Nico P. Verwoerd

The use of peroxidase-mediated deposition of biotin-tyramide in combination with time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization.

The application of europium chelates as delayed fluorescent labels in FISH and immunocytochemistry is hampered by their relatively low quantum yield. To increase the intensity of the delayed fluorescence, we have used a recently introduced peroxidase-mediated amplification system. This system results in a large accumulation of biotin-tyramide, which is detected using streptavidin-europium chelate as label. Optimal staining conditions were evaluated for the immunocytochemical detection of vimentin in cryosections of rat liver, for DNA in situ hybridization (alphoid type probes and 40-KB cosmid probes), and for RNA in situ hybridization (detection of 28S ribosomal RNA, human elongation factor mRNA, and luciferase mRNA). Using a time-resolved fluorescence microscope, intense europium fluorescence was obtained in all these applications when the tyramide amplification system was applied. The signals were strong enough to be observed by eye using the microscope in the time-delayed mode. The routine application of this technique for localization and quantization of antigens or nucleic acid sequences in tissue exhibiting strong autofluorescence is discussed.

A glue for heterochromatin maintenance stable SUV39H1 binding to heterochromatin is reinforced by the SET domain

Trimethylation of histone H3 lysine 9 and the subsequent binding of heterochromatin protein 1 (HP1) mediate the formation and maintenance of pericentromeric heterochromatin. Trimethylation of H3K9 is governed by the histone methyltransferase SUV39H1. Recent studies of HP1 dynamics revealed that HP1 is not a stable component of heterochromatin but is highly mobile (Cheutin, T., A.J. McNairn, T. Jenuwein, D.M. Gilbert, P.B. Singh, and T. Misteli. 2003. Science. 299:721–725; Festenstein, R., S.N. Pagakis, K. Hiragami, D. Lyon, A. Verreault, B. Sekkali, and D. Kioussis. 2003. Science. 299:719–721). Because the mechanism by which SUV39H1 is recruited to and interacts with heterochromatin is unknown, we studied the dynamic properties of SUV39H1 in living cells by using fluorescence recovery after photobleaching and fluorescence resonance energy transfer. Our results show that a substantial population of SUV39H1 is immobile at pericentromeric heterochromatin, suggesting that, in addition to its catalytic activity, SUV39H1 may also play a structural role at pericentromeric regions. Analysis of SUV39H1 deletion mutants indicated that the SET domain mediates this stable binding. Furthermore, our data suggest that the recruitment of SUV39H1 to heterochromatin is at least partly independent from that of HP1 and that HP1 transiently interacts with SUV39H1 at heterochromatin.

Platinum Porphyrins as Phosphorescent Label for Time-resolved Microscopy

We investigated phosphorescent metalloporphyrins as potential labels for time-resolved microscopy. On the basis of spectroscopic analysis of their physicochemical properties (quantum yield, molar absorption coefficient, decay times) the best candidates were selected. Next, we synthesized antibody and avidin metalloporphyrin conjugates. The optimal F/P ratio with respect to quantum yield, decay time, and retention of biological activity of these immunoreagents was determined. The reagents were then evaluated by in situ hybridization and immunocytochemical procedures for demonstration of hapten-labeled DNA probes, membrane antigens (CD type), and 28S rRNA. All stained samples exhibited bright phosphorescence that could be selectively detected using time-resolved microscopy, especially when glucose/glucose oxidase was added to the embedding medium to deplete oxygen. Applications of time-resolved detection of phosphorescent porphyrins in strongly autofluorescent material (histological sections) are discussed.

Phosphorescent Platinum/Palladium Coproporphyrins for Time-resolved Luminescence Microscopy:

Streptavidin and antibodies were labeled with phosphorescent platinum and palladium coproporphyrin. The optimal conjugates were selected on the basis of spectroscopic analysis (molar extinction coefficient, quantum yield, lifetime) and using ELISA assays to determine the retention of biological activity and immunospecificity. They were subsequently tested for the detection of prostate-specific antigen, glucagon, human androgen receptor, p53, and glutathione transferase in strongly autofluorescent tissues. Furthermore, platinum and palladium coproporphyrin-labeled dUTPs were synthesized for the enzymatic labeling of DNA probes. Porphyrin-labeled DNA probes and porphyrin-labeled streptavidin conjugates were evaluated for DNA in situ hybridization on metaphase spreads, using direct and indirect methods, respectively. The developed in situ detection technology is shown to be applicable not only in mammals but also in plants. A modular-based time-resolved microscope was constructed and used for the evaluation of porphyrinstained samples. The time-resolved module was found suitable for detection of antigens and DNA targets in an autofluorescent environment. Higher image contrasts were generally obtained in comparison with conventional detection systems (e.g., fourfold improvement in detection of glutathione transferase).

Evaluation of a time‐resolved fluorescence microscope using a phosphorescent Pt‐porphine model system

A time-resolved fluorescence microscope is presented that allows the sensitive detection of delayed luminescent labels with decay times from one microsecond to several milliseconds. The microscope utilizes an argon ion laser chopped with an acoustooptical modulator as excitation light source in combination with a gated multichannel plate image intensifier in the image plane. A theoretical model for the detection efficiency of practically any time-resolved fluorescence microscope is verified using phosphorescent Pt-porphine-stained Sephadex beads. The detection efficiency of the presented setup was shown to be 42%, which is near the theoretical limit of 50% for non-saturated luminescent dyes. The suppression of prompt fluorescence signals was found to be 1:5,500. The Pt-porphine beads proved to be an excellent model system for time-resolved fluorescence microscopy, showing a high extinction coefficient and high phosphorescence quantum yield in aqueous environment under room temperature conditions. Furthermore, for the microscope described the decay time of the Pt-porphine beads of 47 microseconds is long enough to enable efficient suppression of the prompt fluorescence while maintaining a high excitation and emission duty cycle. This is considered to be of vital importance in order not to saturate the luminescence with the excitation intensities commonly used in fluorescence microscopy.

The use of computers for quantitative cell analysis

SummaryComputers are extensively employed in the quantitative analysis of cells and tissues using flow cytometry and/or image analysis. The first issue discussed in this paper is the use of computer-controlled multiparameter flow cytometry, with special emphasis on the use of pulse-processing techniques for DNA ploidy analysis. Subsequently, modern image analysis using a solid-state camera is discussed. Examples of automated analysis of Feulgen-DNA stained cells (absorption) or cells stained by in situ hybridization procedures (fluorescence) for defined DNA or RNA sequences are given. Finally, a new user-friendly microscope-computer interface (HOME=highly optimized microscope environment) is presented, in which the monitor of a personal computer is optically overlaid with the microscopic image, enabling the immediate reading of patient record data while viewing through the microscope. When equipped with a simple encoding stage for position tracking, this system enables more accurate screening of cytological specimens and provides rapid interactive image analysis and morphometry.