Innovative use of liquid crystalline acids as color developers in leuco dye-based temperature sensors.

Novel temperature sensors with unique optical properties, based on 4-alkylbenzoic acid developers (CnBA, where n is the number of carbon atoms in the alkyl chain ranging from 4 to 6), which exhibit a liquid crystalline phase, and 6'-(diethylamino)-1',2'-benzofluoran (BF) leuco dye are reported. The main aim of this work is to investigate how the molecular packing of CnBA in different phases affects the development of BF. Various techniques were used to study the prepared temperature sensors' phase transitions and thermal stability. The spectroscopic properties of BF : CnBA (1 : 3) were investigated, using temperature-dependent UV-Vis absorption and emission spectroscopy, and the results show that the sensors demonstrate reversible color-changing properties. When the CnBA developers are at room temperature, the materials are pink and emit orange light, while at approximately 105 °C they turn white and emit yellow light. Above that temperature, the sensors return to a pink and orange light emission. Therefore the prepared materials can serve as indicators that inform about not only exceeding a certain temperature threshold but also reaching temperature ranges.

Image division technique in pre-acquisition analysis of information content for automated microscopy.

This article presents a method that allows for reliable automated image acquisition of specimens with high information content in light microscopy with emphasis on fluorescence microscopy applications. Automated microscopy typically relies on autofocusing used for the analysis of information content behaviour along the z-axis within each field of view. However, in the case of a field of view containing more objects that do not lie precisely in one z-plane, traditional autofocusing methods fail due to their principle of operation. We avoid this issue by reducing the original problem to a set of simple and performable tasks: we divide the field of view into a small number of tiles and process each of them individually. The obtained results enable discovering z-planes with rich information content that remain hidden during global analysis of the whole field of view. Our approach therefore outperforms other acquisition methods including the manual one. A large part of the contribution is oriented towards practical application.

Information content analysis in automated microscopy imaging using an adaptive autofocus algorithm for multimodal functions.

We present a new algorithm to analyse information content in images acquired using automated fluorescence microscopy. The algorithm belongs to the group of autofocusing methods, but differs from its predecessors in that it can handle thick specimens and operate also in confocal mode. It measures the information content in images using a 'content function', which is essentially the same concept as a focus function. Unlike previously presented algorithms, this algorithm tries to find all significant axial positions in cases where the content function applied to real data is not unimodal, which is often the case. This requirement precludes using algorithms that rely on unimodality. Moreover, choosing a content function requires careful consideration, because some functions suppress local maxima. First, we test 19 content functions and evaluate their ability to show local maxima clearly. The results show that only six content functions succeed. To save time, the acquisition procedure needs to vary the step size adaptively, because a wide range of possible axial positions has to be passed so as not to miss a local maximum. The algorithm therefore has to assess the steepness of the content function online so that it can decide to use a bigger or smaller step size to acquire the next image. Therefore, the algorithm needs to know about typical behaviour of content functions. We show that for normalized variance, one of the most promising content functions, this knowledge can be obtained after normalizing with respect to the theoretical maximum of this function, and using hierarchical clustering. The resulting algorithm is more reliable and efficient than a simple procedure with constant steps.

Combination of mRNA and protein microarray analysis in complex cell profiling.

UNLABELLED: This study combines mRNA and protein analysis using cDNA and antibody microarray techniques, respectively. These create a novel, integrated perspective into cellular molecular profiles. The aims of this study were to establish a reliable way of integrating these two approaches in order to obtain complex molecular profiles of the cell and to find suitable methods to normalize the data obtained using these approaches.

Antibody microarray and cDNA microarray techniques were used to study expression alterations in HL-60 cells that were differentiated into granulocytes using all-trans retinoic acid (ATRA). We selected this model to evaluate this combined profiling technique because the expression levels of most of the mRNA and protein species in these cells are not altered; therefore it is easier to track and define those species that are changed. The proteins whose levels were altered included c-myc, c-jun, Pyk2, FAK, PKC, TRF1, NF-kappaB and certain caspase types. These proteins are involved in apoptosis and hematopoietic differentiation pathways, and some have also been reported to have oncogenic potential. We compared the results obtained using the two methods, verified them by immunoblotting analysis, and devised normalization approaches.

This is one of the first demonstrations that a combination of antibody microarray and cDNA microarray techniques is required for complex molecular profiling of cells based on multiple parameters. This approach allows a more detailed molecular phenotype of the given sample to be obtained. The results obtained using a combination of the two profiling methods are consistent with those from previous studies that used more traditional methods.

Microarray analysis using a limited amount of cells.

cDNA microarray technology is widely used in various biological and medical disciplines to determine gene expression profiles. Unfortunately, this technology requires a large quantity of input RNA. Since there is an increasing need for more precise analyses of defined cell subpopulations with low cell counts, working protocols using a minimal number of input cells are required. Optimal RNA isolation and its accurate amplification are crucial to the success of these protocols. The HL-60 cell line was used in the search for a suitable protocol that can be used for clinical samples of CD34+ haematopoietic cells obtained from bone marrow. The goal was to discover the best method for isolating and amplifying RNA from a small number of cells. Our evaluation of various methods and kits available in the market revealed that the combination of RNAqueous Kit for RNA isolation and the SenseAmp Plus Kit for one-round RNA amplification produced the best results. This article presents a verified protocol describing a reliable and reproducible method for obtaining enough input RNA for microarray experiments when the number of cells is limited.

Distinct patterns of histone methylation and acetylation in human interphase nuclei.

To study 3D nuclear distributions of epigenetic histone modifications such as H3(K9) acetylation, H3(K4) dimethylation, H3(K9) dimethylation, and H3(K27) trimethylation, and of histone methyltransferase Suv39H1, we used advanced image analysis methods, combined with Nipkow disk confocal microscopy. Total fluorescence intensity and distributions of fluorescently labelled proteins were analyzed in formaldehyde-fixed interphase nuclei. Our data showed reduced fluorescent signals of H3(K9) acetylation and H3(K4) dimethylation (di-me) at the nuclear periphery, while di-meH3(K9) was also abundant in chromatin regions closely associated with the nuclear envelope. Little overlapping (intermingling) was observed for di-meH3(K4) and H3(K27) trimethylation (tri-me), and for di-meH3(K9) and Suv39H1. The histone modifications studied were absent in the nucleolar compartment with the exception of H3(K9) dimethylation that was closely associated with perinucleolar regions which are formed by centromeres of acrocentric chromosomes. Using immunocytochemistry, no di-meH3(K4) but only dense di-meH3(K9) was found for the human acrocentric chromosomes 14 and 22. The active X chromosome was observed to be partially acetylated, while the inactive X was more condensed, located in a very peripheral part of the interphase nuclei, and lacked H3(K9) acetylation. Our results confirmed specific interphase patterns of histone modifications within the interphase nuclei as well as within their chromosome territories.

Nuclear topography of the c-myc gene in human leukemic cells.

The c-myc gene plays an essential role in the regulation of the cell cycle and differentiation. Therefore, changes of the c-myc positioning during differentiation are of great interest. As a model system of cell differentiation, the HL-60 and U-937 human leukemic cell lines were used in our experiments. These cells can be induced to differentiation into granulocytes that represent one of the pathways of blood cell maturation. In this study, changes of the topographic characteristics of the c-myc gene (8q24), centromeric region of chromosome 8 and chromosome 8 domain during differentiation of HL-60 and U-937 cells were detected using fluorescence in-situ hybridisation (FISH). FISH techniques and fluorescence microscopy combined with image acquisition and analysis (high-resolution cytometry) were used in order to detect the topographic features of nuclear chromatin. Increased centre of nucleus-to-gene and gene-to-gene distances of c-myc genes, centromeric region of chromosome 8 and chromosome 8 domains were found early after the induction of granulocytic differentiation by dimethyl sulfoxide (DMSO) or retinoic acid (RA); the size of the chromosome 8 domains was rapidly reduced. In differentiated cells, c-myc is located at greater distances from the centromeric regions of chromosome 8. These results support the idea that relocation of the c-myc gene to the nuclear periphery and the condensation of the chromosome 8 domain might be associated with the c-myc gene expression due to common kinetics during granulocytic differentiation.

The influence of the cell cycle, differentiation and irradiation on the nuclear location of the abl, bcr and c-myc genes in human leukemic cells.

abl and bcr genes play an important role in the diagnostics of chronic myelogenous leukemia (CML). The translocation of these genes results in an abnormal chromosome 22 called the Philadelphia chromosome (Ph). The chimeric bcr-abl gene is a fundamental phenomenon in the pathogenesis of CML. Malignant transformation of hematopoietic cells is also accompanied by the c-myc gene changes (translocation, amplification). Nuclear topology of the abl, bcr and c-myc genes was determined in differentiated as well as in irradiated HL-60 cells using dual-colour fluorescence in situ hybridisation and image analysis by means of a high resolution cytometer. After the induction of the granulocytic differentiation of HL-60 cells with all trans retinoic acid (ATRA) or dimethylsulfoxide (DMSO), the abl and bcr homologous genes were repositioned closer to the nuclear periphery and the average distances between homologous abl-abl and bcr-bcr genes as well as between heterologous abl-bcr genes were elongated as compared with untreated human leukemic promyelocytic HL-60 cells. Elongated gene-to-gene and centre-to-gene distances were also found for the c-myc gene during granulocytic differentiation. In the case of the monocytic maturation of HL-60 cells treated with phorbol esters (PMA), the abl and bcr homologous genes were repositioned closer to each other and closer to the nuclear centre. The position of the c-myc gene did not change significantly after the PMA stimulus. The proximity of the abl and bcr genes was also found after gamma irradiation using 60Co (5 Gy). Immediately after the gamma irradiation c-myc was repositioned closer to the nuclear centre, but 24 h after radiation exposure the c-myc position returned back to the pretreatment level. The c-myc gene topology after gamma irradiation (when the cells are blocked in G2 phase) was different from that detected in the G2 sorted control population. We suggest that changes in the abl, bcr and c-myc topology in the case of gamma irradiation are not the effects of the cell cycle. It is possible, that differences in the cell cycle of hematopoietic cells after the gamma irradiation and concurrent proximity of the abl, bcr and c-myc genes could be important from the point of view of contingent gene translocations, that are responsible for malignant transformation of cells.

Chromosomes participating in translocations typical of malignant hemoblastoses are also involved in exchange aberrations induced by fast neutrons.

Repeated triple-color fluorescence in situ hybridization was used for the detection of exchange aberrations among 10 selected chromosomes of human lymphocytes irradiated with three doses of fast neutrons with a mean energy of 7 MeV. In each hybridization two different pairs of chromosomes were stained. Defined stage positions of metaphases on a slide were stored on a hard disk and an automatic scan of images according to these positions was performed after six successive hybridizations. In this way we obtained six different images of the same metaphase with differently stained pairs of chromosomes and centromeres. The comparison of these images enabled the identification of mutual exchanges between chromosomes 1, 2, 3, 4, 8, 9, 12, 14, 18 and 22. The frequencies of exchanges were not linearly proportional to the molecular weight of interacting chromosomes. The most significant were exchanges between chromosomes 14/18, 14/8, 18/8, 8/3, 1/14, 1/8, 3/18, 3/14 and 9/22. The results indicate significant interactions between chromosomes involved in translocations in B-cell non-Hodgkin's lymphoma and chronic myeloid leukemia. We propose that the reason for the high frequency of exchanges between these chromosomes is their proximity in the cell nucleus. It may also be one of the reasons for the induction of specific translocations leading to malignant transformation of cells.

Spatial distribution of selected genetic loci in nuclei of human leukemia cells after irradiation.

Fluorescence in situ hybridization (FISH) combined with high-resolution cytometry was used to determine the topographic characteristics of the centromeric heterochromatin (of the chromosomes 6, 8, 9, 17) and the tumor suppressor gene TP53 (which is located on chromosome 17) in cells of the human leukemia cell lines ML-1 and U937. Analysis was performed on cells that were either untreated or irradiated with gamma rays and incubated for different intervals after exposure. Compared to untreated cells, homologous centromeres and the TP53 genes were found closer to each other and also closer to the nuclear center 2 h after irradiation. The spatial relationship between genetic elements returned to that of the unirradiated controls during the next 2-3 h. Statistical evaluation of our experimental results shows that homologous centromeres and the homologous genes are positioned closer to each other 2 h after irradiation because they are localized closer to the center of the nucleus (probably due to more pronounced decondensation of the chromatin related to repair). This radial movement of genetic loci, however, is not connected with repair of DSBs by processes involving homologous recombination, because the angular distribution of homologous sequences remains random after irradiation.

Theoretical versus experimental resolution in optical microscopy.

The aim of this article is to compare experimental resolution under different conditions with theoretical resolution predicted using electromagnetic diffraction theory. Imaging properties of fluorescent beads of three different diameters (0.1 microm, 0.2 microm, and 0.5 microm) as well as imaging properties of four different fluorescence-stained DNA targets (ABL gene, BCR gene, centromere 6, and centromere 17) are studied. It is shown how the dependence of the resolution on object size varies with wavelength (520 nm versus 580 nm), type of microscopy (wide-field, confocal using Nipkow disk, confocal laser scanning) and basic image processing steps (median and gaussian filters). Furthermore, specimen influence on the resolution was studied (the influence of embedding medium, coverglass thickness, and depth below the coverglass). Both lateral and axial resolutions are presented. The results clearly show that real objects are far from being points and that experimental resolution is often much worse than the theoretical one. Although the article concentrates on fluorescence imaging using high NA objectives, similar dependence can also be expected for other optical arrangements.

Exchange aberrations among 11 chromosomes of human lymphocytes induced by gamma-rays.

PURPOSE: To detect the frequencies of interchanges among 11 chromosomes in lymphocytes irradiated with gamma-rays and to find out whether these frequencies reflect the proximity of some of these chromosomes within the interphase nucleus. MATERIAL AND METHODS: Exchange aberrations were detected in the first mitosis after irradiation of human lymphocytes with 3 and 5 Gy gamma-rays of 60Co. Two-colour repeated FISH with two differently chemically modified probes in each hybridization was applied. The microscope stage positions of each mitosis were recorded after the first hybridization and used for the automatic scanning of images after all successive experiments. Five images were obtained for each mitosis differing in visualized pairs of chromosomes. Comparing these images, exchanges among 10 chromosomes could be detected. Painting of the p arm of chromosome 21 with the painting probe for chromosome 22 also made it possible to detect exchanges of this chromosome with other chromosomes of the selected group. RESULTS: Frequencies of exchange aberrations induced in chromosomes of the selected group as well as interchanges between many pairs of chromosomes of this group were roughly proportional to the DNA content of chromosomes. Higher frequencies of interchanges than expected according to the model of linear proportionality were found between several chromosomes involved in translocations frequent in different subtypes of leukaemia. CONCLUSIONS: Frequencies of interchanges among 11 chromosomes of human lymphocytes induced by gamma-rays do not indicate as clearly as fast neutrons the non-random arrangement of chromosomes in the cell nucleus. The interaction of a large number of chromosomes in exchange aberrations suggests that the chromatin in the territory of one chromosome is accessible for several other chromosomes.

Automated microaxial tomography of cell nuclei after specific labelling by fluorescence in situ hybridisation.

Microaxial tomography provides a good means for microscopic image acquisition of cells or sub-cellular components like cell nuclei with an improved resolution, because shortcomings of spatial resolution anisotropy in optical microscopy can be overcome. Thus, spatial information of the object can be obtained without the necessity of confocal imaging. Since the very early developments of microaxial tomography, a considerable drawback of this method was a complicated image acquisition and processing procedure that requires much operator time. In order to solve this problem the Heidelberg 2pi-tilting device has been mounted on the Brno high-resolution cytometer as an attempt to bring together advanced microscopy and fast automated computer image acquisition and analysis. A special software module that drives all hardware components required for automated microaxial tomography and performs image acquisition and processing has been developed. First, a general image acquisition strategy is presented. Then the procedure for automation of axial tomography and the developed software module are described. The rotation precision has been experimentally proved followed by experiments with a specific biological example. For this application, also a method for the preparation of cell nuclei attached to glass fibres has been developed that allows for the first time imaging of three-dimensionally conserved, fluorescence in situ hybridisation-stained cell nuclei fixed to a glass fibre.

Comparative transcriptome maps: a new approach to the diagnosis of colorectal carcinoma patients using cDNA microarrays.

The progression of colorectal cancer involves accumulation of various genetic and epigenetic events that dramatically change gene expression. The aim of this study was to investigate a possible new approach to the diagnosis of colorectal carcinoma patients, based on their gene expression profiles. Human 19K cDNA microarrays were used to analyze the gene expression profiles of 18 colorectal carcinoma patients. Transcriptome maps (TMs) were analyzed to detect chromosomal regions that could serve as potential diagnostic markers for colon cancer. A comparison of TMs showed chromosome regions with conserved changes of gene expression typical of colorectal cancer in general, and also patient-specific variable regions. We identified 195 genes with significantly altered expression in colon cancer. Functional analysis of the regulated genes distinguished three main categories: biological processes, cellular components, and molecular functions. We found that different patients had chromosome regions characterized by very similar changes of gene expression, probably linked to the most fundamental events in carcinogenesis. On the other hand, variable chromosome regions can be patient-specific. The variable regions may provide further information on the individual pathogenesis and prognosis of the patient. Comparison of TMs is proposed as a tool to facilitate diagnosis and treatment planning for individual patients.

An efficient algorithm for measurement and correction of chromatic aberrations in fluorescence microscopy.

Even the best optical microscopes available on the market exhibit chromatic aberrations to some extent. In some types of study, chromatic aberrations of current optics cannot be neglected and a software correction is highly desirable. This paper describes a novel method of chromatic aberration measurement and software correction using sub-resolution bead imaging and computer image analysis. The method is quick, precise and enables the determination of both longitudinal and lateral chromatic aberrations. Correction function can be computed in about half an hour, including image acquisition. Using this approach, chromatic aberrations can be reduced to 10-20 nm laterally and 10-60 nm axially depending on the type of optical set-up. The method is especially suitable for fluorescence microscopy, where a limited number of wavelengths are observed.

Efficient real-time confocal microscopy with white light sources.

The main advantage of confocal microscopes over their conventional counterparts arises from their ability to optically 'section' nearly transparent materials; the thin image slices thus obtained can be used to reconstruct three-dimensional images, a capability which is particularly useful for the study of biological specimens. Confocal microscopes have previously used either a single laser-illuminated point-source and single point-detector (which are scanned in tandem across the object) or white-light illumination with multiple point-sources and detectors. Single-point-source systems, however, do not usually form images in real time and are restricted to using available laser wavelengths. Multiple-point-source systems, on the other hand, produce images in real time but use light very inefficiently--typically 1% or less is used for imaging. Here we demonstrate a white-light, multiple-point-source method which can in principle produce images in real time with light efficiencies as high as 50%. This system is likely to find broad practical application, particularly in the imaging of weakly reflecting or weakly fluorescent specimens.

Precise 3D image alignment in micro-axial tomography.

Micro (micro-) axial tomography is a challenging technique in microscopy which improves quantitative imaging especially in cytogenetic applications by means of defined sample rotation under the microscope objective. The advantage of micro-axial tomography is an effective improvement of the precision of distance measurements between point-like objects. Under certain circumstances, the effective (3D) resolution can be improved by optimized acquisition depending on subsequent, multi-perspective image recording of the same objects followed by reconstruction methods. This requires, however, a very precise alignment of the tilted views. We present a novel feature-based image alignment method with a precision better than the full width at half maximum of the point spread function. The features are the positions (centres of gravity) of all fluorescent objects observed in the images (e.g. cell nuclei, fluorescent signals inside cell nuclei, fluorescent beads, etc.). Thus, real alignment precision depends on the localization precision of these objects. The method automatically determines the corresponding objects in subsequently tilted perspectives using a weighted bipartite graph. The optimum transformation function is computed in a least squares manner based on the coordinates of the centres of gravity of the matched objects. The theoretically feasible precision of the method was calculated using computer-generated data and confirmed by tests on real image series obtained from data sets of 200 nm fluorescent nano-particles. The advantages of the proposed algorithm are its speed and accuracy, which means that if enough objects are included, the real alignment precision is better than the axial localization precision of a single object. The alignment precision can be assessed directly from the algorithm's output. Thus, the method can be applied not only for image alignment and object matching in tilted view series in order to reconstruct (3D) images, but also to validate the experimental performance (e.g. mechanical precision of the tilting). In practice, the key application of the method is an improvement of the effective spatial (3D) resolution, because the well-known spatial anisotropy in light microscopy can be overcome. This allows more precise distance measurements between point-like objects.

Optical transfer functions of 4Pi confocal microscopes: theory and experiment.

We measure the point-spread function in the two main configurations of 4Pi confocal microscopy as well as in the traditional confocal arrangement and derive the optical transfer functions from the experimental data. The optical transfer functions are in good agreement with their theoretical counterparts. We find a 3.5- to 5-fold increased axial bandwidth of the 4Pi confocal microscope and hence confirm the enhanced spatial-frequency content of 4Pi images.

Confocal microscopy by aperture correlation.

Most confocal microscopes do not produce images in real time with nonlaser light sources. The tandem scanning confocal microscope does produce such images but, because the pinhole apertures of the Nipkov disk must be placed far apart to reduce cross talk between neighboring pinholes, only 1% or less of the light available for imaging is used. We show that, by using aperture correlation techniques and relaxing the requirement to obtain a pure confocal image directly, one can obtain real-time confocal images with a dramatically increased (25% or even 50%) light budget.