Preparation of homogeneous samples of double-labelled protein suitable for single-molecule FRET measurements.

Preparation of pure and homogenous site specifically single- and double-labelled biopolymers suitable for spectroscopic determination of structural characteristics is a major current challenge in biopolymers chemistry. In particular, proper analysis of single-molecule Förster resonance energy transfer measurements is based on the spectral characteristics of the probes. Heterogeneity of any of the probes may introduce errors in the analysis, and hence, care must be taken to avoid preparation of inhomogeneous labelled biopolymer samples. When we prepared samples of Escherichia coli adenylate kinase (AK) mutants labelled with either Atto 488 or Atto 647N, the products were spectrally inhomogeneous and the composition of the mixture changed gradually over time. We show here that the inhomogeneity was not a result of variation in the dye interaction with neighbouring side chains. Rather, the slow drift of the spectral characteristics of the probes was a characteristic of an irreversible chemical transformation probably due to the hydrolysis of the succinimide ring of the attached dye into its succinamic acid form. Overnight incubation of the labelled protein in mild basic solution accelerated the interconversion, yielding homogeneous labelled samples. Using this procedure, we obtained stable homogenous AK mutant labelled at residues 142 and 188.

Multicolor spectral karyotyping of human chromosomes.

The simultaneous and unequivocal discernment of all human chromosomes in different colors would be of significant clinical and biologic importance. Whole-genome scanning by spectral karyotyping allowed instantaneous visualization of defined emission spectra for each human chromosome after fluorescence in situ hybridization. By means of computer separation (classification) of spectra, spectrally overlapping chromosome-specific DNA probes could be resolved, and all human chromosomes were simultaneously identified.

Sleep increases chromosome dynamics to enable reduction of accumulating DNA damage in single neurons.

Sleep is essential to all animals with a nervous system. Nevertheless, the core cellular function of sleep is unknown, and there is no conserved molecular marker to define sleep across phylogeny. Time-lapse imaging of chromosomal markers in single cells of live zebrafish revealed that sleep increases chromosome dynamics in individual neurons but not in two other cell types. Manipulation of sleep, chromosome dynamics, neuronal activity, and DNA double-strand breaks (DSBs) showed that chromosome dynamics are low and the number of DSBs accumulates during wakefulness. In turn, sleep increases chromosome dynamics, which are necessary to reduce the amount of DSBs. These results establish chromosome dynamics as a potential marker to define single sleeping cells, and propose that the restorative function of sleep is nuclear maintenance.

Telomeric aggregates and end-to-end chromosomal fusions require myc box II.

Telomeres of tumor cells form telomeric aggregates (TAs) within the three-dimensional (3D) interphase nucleus. Some of these TAs represent end-to-end chromosomal fusions and may subsequently initiate breakage-bridge-fusion cycles. Wild-type (wt) and myc box II mutant (mt) Myc induce different types of genomic instability when conditionally expressed in mouse proB cells (Ba/F3). Only wt Myc overexpressing Ba/F3 cells are capable of tumor formation in severe combined immunodeficient mice. In this study, we investigated whether telomere dysfunction leading to TA formation is linked to the genetic changes that permit wt c-Myc-dependent transformation of Ba/F3 cells. To this end, we examined the 3D organization of telomeres after the deregulated expression of deletion myc boxII mutant (Delta106) or wt Myc. Delta106-Myc overexpression did not induce TAs, whereas wt-Myc deregulation did. Instead, Delta106-Myc remodelled the 3D telomeric organization such that telomeres aligned in the center of the 3D interphase nucleus forming a telomeric disk owing to a Delta106-induced G1/S cell cycle arrest. In contrast, wt-Myc overexpression led to distorted telomere distribution and TA formation. Analysis of chromosomal alterations using spectral karyotyping confirmed Delta106-Myc and wt-Myc-associated genomic instability. A significant number of chromosomal end-to-end fusions indicative of telomere dysfunction were noted in wt-Myc-expressing cells only. This study suggests that TAs may play a fundamental role in Myc-induced tumorigenesis and provides a novel way to dissect tumor initiation.

Measuring the wavelength-dependent divergence of transmission through sub-wavelength hole-arrays by spectral imaging.

We present a study on the far-field patterns of light transmitted through sub-wavelength metallic hole-arrays. Spectral imaging measurements are used here on hole arrays for the first time. It provides both spatial and spectral information of the transmission in far-field. The visibility of the images, measured in two illumination modes: Köhler and collimated, is calculated for different planes in and out of focus. The transmission under collimated illumination reveals that 75% of the beam if non-divergent. The results are in agreement with the low divergence measured by Lezec [Science 297, 820 (2002)].

Loss of lamin A function increases chromatin dynamics in the nuclear interior.

Chromatin is organized in a highly ordered yet dynamic manner in the cell nucleus, but the principles governing this organization remain unclear. Similarly, it is unknown whether, and how, various proteins regulate chromatin motion and as a result influence nuclear organization. Here by studying the dynamics of different genomic regions in the nucleus of live cells, we show that the genome has highly constrained dynamics. Interestingly, depletion of lamin A strikingly alters genome dynamics, inducing a dramatic transition from slow anomalous diffusion to fast and normal diffusion. In contrast, depletion of LAP2α, a protein that interacts with lamin A and chromatin, has no such effect on genome dynamics. We speculate that chromosomal inter-chain interactions formed by lamin A throughout the nucleus contribute to chromatin dynamics, and suggest that the molecular regulation of chromatin diffusion by lamin A in the nuclear interior is critical for the maintenance of genome organization.

3D restoration with multiple images acquired by a modified conventional microscope.

A problem in high magnification microscopy is the blurring in the imaging of an object. In this article, we demonstrate a restoration technique that simultaneously makes use of the confocal image and the wide-field image. These images can be acquired by a modified conventional microscope. In front of the light-source, there is an array of pinholes. There are no pinholes at the detection plane. Instead, one or more pixels from the CCD camera are used, where the pinholes would have been. Using all pixels gives the wide-field image, but using a selected subset can give a confocal image. The array is used to speed up the process of acquiring the image. Note that the speed of acquisition is proportional to the number of pinholes. We show that the restoration from the two images can lead to a better result than using only one of the images. If this is the case, we show that a distance of 5 times the diameter of the pinholes can give the same results as a distance of 20 times after deconvolution. This offers an increase in acquisition time of a factor 16.

Fourier transform multipixel spectroscopy and spectral imaging of protoporphyrin in single melanoma cells.

Fourier transform multipixel spectroscopy was applied to subcellular localization of endogenous protoporphyrin (endo-PP) in single living B16 melanoma cells during photosensitization. Continuous fluorescence spectra for each pixel were recorded using a Sagnac interferometer coupled to a charge-coupled device camera. Multiple frames of data were acquired for each pixel composing the image, then they were stored as interferometric data and resolved as spectra for every pixel (10(3)-4 x 10(3) point pixels in a single cell). The net result was the intensity I (x, y, gamma), for each pixel of the image (x,y), at any wave-length (gamma). The present study demonstrates the application of Fourier transformed multipixel spectroscopy for spectral imaging of melanoma cells incubated with 5-aminolevulinic acid (ALA). The fluorescence image of ALA-treated cells revealed endo-PP all over the cytosol with a vesicular distribution, which represent mitochondria and endoplasmic reticulum compartments. Two main spectral fluorescence peaks were demonstrated at 630 and 670 nm, of monomeric and aggregated protoporphyrin, with intensities that differed from one subcellular site to another. Photoirradiation of the cells induced point-specific subcellular fluorescence spectrum changes and demonstrated photoproduct formation. Spectral-image reconstruction revealed the subcellular distribution of porphyrin species in single photosensitized cells. Multipixel spectroscopy of exogenous protoporphyrin revealed an endosomal-lysosomal compartment in aggregated states, whereas monomeric porphyrin species were localized mainly on the outer membrane. Photo-products could be visualized at sites of formation in subcellular compartments.

Ergodicity convergence test suggests telomere motion obeys fractional dynamics.

Anomalous diffusion, observed in many biological processes, is a generalized description of a wide variety of processes, all obeying the same law of mean-square displacement. Identifying the basic mechanisms of these observations is important for deducing the nature of the biophysical systems measured. We implement a previously suggested method for distinguishing between fractional Langevin dynamics, fractional Brownian motion, and continuous time random walk based on the ergodic nature of the data. We apply the method together with the recently suggested P-variation test and the displacement correlation to the lately measured dynamics of telomeres in the nucleus of mammalian cells and find strong evidence that the telomeres motion obeys fractional dynamics. The ergodic dynamics are observed experimentally to fit fractional Brownian or Langevin dynamics.

Transient anomalous diffusion of telomeres in the nucleus of mammalian cells.

We measured individual trajectories of fluorescently labeled telomeres in the nucleus of eukaryotic cells in the time range of 10(-2)-10(4)sec by combining a few acquisition methods. At short times the motion is subdiffusive with r2 approximately talpha and it changes to normal diffusion at longer times. The short times diffusion may be explained by the reptation model and the transient diffusion is consistent with a model of telomeres that are subject to a local binding mechanism with a wide but finite distribution of waiting times. These findings have important biological implications with respect to the genome organization in the nucleus.

Characterizing the three-dimensional organization of telomeres.

BACKGROUND: Quantitative analysis can be used in combination with fluorescence microscopy. Although the human eye is able to obtain good qualitative results, when analyzing the spatial organization of telomeres in interphase nuclei, there is a need for quantitative results based on image analysis. METHODS: We developed a tool for analyzing three-dimensional images of telomeres stained by fluorescence in situ hybridization in interphase nuclei with DNA counterstained with 4',6-diamidino-2-phenylindole. After deconvolution of the image, we segmented individual telomeres. From the location of the telomeres we derived a distribution parameter rhoT, which indicated whether the telomeres were in a disk (rhoT >> 1) or not (rhoT approximately 1). We sorted mouse lymphocyte nuclei and measured rhoT. We also performed a bromodeoxyuridine synchronous cell sorting experiment on live cells and measured rhoT at several instances. RESULTS: Measuring rhoT for nuclei in G0/G1, S, and G2 produced 1.4 +/- 0.1, 1.5 +/- 0.2, and 14 +/- 2, respectively, showing a significant difference between G2 and G0/G1 or S. For the bromodeoxyuridine synchronous cell sorting experiment, we found a cell cycle dependency of rhoT and a correlation between rhoT and an observer. CONCLUSIONS: In this study we present a quantitative method to characterize the organization of telomeres using three-dimensional imaging, image processing, and image analysis.

Signal to noise analysis of multiple color fluorescence imaging microscopy.

BACKGROUND: Various approaches that were recently developed demonstrate the ability to simultaneously detect all human (or other species) chromosomes by using combinatorial labeling and fluorescence in situ hybridization (FISH). With the growing interest in this field, it is important to develop tools for optimizing and estimating the accuracy of different experimental methods. METHODS: We have analyzed the principles of multiple color fluorescence imaging microscopy. First, formalism based on the physical principles of fluorescence microscopy and noise analysis is introduced. Next, a signal to noise (S/N) analysis is performed and summarized in a simple accuracy criterion. The analysis assumes shot noise to be the dominant source of noise. RESULTS: The accuracy criterion was used to calculate the S/N of multicolor FISH (M-FISH), spectral karyotyping, ratio imaging, and a method based on using a set of broad band filters. Spectral karyotyping is tested on various types of samples and shows accurate classifications. We have also tested classification accuracy as a function of total measurement time. CONCLUSIONS: The accuracy criterion that we have developed can be used for optimizing and analyzing different multiple color fluorescence microscopy methods. The assumption that shot noise is dominant in these measurements is supported by our measurements.

Spectral imaging of multi-color chromogenic dyes in pathological specimens.

We have investigated the use of spectral imaging for multi-color analysis of permanent cytochemical dyes and enzyme precipitates on cytopathological specimens. Spectral imaging is based on Fourier-transform spectroscopy and digital imaging. A pixel-by-pixel spectrum-based color classification is presented of single-, double-, and triple-color in situ hybridization for centromeric probes in T24 bladder cancer cells, and immunocytochemical staining of nuclear antigens Ki-67 and TP53 in paraffin-embedded cervical brush material (AgarCyto). The results demonstrate that spectral imaging unambiguously identifies three chromogenic dyes in a single bright-field microscopic specimen. Serial microscopic fields from the same specimen can be analyzed using a spectral reference library. We conclude that spectral imaging of multi-color chromogenic dyes is a reliable and robust method for pixel color recognition and classification. Our data further indicate that the use of spectral imaging (a) may increase the number of parameters studied simultaneously in pathological diagnosis, (b) may provide quantitative data (such as positive labeling indices) more accurately, and (c) may solve segmentation problems currently faced in automated screening of cell- and tissue specimens.