein immobilization, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) was used as a cross-linker. The immobilization of the protein to the PPEDA-coated slide was carried out by consecutive incubations with 1 mg/ml EDC for 5 min and 0.1 mg/ml IgG for 1 h. This efficiently produced the functionally active protein-immobilized slide. Therefore, this work shows that the plasma technique can be applied to produce a high-quality glass surface for the immobilization of proteins and other materials.     

Enhanced fluorescence cell imaging with metal-coated slides

Fluorescence labeling is the prevailing imaging technique in cell biology research. When they involve statistical investigations on a large number of cells, experimental studies require both low magnification to get a reliable statistical population and high contrast to achieve accurate diagnosis on the nature of the cells' perturbation. Because microscope objectives of low magnification generally yield low collection efficiency, such studies are limited by the fluorescence signal weakness. To overcome this technological bottleneck, we proposed a new method based on metal-coated substrates that enhance the fluorescence process and improve collection efficiency in epifluorescence observation and that can be directly used with a common microscope setup. We developed a model based on the dipole approximation with the aim of simulating the optical behavior of a fluorophore on such a substrate and revealing the different mechanisms responsible for fluorescence enhancement. The presence of a reflective surface modifies both excitation and emission processes and additionally reshapes fluorescence emission lobes. From both theoretical and experimental results, we found the fluorescence signal emitted by a molecular cyanine 3 dye layer to be amplified by a factor approximately 30 when fluorophores are separated by a proper distance from the substrate. We then adapted our model to the case of homogeneously stained micrometer-sized objects and demonstrated mean signal amplification by a factor approximately 4. Finally, we applied our method to fluorescence imaging of dog kidney cells and verified experimentally the simulated results.     

Enumerating bacterial cells on bioadhesive coated slides

Quantifying bacterial abundance and biomass is fundamental to many microbiological studies. Directly counting via epifluorescence microscopy has become the method of choice, especially for environmental samples, and conventional techniques require filtration of cells onto black polycarbonate membrane filters. We investigated the utility of instead capturing stained bacterial suspensions on bioadhesive slides, performing tests using pure cultures of bacteria, mixtures of cultured bacteria, and environmental samples from five habitat types. When compared to the standard filtration and flow cytometric approaches, bioadhesive slides were found to be an accurate and precise platform for rapid enumeration of bacteria. Total bacterial counts made using the three methods were positively correlated for acridine orange and Live/Dead® (L/D) staining (0.81 ≤ r ≤ 0.95, all p ≤ 0.002). All platforms had similar precision, though counts obtained using bioadhesive slides were significantly higher than those made with polycarbonate filters and flow cytometry. The specific bioadhesive slides we used resulted in substantial cell mortality for certain pure cultures and river water samples, limiting their use for L/D determination. Cell enumeration using bioadhesive slides is particularly effective because it is highly precise at a wide range of cell concentrations, allows observation of cells that are not readily discernible on filters, reduces the number of steps and processing materials associated with sample analysis, and increases throughput.