Combining multicolor FISH with fluorescence lifetime imaging for chromosomal identification and chromosomal sub structure investigation

Understanding the structure of chromatin in chromosomes during normal and diseased state of cells is still one of the key challenges in structural biology. Using DAPI staining alone together with Fluorescence lifetime imaging (FLIM), the environment of chromatin in chromosomes can be explored. Fluorescence lifetime can be used to probe the environment of a fluorophore such as energy transfer, pH and viscosity. Multicolor FISH (M-FISH) is a technique that allows individual chromosome identification, classification as well as assessment of the entire genome. Here we describe a combined approach using DAPI as a DNA environment sensor together with FLIM and M-FISH to understand the nanometer structure of all 46 chromosomes in the nucleus covering the entire human genome at the single cell level. Upon DAPI binding to DNA minor groove followed by fluorescence lifetime measurement and imaging by multiphoton excitation, structural differences in the chromosomes can be studied and observed. This manuscript provides a blow by blow account of the protocol required to perform M-FISH-FLIM of whole chromosomes

Combining Multicolor FISH with Fluorescence Lifetime Imaging for Chromosomal Identification and Chromosomal Sub Structure Investigation

Understanding the structure of chromatin in chromosomes during normal and diseased state of cells is still one of the key challenges in structural biology. Using DAPI staining alone together with Fluorescence lifetime imaging (FLIM), the environment of chromatin in chromosomes can be explored. Fluorescence lifetime can be used to probe the environment of a fluorophore such as energy transfer, pH and viscosity. Multicolor FISH (M-FISH) is a technique that allows individual chromosome identification, classification as well as assessment of the entire genome. Here we describe a combined approach using DAPI as a DNA environment sensor together with FLIM and M-FISH to understand the nanometer structure of all 46 chromosomes in the nucleus covering the entire human genome at the single cell level. Upon DAPI binding to DNA minor groove followed by fluorescence lifetime measurement and imaging by multiphoton excitation, structural differences in the chromosomes can be studied and observed. This manuscript provides a blow by blow account of the protocol required to perform M-FISH-FLIM of whole chromosomes

In-vivo anterior segment OCT imaging provides unique insight into cerulean blue-dot opacities and cataracts in Down syndrome

Down syndrome (DS) is frequently associated with cataract, but there remains scant information about DS cataract morphology. Supra-nuclear cataracts in DS have been proposed as indicative of beta-amyloid (Aβ) aggregation and thus potential biomarkers for Alzheimer’s (AD). This study employed anterior segment OCT (AS-OCT) and slit-lamp (SL) photography to image the crystalline lens in DS, compared with adult controls. Lens images were obtained post-dilation. Using MATLAB, AS-OCT images were analysed and lens opacities calculated as pixel intensity and area ratios. SL images were classified using LOCS III. Subjects were n = 28 DS (mean ± SD 24.1 ± 14.3years), and n = 36 controls (54.0 ± 3.4years). For the DS group, AS-OCT imaging revealed the frequent presence of small dot opacities (27 eyes, 50%) in the cortex and nucleus of the lens, covering an area ranging from 0.2–14%. There was no relation with age or visual acuity and these dot opacities (p > 0.5) and they were not present in any control lenses. However, their location and morphology does not coincide with previous reports linking these opacities with Aβ accumulation and AD. Four participants (14%) in the DS group had clinically significant age-related cataracts, but there was no evidence of early onset of age-related cataracts in DS

In-vivo anterior segment OCT imaging provides unique insight into cerulean blue-dot opacities and cataracts in Down syndrome

Down syndrome (DS) is frequently associated with cataract, but there remains scant information about DS cataract morphology. Supra-nuclear cataracts in DS have been proposed as indicative of betaamyloid (Aß) aggregation and thus potential biomarkers for Alzheimer’s (AD). This study employed anterior segment OCT (AS-OCT) and slit-lamp (SL) photography to image the crystalline lens in DS, compared with adult controls. Lens images were obtained post-dilation. Using MATLAB, AS-OCT images were analysed and lens opacities calculated as pixel intensity and area ratios. SL images were classifed using LOCS III. Subjects were n=28 DS (mean±SD 24.1±14.3years), and n=36 controls (54.0±3.4years). Forthe DS group,AS-OCT imaging revealed the frequent presence of small dot opacities (27 eyes, 50%) in the cortex and nucleus ofthe lens, covering an area ranging from 0.2–14%. There was no relation with age or visual acuity and these dot opacities (p>0.5) and they were not present in any control lenses. However, their location and morphology does not coincide with previous reports linking these opacities with Aß accumulation andAD. Four participants (14%) in the DS group had clinically signifcant age-related cataracts, butthere was no evidence of early onset of age-related cataracts in DS.Peer ReviewedPostprint (author's final draft

Structural and biochemical studies of the S. cerevisiae condensin complex

The Structural Maintenance of Chromosomes (SMC) complex family forms an essential part of the genome’s organisational machinery in all domains of life. In eukaryotes condensin is one such complex that mediates the packaging of DNA for segregation during cell division. This pentameric ATPase is comprised of two elongated Smc proteins (Smc2 and Smc4), a kleisin type protein (Brn1) and two HEAT repeat proteins (Ycg1 and Ycs4). Despite the important role of condensin in such fundamental cell processes its overall structure, and consequently its molecular mechanism, are currently unknown. This study describes the processes leading to production of the first atomic resolution model of the Saccharomyces cerevisiae condensin complex in the ATP free apo state by cryo electronmicroscopy. This model demonstrates that several features such as a folded conformation in the Smc protein arms are common to multiple members of the SMC complex family. Furthermore, previously described structural features such as the location of the Ycs4 subunit in contact with the Smc protein head domains, and a discontinuity in the arms of the Smc proteins called the joint, are also described as part of the whole complex. Finally, the position of the Ycg1 subunit is also described as sitting flexibly under the Smc head domains, anchored by the Brn1 subunit to the rest of the complex. Also described in this study is preliminary work to further characterise the biochemical properties of the condensin complex in the context of chromatin using the Lumix C-trap optical tweezer system. Condensins’ ability to compact DNA in real time and to translocate on a fixed DNA substrate in the C-trap system are demonstrated, as is ongoing work to chromatinise the same DNA substrate in situ. This chromatinisation involves the purification of recombinant S. cerevisiae histone octamers and histone chaperone Nap1 which have been successfully applied to DNA within the C-trap to create a chromatin substrate suitable for future experimentation.Open Acces

Human Metaphase Chromosome Analysis using Image Processing

Development of an effective human metaphase chromosome analysis algorithm can optimize expert time usage by increasing the efficiency of many clinical diagnosis processes. Although many methods exist in the literature, they are only applicable for limited morphological variations and are specific to the staining method used during cell preparation. They are also highly influenced by irregular chromosome boundaries as well as the presence of artifacts such as premature sister chromatid separation. Therefore an algorithm is proposed in this research which can operate with any morphological variation of the chromosome across images from multiple staining methods. The proposed algorithm is capable of calculating the segmentation outline, the centerline (which gives the chromosome length), partitioning of the telomere regions and the centromere location of a given chromosome. The algorithm also detects and corrects for the sister chromatid separation artifact in metaphase cell images. A metric termed the Candidate Based Centromere Confidence (CBCC) is proposed to accompany each centromere detection result of the proposed method, giving an indication of the confidence the algorithm has on a given localization. The proposed method was first tested for the ability of calculating an accurate width profile against a centerline based method [1] using 226 chromosomes. A statistical analysis of the centromere detection error values proved that the proposed method can accurately locate centromere locations with statistical significance. Furthermore, the proposed method performed more consistently across different staining methods in comparison to the centerline based approach. When tested with a larger data set of 1400 chromosomes collected from a set of DAPI (4\u27,6-diamidino-2-phenylindole) and Giemsa stained cell images, the proposed candidate based centromere detection algorithm was able to accurately localize 1220 centromere locations yielding a detection accuracy of 87%

Biometrics

Biometrics uses methods for unique recognition of humans based upon one or more intrinsic physical or behavioral traits. In computer science, particularly, biometrics is used as a form of identity access management and access control. It is also used to identify individuals in groups that are under surveillance. The book consists of 13 chapters, each focusing on a certain aspect of the problem. The book chapters are divided into three sections: physical biometrics, behavioral biometrics and medical biometrics. The key objective of the book is to provide comprehensive reference and text on human authentication and people identity verification from both physiological, behavioural and other points of view. It aims to publish new insights into current innovations in computer systems and technology for biometrics development and its applications. The book was reviewed by the editor Dr. Jucheng Yang, and many of the guest editors, such as Dr. Girija Chetty, Dr. Norman Poh, Dr. Loris Nanni, Dr. Jianjiang Feng, Dr. Dongsun Park, Dr. Sook Yoon and so on, who also made a significant contribution to the book

Development of polarization-resolved optical scanning microscopy imaging techniques to study biomolecular organizations

Light, as electromagnetic radiation, conveys energy through space and time via fluctuations in electric and magnetic fields. This thesis explores the interaction of light and biological structures through polarization-resolved imaging techniques. Light microscopy, and polarization analysis enable the examination of biological entities. Biological function often centers on chromatin, the genetic material composed of DNA wrapped around histone proteins within cell nuclei. This structure's chiral nature gives rise to interactions with polarized light. This research encompasses three main aspects. Firstly, an existing multimodal Circular Intensity Differential Scattering (CIDS) and fluorescence microscopy are upgraded into an open configuration to be integrated with other modalities. Secondly, a novel cell classification method employing CIDS and a phasor representation is introduced. Thirdly, polarization analysis of fluorescence emission is employed for pathological investigations. Accordingly, the thesis is organized into three chapters. Chapter 1 lays the theoretical foundation for light propagation and polarization, outlining the Jones and Stokes-Mueller formalisms. The interaction between light and optical elements, transmission, and reflection processes are discussed. Polarized light's ability to reveal image contrast in polarizing microscopes, linear and nonlinear polarization-resolved microscopy, and Mueller matrix microscopy as a comprehensive technique for studying biological structures are detailed. Chapter 2 focuses on CIDS, a label-free light scattering method, including a single point angular spectroscopy mode and scanning microscopy imaging. A significant upgrade of the setup is achieved, incorporating automation, calibration, and statistical analysis routines. An intuitive phasor approach is proposed, enabling image segmentation, cell discrimination, and enhanced interpretation of polarimetric contrast. As a result, image processing programs have been developed to provide automated measurements using polarization-resolved laser scanning microscopy imaging integrated with confocal fluorescence microscopy of cells and chromatin inside cell nuclei, including the use of new types of samples such as progeria cells. Chapter 3 applies a polarization-resolved two-photon excitation fluorescence (2PEF) microscopy to study multicellular cancerous cells. A homemade 2PEF microscope is developed for colon cancer cell analysis. The integration of polarization and fluorescence techniques leads to a comprehensive understanding of the molecular orientation within samples, particularly useful for cancer diagnosis. Overall, this thesis presents an exploration of polarization-resolved imaging techniques for studying biological structures, encompassing theory, experimental enhancements, innovative methodologies, and practical applications