Towards many colors in FISH on 3D-preserved interphase nuclei

The article reviews the existing methods of multicolor FISH on nuclear targets, first of all, interphase chromosomes. FISH proper and image acquisition are considered as two related components of a single process. We discuss (1) M-FISH (combinatorial labeling + deconvolution + widefield microscopy); (2) multicolor labeling + SIM (structured illumination microscopy); (3) the standard approach to multicolor FISH + CLSM (confocal laser scanning microscopy; one fluorochrome - one color channel); (4) combinatorial labeling + CLSM; (5) non-combinatorial labeling + CLSM + linear unmixing. Two related issues, deconvolution of images acquired with CLSM and correction of data for chromatic Z-shift, are also discussed. All methods are illustrated with practical examples. Finally, several rules of thumb helping to choose an optimal labeling + microscopy combination for the planned experiment are suggested. Copyright (c) 2006 S. Karger AG, Basel

A Computational Framework for the Structural Change Analysis of 3D Volumes of Microscopic Specimens

Glaucoma, commonly observed with an elevation in the intraocular pressure level (IOP), is one of the leading causes of blindness. The lamina cribrosa is a mesh-like structure that provides axonal support for the optic nerves leaving the eye. The changes in the laminar structure under IOP elevations may result in the deaths of retinal ganglion cells, leading to vision degradation and loss. We have developed a comprehensive computational framework that can assist the study of structural changes in microscopic structures such as lamina cribrosa. The optical sectioning property of a confocal microscope facilitates imaging thick microscopic specimen at various depths without physical sectioning. The confocal microscope images are referred to as optical sections. The computational framework developed includes: 1) a multi-threaded system architecture for tracking a volume-of-interest within a microscopic specimen in a parallel computation environment using a reliable-multicast for collective-communication operations 2) a Karhunen-Loève (KL) expansion based adaptive noise prefilter for the restoration of the optical sections using an inverse restoration method 3) a morphological operator based ringing metric to quantify the ringing artifacts introduced during iterative restoration of optical sections 4) a l2 norm based error metric to evaluate the performance of optical flow algorithms without a priori knowledge of the true motion field and 5) a Compute-and-Propagate (CNP) framework for iterative optical flow algorithms. The realtime tracking architecture can convert a 2D-confocal microscope into a 4D-confocal microscope with tracking. The adaptive KL filter is suitable for realtime restoration of optical sections. The CNP framework significantly improves the speed and convergence of the iterative optical flow algorithms. Also, the CNP framework can reduce the errors in the motion field estimates due to the aperture problem. The performance of the proposed framework is demonstrated on real-life image sequences and on z-Stack datasets of random cotton fibers and lamina cribrosa of a cow retina with an experimentally induced glaucoma. The proposed framework can be used for routine laboratory and clinical investigation of microstructures such as cells and tissues, for the evaluation of complex structures such as cornea and has potential use as a surgical guidance tool

Efficient Bayesian-based Multi-View Deconvolution

Light sheet fluorescence microscopy is able to image large specimen with high resolution by imaging the sam- ples from multiple angles. Multi-view deconvolution can significantly improve the resolution and contrast of the images, but its application has been limited due to the large size of the datasets. Here we present a Bayesian- based derivation of multi-view deconvolution that drastically improves the convergence time and provide a fast implementation utilizing graphics hardware.Comment: 48 pages, 20 figures, 1 table, under review at Nature Method

SR-FTiR microscopy and FTIR imaging in the earth sciences

During the last decades, several books have been devoted to the application of spectroscopic methods in mineralogy. Several short courses and meetings have addressed particular aspects of spectroscopy, such as the analysis of hydrous components in minerals and Earth materials. In these books, complete treatment of the infrared theory and practical aspects of instrumentation and methods, along with an exhaustive list of references, can be found. The present chapter is intended to cover those aspects of infrared spectroscopy that have been developed in the past decade and are not included in earlier reviews such as Volume 18 of Reviews in Mineralogy. These new topics involve primarily: (1) the use of synchrotron radiation (SR), which, although not a routine method, is now rather extensively applied in infrared studies, in particular those requiring ultimate spatial and time resolution and the analysis of extremely small samples (a few tens of micrometers); (2) the development of imaging techniques also for foreseen time resolved studies of geo-mineralogical processes and environmental studies.Comment: 36 pages, 24 figures - Reviews in Mineralogy & Geochemistry - Vol. 78 (2013) in pres

Tandem Scanning Reflected Light Microscopy: Applications in Clinical Dental Research

The Tandem Scanning Reflected Light Microscope (TSRLM) enables the investigation of microscopic structures both at and deep to the surface of intact objects. The present paper reviews studies undertaken to determine whether the TSRLM would be usable and useful in the investigation of natural and restorative dental materials in vitro and in vivo. It was found that the TSRLM could be used to study normal and diseased dental tissues and the new materials which are used to replace lost substance. More importantly, it could be used to characterize the interface between tooth and optically translucent materials in bulk samples, giving high resolution information from not only a shallow depth of field, but at planes below cut surfaces. This makes it possible to study interfacial regions in three dimensions without the risk of delamination that must accompany the preparation of a microscopic section. The use of fluorescent markers enables more information to be derived from the tooth/adhesive interface. Studies to date indicate the need for the development of adhesion promoting agents which incorporate a fluorescent radical in their molecular structure. Preliminary work using the instrument for observation of cutting interactions between a high speed bur and a tooth indicates some useful potential in the study of cavity preparation techniques and tissue failure mechanisms. Recent developments of the TSRLM for three dimensional imaging in other dental applications are outlined. This microscope is an important advance in the microscopic assessment of adaptation of biomaterials to hard tissues

A virtual world of paleontology

Computer-aided visualization and analysis of fossils has revolutionized the study of extinct organisms. Novel techniques allow fossils to be characterized in three dimensions and in unprecedented detail. This has enabled paleontologists to gain important insights into their anatomy, development, and preservation. New protocols allow more objective reconstructions of fossil organisms, including soft tissues, from incomplete remains. The resulting digital reconstructions can be used in functional analyses, rigorously testing long-standing hypotheses regarding the paleobiology of extinct organisms. These approaches are transforming our understanding of long-studied fossil groups, and of the narratives of organismal and ecological evolution that have been built upon them

Bioimage informatics in STED super-resolution microscopy

Optical microscopy is living its renaissance. The diffraction limit, although still physically true, plays a minor role in the achievable resolution in far-field fluorescence microscopy. Super-resolution techniques enable fluorescence microscopy at nearly molecular resolution. Modern (super-resolution) microscopy methods rely strongly on software. Software tools are needed all the way from data acquisition, data storage, image reconstruction, restoration and alignment, to quantitative image analysis and image visualization. These tools play a key role in all aspects of microscopy today – and their importance in the coming years is certainly going to increase, when microscopy little-by-little transitions from single cells into more complex and even living model systems. In this thesis, a series of bioimage informatics software tools are introduced for STED super-resolution microscopy. Tomographic reconstruction software, coupled with a novel image acquisition method STED< is shown to enable axial (3D) super-resolution imaging in a standard 2D-STED microscope. Software tools are introduced for STED super-resolution correlative imaging with transmission electron microscopes or atomic force microscopes. A novel method for automatically ranking image quality within microscope image datasets is introduced, and it is utilized to for example select the best images in a STED microscope image dataset.Siirretty Doriast

Determining safe laser conditions for the cleaning of historic stained-glasses by laser ablation

New ultra-short pulsed lasers open new opportunities in the developing of new safe restoration techniques to be applied in stained-glass samples. The pulse duration that are available limit the volume of the sample that it is affected when these lasers are focused on a surface. This allows developing laser cleaning protocols that facilitates the elimination of external layers without deteriorating the layers that are below them, in this case, the glass or the grisaille. In this work, the use of a 238 fs UV laser has been studied. It has been determined that this laser can induced two types of damage on the glass. A mechanical one associated with the ablation of the sample surface and an optical one, associated with a change in color that modifies the transmittance spectra of the glasses and increases the absorption level in the UV range of the spectra. The mechanical and optical damage thresholds have been determined on microscope slides and on contemporary stained-glass samples with different colors. In colorless samples, the damage thresholds can be measured using the Lui method that allows to measure the size of the laser beam and the level of energy to damage the sample. Using these results, it has been possible to define some laser cleaning protocols on stained-glass samples painted with permanent ink. Good results are obtained in a multi-step process in which the initial step is performed with a higher level of energy in order to remove a high percentage of the ink layer and after the cleaning is finished using several steps with energy levels below the optical damage threshold; Determinação de condições seguras de laser para a limpeza de vitrais históricos por ablação a laser RESUMO: Novos lasers pulsados ultracurtos abrem novas oportunidades no desenvolvimento de novas técnicas seguras de restauração a serem aplicadas em amostras de vitrais. A duração do pulso disponível limita o volume da amostra que é afetado quando esses lasers são focados em uma superfície. Isso permite desenvolver protocolos de limpeza a laser que facilitam a eliminação de camadas externas sem deteriorar as camadas que estão abaixo delas, neste caso, o vidro ou a grisalha. Neste trabalho, foi estudado o uso de um laser UV de 238 fs. Foi determinado que este laser pode induzir dois tipos de danos no vidro. Uma mecânica associada à ablação da superfície da amostra e uma óptica, associada a uma mudança de cor que modifica os espectros de transmitância dos vidros e aumenta o nível de absorção na faixa UV dos espectros. Os limites de danos mecânicos e ópticos foram determinados em lâminas de microscópio e em amostras de vitrais contemporâneos com cores diferentes. Em amostras incolores, os limites de dano podem ser medidos usando o método Lui que permite medir o tamanho do feixe de laser e o nível de energia para danificar a amostra. A partir desses resultados, foi possível definir alguns protocolos de limpeza a laser em amostras de vitrais pintados com tinta permanente. Bons resultados são obtidos em um processo de várias etapas em que a etapa inicial é realizada com um nível de energia maior para remover uma alta porcentagem da camada de tinta e após a limpeza ser finalizada usando várias etapas com níveis de energia abaixo do dano óptico limite

Applications of Surface Metrology to Issues in Art

This work investigates applying surface metrology techniques to issues in art, such as, restoration and identification. It seeks possible areas of collaboration between the Worcester Art Museum and Worcester Polytechnic Institute\u27s Surface Metrology Lab. Surface metrology is the study of measurement and analysis of surface textures, or roughness. We have completed an experiment to demonstrate that scanning laser microscopy and scale-sensitve fractal analysis used on paintings can discriminate brush types and paints