Superresolution imaging of biological nanostructures by spectral precision distance microscopy.

For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far-field methods have been developed allowing a substantial increase of resolution ("superresolution microscopy", or "lightoptical nanoscopy"). This opens an avenue to 'nano-image' intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses and the molecular interaction information provided by molecular cell biology. In this review, we describe the principles of spectrally assigned localization microscopy (SALM) of biological nanostructures, focusing on a special SALM approach, spectral precision distance/position determination microscopy (SPDM) with physically modified fluorochromes (SPDM(Phymod) . Generally, this SPDM method is based on high-precision localization of fluorescent molecules, which can be discriminated using reversibly bleached states of the fluorophores for their optical isolation. A variety of application examples is presented, ranging from superresolution microscopy of membrane and cytoplasmic protein distribution to dual-color SPDM of nuclear proteins. At present, we can achieve an optical resolution of cellular structures down to the 20-nm range, with best values around 5 nm (∼1/100 of the exciting wavelength).

Combining FISH with localisation microscopy: Super-resolution imaging of nuclear genome nanostructures.

The optical resolution of conventional far field fluorescence light microscopy is restricted to about 200 nm laterally and 600 nm axially and has been thought for many decades to be an insurmountable barrier for the quantitative spatial analysis of cellular and hence also nuclear constituents. Novel approaches in light microscopy have now overcome this barrier. Here, we report on a special method of localisation microscopy, spectral precision distance/position determination microscopy and its combination with fluorescence in situ hybridization to analyse the spatial distribution of specific DNA sequences in human cell nuclei at the macromolecular optical resolution level. As an example, repetitive DNA sequence DYZ2 located within the heterochromatin region on human chromosome Yq12 was labelled with clone pHY2.1. Between 300 and 700 single-probe molecules were resolved in individual chromatin domains, corresponding to a detected molecule density around 500/µm(2), i.e., many times higher than resolvable by conventional fluorescence microscopy. A mean localisation accuracy of about 20 nm indicated a mean optical resolution in the 50 nm range. Beyond new perspectives for light microscopic studies of specific chromatin nanostructures, this may open a new avenue towards the general analysis of copy number of specific DNA sequences in small regions of individual interphase nuclei.

Epigenetic regulation of promiscuous gene expression in thymic medullary epithelial cells.

Thymic central tolerance comprehensively imprints the T-cell receptor repertoire before T cells seed the periphery. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process by virtue of promiscuous expression of tissue-restricted autoantigens. The molecular regulation of this unusual gene expression, in particular the involvement of epigenetic mechanisms is only poorly understood. By studying promiscuous expression of the mouse casein locus, we report that transcription of this locus proceeds from a delimited region ("entry site") to increasingly complex patterns along with mTEC maturation. Transcription of this region is preceded by promoter demethylation in immature mTECs followed upon mTEC maturation by acquisition of active histone marks and local locus decontraction. Moreover, analysis of two additional gene loci showed that promiscuous expression is transient in single mTECs. Transient gene expression could conceivably add to the local diversity of self-antigen display thus enhancing the efficacy of central tolerance.

Localization microscopy reveals expression-dependent parameters of chromatin nanostructure.

A combined approach of 2D high-resolution localization light microscopy and statistical methods is presented to infer structural features and density fluctuations at the nuclear nanoscale. Hallmarks of nuclear nanostructure are found on the scale below 100 nm for both human fibroblast and HeLa cells. Mechanical measures were extracted as a quantitative tool from the histone density fluctuations inside the cell to obtain structural fluctuations on the scale of several micrometers. Results show that different mechanisms of expression of the same nuclear protein type lead to significantly different patterns on the nanoscale and to pronounced differences in the detected compressibility of chromatin. The observed fluctuations, including the experimental evidence for dynamic looping, are consistent with a recently proposed chromatin model.

Visualization, analysis, and design of COMBO-FISH probes in the grid-based GLOBE 3D genome platform.

The genome architecture in cell nuclei plays an important role in modern microscopy for the monitoring of medical diagnosis and therapy since changes of function and dynamics of genes are interlinked with changing geometrical parameters. The planning of corresponding diagnostic experiments and their imaging is a complex and often interactive IT intensive challenge and thus makes high-performance grids a necessity. To detect genetic changes we recently developed a new form of fluorescence in situ hybridization (FISH) - COMBinatorial Oligonucleotide FISH (COMBO-FISH) - which labels small nucleotide sequences clustering at a desired genomic location. To achieve a unique hybridization spot other side clusters have to be excluded. Therefore, we have designed an interactive pipeline using the grid-based GLOBE 3D Genome Viewer and Platform to design and display different labelling variants of candidate probe sets. Thus, we have created a grid-based virtual "paper" tool for easy interactive calculation, analysis, management, and representation for COMBO-FISH probe design with many an advantage: Since all the calculations and analysis run in a grid, one can instantly and with great visual ease locate duplications of gene subsequences to guide the elimination of side clustering sequences during the probe design process, as well as get at least an impression of the 3D architectural embedding of the respective chromosome region, which is of major importance to estimate the hybridization probe dynamics. Beyond, even several people at different locations could work on the same process in a team wise manner. Consequently, we present how a complex interactive process can profit from grid infrastructure technology using our unique GLOBE 3D Genome Platform gateway towards a real interactive curative diagnosis planning and therapy monitoring.

Nanostructure analysis using spatially modulated illumination microscopy.

We describe the usage of the spatially modulated illumination (SMI) microscope to estimate the sizes (and/or positions) of fluorescently labeled cellular nanostructures, including a brief introduction to the instrument and its handling. The principle setup of the SMI microscope will be introduced to explain the measures necessary for a successful nanostructure analysis, before the steps for sample preparation, data acquisition and evaluation are given. The protocol starts with cells already attached to the cover glass. The protocol and duration outlined here are typical for fixed specimens; however, considerably faster data acquisition and in vivo measurements are possible.

Inheritance of gene density-related higher order chromatin arrangements in normal and tumor cell nuclei.

A gene density-related difference in the radial arrangement of chromosome territories (CTs) was previously described for human lymphocyte nuclei with gene-poor CT #18 located toward the nuclear periphery and gene-dense CT #19 in the nuclear interior (Croft, J.A., J.M. Bridger, S. Boyle, P. Perry, P. Teague, and W.A. Bickmore. 1999. J. Cell Biol. 145:1119-1131). Here, we analyzed the radial distribution of chromosome 18 and 19 chromatin in six normal cell types and in eight tumor cell lines, some of them with imbalances and rearrangements of the two chromosomes. Our findings demonstrate that a significant difference in the radial distribution of #18 and #19 chromatin is a common feature of higher order chromatin architecture in both normal and malignant cell types. However, in seven of eight tumor cell lines, the difference was less pronounced compared with normal cell nuclei due to a higher fraction of nuclei showing an inverted CT position, i.e., a CT #18 located more internally than a CT #19. This observation emphasizes a partial loss of radial chromatin order in tumor cell nuclei.