Vertebrate chromosomes terminate in variable numbers of T2AG3 nucleotide repeats. In order to study telomere repeats at individual chromosomes, we developed novel, quantitative fluorescence in situ hybridization procedures using labeled (C3TA2)3 peptide nucleic acid and digital imaging microscopy. Telomere fluorescence intensity values from metaphase chromosomes of cultured human hematopoietic cells decreased with the replication history of the cells, varied up to six-fold within a metaphase, and were similar between sister chromatid telomeres. Surprisingly, telomere fluorescence intensity values within normal adult bone marrow metaphases did not show a normal distribution, suggesting that a minimum number of repeats at each telomere is required and/or maintained during normal hematopoiesis.

Chromosome ends are protected from degradation by the presence of the highly repetitive hexanucleotide sequence of TTAGGG and associated proteins. These so-called telomeric complexes are suggested to play an important role in establishing a functional nuclear chromatin organization. Using peptide nucleic acid (PNA) probes, we studied the dynamic behavior of telomeric DNA repeats in living human osteosarcoma U2OS cells. A¯uorescent cy3-labeled PNA probe was introduced in living cells by glass bead loading and was shown to speci®cally associate with telomeric DNA shortly afterwards. Telomere dynamics were imaged for several hours using digital¯uorescence microscopy. While the majority of telomeres revealed constrained diffusive movement, individual telomeres in a human cell nucleus showed signi®cant directional movements. Also, a subfraction of telomeres were shown to associate and dissociate, suggesting that in vivo telomere clusters are not stable but dynamic structures. Furthermore, telomeres were shown to associate with promyelocytic leukemia (PML) bodies in a dynamic manner.

SUMMARYWe 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 haptenlabeled 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.

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.

Key terms: Background correction, CCD camera., filters, fluorochromes, image quality assessment. intensity normalization, pixel shift correction, relative copy number karyotypeComparative Genomic Hybridization (CGH) analysis (4) has rapidly established itself as a powerful method for analyzing whole genomic DNA (3, 5 , 6, 10, 14, 15), and many researchers are interested in acquiring their own CGH analysis capability. Although some chromosomal imbalances, e.g. high copy number amplifications, can be identified by conventional microscopy, comprehensive interpretation of CGH requires digital image analysis. However, at the time of writing there are no comrnercially available software packages which come close to the capabilities of the in-house systems used by the various research teams which have been at the forefront of CGH developments and application (9, 11, 13).At a recent workshop o n the topic of "CGH Imaging" (organised by the EC Concerted Action on Automation of Molecular Cytogenetic Analyses, and held at the MRC Human Genetics Unit, Edinburgh, 2-3 June 1994), three discussion sessions were devoted to specifying what could be considered to be an "adequate provision" of hardware and analysis software, and what guidelines should be used in the interpretation of CGH data. These are respectively the topics of the three sections of this report, which summarises the consensus that was reached at that workshop.We hope that these recommendations will prove to be useful for prospective users of CGH. This technical note is not intended to be an introduction to CGH, nor a sur. vey of the literature on the subject, nor is it concerned with preparative techniques (which are discussed in de.. tail in Refs. 7, 8). Readers are referred to original source articles for detailed information on biological preparation protocols, digital microscopy, and analysis software; and also for further discussion of the issues concerning interpretation of the results of CGH analyses, and limitations of the technique. HARDWARE REQUIREMENTS Microscope OpticsRecent microscope models from the major manufacturers, equipped for epi-fluorescence, are adequate. The objective should be "plan" and apochromatic with a high numerical aperture. Some comniercially available apochromatic objectives are not suitable for CGH analysis since they do not transmit UV excitation. Some recent "fluar" objectivcs have developed high levels of autofluorescence after short periods of use; users should check for lens autofluorescence regularly and replace the objective when necessary. Mercury arc lamp light sources are adequate if they are stable, and can be aligned to give uniform illumination without chromatic variation. Recent microscope models hare improved collector lenses that correct for chromatic aberrations of the illumination, but problems may be experienced with older models. FiltersFilter sets should be selected to minimize the crosstalk betwcen fluorochromes. Ideally, this would be achieved by using fluorochromes with completely separated excitation a...