PMID- 10947902
OWN - NLM
STAT- MEDLINE
DCOM- 20000918
LR  - 20190905
IS  - 0022-2720 (Print)
IS  - 0022-2720 (Linking)
VI  - 199
IP  - Pt 2
DP  - 2000 Aug
TI  - Three-dimensional spectral precision distance microscopy of chromatin 
      nanostructures after triple-colour DNA labelling: a study of the BCR region on 
      chromosome 22 and the Philadelphia chromosome.
PG  - 96-105
AB  - Topological analysis of the three-dimensional (3D) chromatin nanostructure and 
      its function in intact cell nuclei implies the use of high resolution far field 
      light microscopy, e.g. confocal laser scanning microscopy (CLSM). However, 
      experimental evidence indicates that, in practice, under biologically relevant 
      conditions, the spatial resolution of CLSM is limited to about 300 nm in the 
      lateral direction and about 700 nm in the axial direction. To overcome this 
      shortcoming, the use of a recently developed light microscopical approach, 
      spectral precision distance microscopy (SPDM) is established. This approach is 
      based on the precise localization of small labelling sites of a given target in 
      spectrally differential images. By means of quantitative image analysis, the bary 
      centres (intensity weighted centroid analogous to the centre of mass) of these 
      independently registered labelling sites can be used as point markers for 
      distance and angle measurements after appropriate calibration of optical 
      aberrations (here, polychromatic shifts). In combination with specific labelling 
      of very small chromatin target sites with dyes of different spectral signatures 
      by fluorescence in situ hybridization (FISH), SPDM presently allows us to analyse 
      the nuclear topology in three-dimensionally conserved nuclei with a 'resolution 
      equivalent', many times smaller than the conventional optical resolution. Chronic 
      myelogeneous leukaemia (CML) is genetically characterized by the fusion of parts 
      of the BCR and ABL genes on chromosomes 22 and 9, respectively. In most cases, 
      the fusion leads to a translocation t(9; 22) producing the Philadelphia 
      chromosome. SPDM was applied to analyse the 3D chromatin structure of the BCR 
      region on the intact chromosome 22 and the BCR-ABL fusion gene on the 
      Philadelphia chromosome (Ph) by using a new triple-colour FISH protocol: two 
      different DNA probes were used to detect the BCR region and the third DNA probe 
      was used to identify the location of the ABL gene. Consistent 3D distance 
      measurements down to values considerably smaller than 100 nm were performed. The 
      angle distributions between the three labelled sites on the Philadelphia 
      chromosome territory were compared to two state-of-the-art computer models of 
      nuclear chromatin structure. Significant differences between measured and 
      simulated angle distributions were obtained, indicating a complex and non-random 
      angle distribution.
FAU - Esa, A
AU  - Esa A
AD  - Applied Optics and Information Processing, Kirchhoff Institute for Physics (KIP), 
      University of Heidelberg, Albert-Ueberle-Str. 3-5, 6920 Heidelberg, Germany.
FAU - Edelmann, P
AU  - Edelmann P
FAU - Kreth, G
AU  - Kreth G
FAU - Trakhtenbrot, L
AU  - Trakhtenbrot L
FAU - Amariglio, N
AU  - Amariglio N
FAU - Rechavi, G
AU  - Rechavi G
FAU - Hausmann, M
AU  - Hausmann M
FAU - Cremer, C
AU  - Cremer C
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - J Microsc
JT  - Journal of microscopy
JID - 0204522
RN  - 0 (Chromatin)
RN  - 0 (DNA, Neoplasm)
RN  - 0 (Fluorescent Dyes)
SB  - IM
MH  - Bone Marrow Cells/*pathology
MH  - Chromatin/*ultrastructure
MH  - Chromosomes, Human, Pair 22/*ultrastructure
MH  - DNA, Neoplasm/ultrastructure
MH  - Fluorescent Dyes
MH  - Genes, abl/*genetics
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/methods
MH  - Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*genetics/pathology
MH  - Microscopy/methods
MH  - Models, Molecular
MH  - *Philadelphia Chromosome
EDAT- 2000/08/18 11:00
MHDA- 2000/09/23 11:01
CRDT- 2000/08/18 11:00
PHST- 2000/08/18 11:00 [pubmed]
PHST- 2000/09/23 11:01 [medline]
PHST- 2000/08/18 11:00 [entrez]
AID - jmi707 [pii]
AID - 10.1046/j.1365-2818.2000.00707.x [doi]
PST - ppublish
SO  - J Microsc. 2000 Aug;199(Pt 2):96-105. doi: 10.1046/j.1365-2818.2000.00707.x.