Figure 1. Virtual microscopy of optical resolution improvement. (a) 3D visualization and projections to the lateral (x, y) plane and the vertical (x, z) and (y, z) planes of a computer simulation of a 1 Mbp chromatin domain of total diameter 500 nm (Cremer and Cremer, 2001) assumed to comprise 10 condensed 100 kbp domains formed by nucleosome chains, having an individual size in the 100 nm range. The small ‘beads’ represent the individual nucleosomes. Here, a labeling of all 10 domains with the same spectral signature was assumed. (b)–(d) Virtual microscopy representations (3D visualization, projections) of the monospectral computer simulated 1 Mbp domain shown in (a). For this, the simulated 3D data set of (a) was convoluted with the effective point spread function for the optical resolution assumed. (b) Virtual microscopy assuming a theoretical limit of STED microscopy (see text) of 25 nm. The positions and sizes of the ten 100 kbp regions are all resolved. (c) Virtual microscopy assuming an effective optical resolution of 100 nm (as achieved for example by 4Pi, STED, standing-wave excitation microscopy). Some structural resolution is still maintained. (d) Virtual microscopy assuming an effective optical resolution of 250 nm (achieved, for example, by merging confocal images obtained from different angles). All structural information has been lost, except the total size of the 1 Mbp domain. The bar indicates 100 nm. (Figure kindly provided by Dr G. Kreth, Heidelberg.)

Figure 2. Virtual microscopy of improvement of effective structural resolution by multispectral labeling. (a) 3D visualization of a computer simulation of a 1 Mbp chromatin domain (see Figure 1a). In contrast to Figure 1a, however, here only eight of the ten 100 kbp domains are shown. Each of these eight domains has been assumed to be labeled with a different spectral signature. (b)–(d) Virtual microscopy representations (3D visualization, projections) of the multispectral computer simulation of (a). Optical aberrations, for example chromatic deviations, detector and photon noise were neglected. (b) Virtual microscopy assuming a theoretical limit of STED microscopy of 25 nm. The positions and sizes of the eight labeled 100 kbp domains are all resolved; the domains are identified individually due to their distinct spectral signatures. (c) Virtual microscopy assuming an effective optical resolution of 100 nm. In contrast to the monospectral case (Figure 1c), the positions and sizes of the eight labeled 100 kbp domains with an individual size in the 100 nm range are all resolved; the eight labeled domains are also identified individually due to their distinct spectral signature. (d) Virtual microscopy assuming an effective optical resolution of 250 nm. In contrast to the monospectral case (Figure 1d), here the eight labeled domains are identified individually due to their spectral signatures; the positional information as given by the centers of the individual spherical effective diffraction images is also maintained. The size information, however, has been completely lost. The bar indicates 100 nm. (Figure kindly provided by Dr G. Kreth, Heidelberg.)

Figure 3. Principle of improvement of topological resolution by spectral precision distance microscopy (SPDM). The precise measurement of multiple positions, distances and angles between specifically labeled target sites (topology) by SPDM and related methods is based on the use of point-like objects in at least one spatial direction labeled with different spectral signatures. By spectrally discriminating image acquisition, the diffraction-limited intensity distribution of each object is then individually recorded. Thus, each spectral detection channel of the microscope contains only the diffraction-limited intensity distribution. After careful correction for chromatic and monochromatic aberrations, for each spectral channel, the image position corresponding to the geometrical image point may then be determined by appropriate algorithms, such as the maximum of the diffraction image, or the barycenter, the fluorescence intensity-weighted analog to the center of mass. Consequently, topological information about the object can be obtained considerably beyond the limit of optical resolution. (a)–(c) One spectral signature (monospectral) labeling of three point-like fluorescent objects situated on a horizontal straight line, assumed to have a next-neighbor distance of 50 nm each. (a) Object positions in the object space. (b) Diffraction image obtained assuming an optical resolution of 250 nm. The spatial coordinates of the diffraction image have been normalized to the same scale as in the object space. (c) Normalized fluorescence intensity distribution along a horizontal line through the center of the diffraction image shown in (b). No positional contrast has been maintained. (d)–(f) Three spectral signature labeling of the three point-like objects in (a). Here, each of the objects was assumed to have been labeled with a different spectral signature. (e) Diffraction image of (a) after spectrally discriminating image acquisition and correction of optical aberration. The diffraction images of the objects are strongly overlapping. (f) In the fluorescence intensity line scans through the diffraction images for the three spectral signatures, the maximum of fluorescence barycenter position of each of the diffraction images can be determined independently from the others, thus allowing determination of the geometrical image points of the three objects and hence their positions and distances from each other, with a precision much better than the optical resolution. The virtual microscopy diffraction images (b, e) were computed for a confocal microscope with NA=1.4/63×oil immersion objective. (Reproduced with permission from Cremer et al., 1999)

Figure 4. Virtual microscopy of topological resolution improvement by multispectral spectral precision distance microscopy (SPDM). Above: (a)–(c) Virtual microscopy 3D visualization of a computer simulation of an individual 100 kbp chromatin domain, assuming monospectral labeling (see Figure 1a). (a) Original simulation. (b) Assuming an optical resolution of 25 nm. (c) Assuming an optical resolution of 100 nm (compare Figure 1a). Although in (b) and (c) the overall size of the domain has been obtained correctly, in both cases all internal structural information has been lost. Below: Virtual SPDM of two 100 kbp chromatin domains. Here, it was assumed that the linear 100 kbp sequence was labeled in 10 sections of 10 kbp each, using a different spectral signature for each section. Representation of the computer simulation of a ‘condensed’ (above) and a ‘decondensed’ 100 kbp chromatin domain (nucleosome chain) labeled with the 10 spectral signatures as indicated. The small dots represent the individual nucleosomes. The bar is 100 nm. The monospectral (to the left) and the multispectral (to the right) virtual microscopy diffraction images were calculated assuming an optical resolution of 100 nm (half the width of the PSF). While in the monospectral diffraction image all structural information has been lost, following spectrally discriminating registration, in the multispectral image (shown after optical aberration correction) the 10 spectral diffraction images overlie each other. After the SPDM reconstruction (using the fluorescence intensity barycenters/‘gravity centers’ of each of the independently registered diffraction images) the positions of the gravity centers of the ten 10 kbp sections of the two 100 kbp domains can be reconstructed. Thus, the topology of the two 100 kbp domains can be reconstructed to considerable detail. For example, the topology of the ‘condensed’ and the ‘decondensed’ 100 kbp domain can be clearly distinguished. Applying the same procedure to a specific 10-kb section (labeling the ten 1-kb sections of the 10-kb sequence with 10 different spectral signatures; performing SPDM), the topology of a gene region would be reconstructed down to the five-nucleosome level. Repeating the same procedure with multispectral oligonucleotide labeling on the 1 kbp level would allow a topological reconstruction on the molecular level. Such high topological resolution reconstructions, however, would require a positional and distance resolution in the range of 1 nm and below. Virtual microscopy simulations (Failla et al., unpublished data) indicate that such requirements can be realized under fluorescence photon yield conditions corresponding to single-fluorochrome labeling (Knemeyer et al., 2000). (Figure kindly provided by Dr G. Kreth, Heidelberg.)

Figure 5. Analysis of genome nanostructure by three-color confocal microscopy and SPDM. (a) From left to right (schematically): Linear mapping of three probes labeled with different spectral signatures and covering the BCR–ABL fusion region in bone marrow cells of patients with chronic myelogeneous leukemia (CLM); a complex folding of the 3D chromatin structure of this region in nuclei carrying the fusion region; the triangles resulting from the intensity barycenters of the three sequence sites labeled with three spectral signatures, after application of the SPDM procedure (from Esa et al., 2000). (b) From left to right (schematically): Linear mapping of two probes covering the BCR region on the intact chromosome 22; a complex folding of the 3D chromatin structure of this region. The topology of three sequences in the BCR–ABL fusion gene region in bone marrow cell nuclei from leukemia patients determined by confocal SPDM. Shown are the relative mean positions of the fluorescence intensity barycenters, in relation to the observation volume of the CLSM used (transparent ellipsoid) (from Esa et al., 2000). (a) Left: The relative mean positions and distances (open dots) obtained by experimental three-color confocal SPDM of two BCR regions and one ABL region in t(9;22) Philadelphia chromosome territory regions of bone marrow cells of CML patients according to (a), measured in 75 nuclei (from Esa et al., 2000). Right: Confocal monospectral virtual microscopy diffraction image calculated for the experimental mean positions shown on the left (vertical axis denotes optical axis of the confocal microscope), using an experimental confocal point spread function (kindly provided by Dr H. Bornfleth, Heidelberg). In the case of monospectral labeling, all topological information about the BCR–ABL fusion region has been lost (virtual microscopy image kindly provided by J. von Hase, Heidelberg.)