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Spectral imaging in preclinical research and clinical pathology.
Spectral imaging methods are attracting increased interest from researchers and practitioners in basic science, pre-clinical and clinical arenas. A combination of better labeling reagents and better optics creates opportunities to detect and measure multiple parameters at the molecular and cellular level. These tools can provide valuable insights into the basic mechanisms of life, and yield diagnostic and prognostic information for clinical applications. There are many multispectral technologies available, each with its own advantages and limitations. This chapter will present an overview of the rationale for spectral imaging, and discuss the hardware, software and sample labeling strategies that can optimize its usefulness in clinical settings
Development of a complete advanced computational workflow for high-resolution LDI-MS metabolomics imaging data processing and visualization
La imatge per espectrometria de masses (MSI) mapeja la distribució espacial de les molècules en una mostra. Això permet extreure informació Metabolòmica espacialment corralada d'una secció de teixit. MSI no s'usa àmpliament en la metabolòmica espacial a causa de diverses limitacions relacionades amb les matrius MALDI, incloent la generació d'ions que interfereixen en el rang de masses més baix i la difusió lateral dels compostos. Hem desenvolupat un flux de treball que millora l'adquisició de metabòlits en un instrument MALDI utilitzant un "sputtering" per dipositar una nano-capa d'Au directament sobre el teixit. Això minimitza la interferència dels senyals del "background" alhora que permet resolucions espacials molt altes. S'ha desenvolupat un paquet R per a la visualització d'imatges i processament de les dades MSI, tot això mitjançant una implementació optimitzada per a la gestió de la memòria i la programació concurrent. A més, el programari desenvolupat inclou també un algoritme per a l'alineament de masses que millora la precisió de massa.La imagen por espectrometría de masas (MSI) mapea la distribución espacial de las moléculas en una muestra. Esto permite extraer información metabolòmica espacialmente corralada de una sección de tejido. MSI no se usa ampliamente en la metabolòmica espacial debido a varias limitaciones relacionadas con las matrices MALDI, incluyendo la generación de iones que interfieren en el rango de masas más bajo y la difusión lateral de los compuestos. Hemos desarrollado un flujo de trabajo que mejora la adquisición de metabolitos en un instrumento MALDI utilizando un “sputtering” para depositar una nano-capa de Au directamente sobre el tejido. Esto minimiza la interferencia de las señales del “background” a la vez que permite resoluciones espaciales muy altas. Se ha desarrollado un paquete R para la visualización de imágenes y procesado de los datos MSI, todo ello mediante una implementación optimizada para la gestión de la memoria y la programación concurrente. Además, el software desarrollado incluye también un algoritmo para el alineamiento de masas que mejora la precisión de masa.Mass spectrometry imaging (MSI) maps the spatial distributions of molecules in a sample. This allows extracting spatially-correlated metabolomics information from tissue sections. MSI is not widely used in spatial metabolomics due to several limitations related with MALDI matrices, including the generation of interfering ions and in the low mass range and the lateral compound delocalization. We developed a workflow to improve the acquisition of metabolites using a MALDI instrument. We sputter an Au nano-layer directly onto the tissue section enabling the acquisition of metabolites with minimal interference of background signals and ultra-high spatial resolution. We developed an R package for image visualization and MSI data processing, which is optimized to manage datasets larger than computer’s memory using a mutli-threaded implementation. Moreover, our software includes a label-free mass alignment algorithm for mass accuracy enhancement
Sparse Proteomics Analysis - A compressed sensing-based approach for feature selection and classification of high-dimensional proteomics mass spectrometry data
Background: High-throughput proteomics techniques, such as mass spectrometry
(MS)-based approaches, produce very high-dimensional data-sets. In a clinical
setting one is often interested in how mass spectra differ between patients of
different classes, for example spectra from healthy patients vs. spectra from
patients having a particular disease. Machine learning algorithms are needed to
(a) identify these discriminating features and (b) classify unknown spectra
based on this feature set. Since the acquired data is usually noisy, the
algorithms should be robust against noise and outliers, while the identified
feature set should be as small as possible.
Results: We present a new algorithm, Sparse Proteomics Analysis (SPA), based
on the theory of compressed sensing that allows us to identify a minimal
discriminating set of features from mass spectrometry data-sets. We show (1)
how our method performs on artificial and real-world data-sets, (2) that its
performance is competitive with standard (and widely used) algorithms for
analyzing proteomics data, and (3) that it is robust against random and
systematic noise. We further demonstrate the applicability of our algorithm to
two previously published clinical data-sets
Mass Spectrometry-Based Proteomics Workflows in Cancer Research: The Relevance of Choosing the Right Steps
Mass spectrometry; Proteomics; WorkflowsEspectrometría de masas; Proteómica; Flujos de trabajoEspectrometria de masses; Proteòmica; Fluxos de treballThe qualitative and quantitative evaluation of proteome changes that condition cancer development can be achieved with liquid chromatography–mass spectrometry (LC-MS). LC-MS-based proteomics strategies are carried out according to predesigned workflows that comprise several steps such as sample selection, sample processing including labeling, MS acquisition methods, statistical treatment, and bioinformatics to understand the biological meaning of the findings and set predictive classifiers. As the choice of best options might not be straightforward, we herein review and assess past and current proteomics approaches for the discovery of new cancer biomarkers. Moreover, we review major bioinformatics tools for interpreting and visualizing proteomics results and suggest the most popular machine learning techniques for the selection of predictive biomarkers. Finally, we consider the approximation of proteomics strategies for clinical diagnosis and prognosis by discussing current barriers and proposals to circumvent them.This research was funded by the Research Council of Norway INFRASTRUKTUR-program (project number: 295910)
Fluorescence Multiplexing with Combination Probes for Biological and Diagnostic Applications
Cancer refers to a group of diseases containing more than 200 different subtypes. Cancer is a heterogeneous disease by nature, meaning that there are differences among tumors of the same type in different patients, and there are differences among cancer cells within a single tumor of one patient. Since cancer is not a single disease, nor does it have a single cause, it proves to be incredibly hard to diagnose and treat. The ability to study cellular markers, cell and tissue spatial arrangement, and gene function are all integral parts of cancer diagnostic and treatment efforts.
Here, I first present a review of current techniques for quantitative tissue imaging at cellular resolution. I broadly divide current imaging techniques into three categories: fluorescence-based, mass spectrometry-based, and sequencing-based. In this work, I primarily concentrate on fluorescence-based methods, with the focus being on our recently developed theory Multiplexing using Spectral Imaging and Combinatorics (MuSIC). The basis for MuSIC is to create combinations of fluorescent molecules (whether it be small molecule fluorophores or fluorescent proteins) to create unique spectral signatures.
I then present a protocol for labeling antibodies with combinations of small molecule fluorophores, which I refer to as MuSIC probes. I use fluorescent oligonucleotides (oligos) to arrange the fluorophores at specified distances and orientations from one another in order to produce complex fluorescence spectra when the probe is excited. This labeling protocol is demonstrated using a 3-probe experimental setup, bound to Protein A beads, and analyzed via spectral flow cytometry. When translating this method to staining human cells, our staining intensity was not comparable to that of a conventional antibody labeling kit. Therefore, next I present an improved method to label antibodies with MuSIC probes with increased signal intensity. I re-arrange the oligo-fluorophore arrangement of the MuSIC probe to emit an increased fluorescent signal. Then I validate this approach by comparing the staining intensity of MuSIC probe-labeled antibodies to a conventional antibody labeling kit using human peripheral blood mononuclear cells.
Lastly, I present simulation theories for the multiplexing capabilities of MuSIC probes for various biological and diagnostic applications. First, I present a theory for high-throughput genetic interaction screening using MuSIC probes generated from 18 currently available fluorescent proteins. Simulation studies based on constraints of current spectral flow cytometry equipment suggest our ability to perform genetic interaction screens at the human genome-scale. Finally, I adapt this simulation protocol to generate MuSIC probes from 30 currently available small-molecule fluorophores. Using the same constraints as before, I predict that I can perform cell-type profiling of 200+ analytes.
I hope that the work presented here provides a foundation for the use of combination probes for various biological and disease applications and ultimately help to better diagnose and treat different types of cancer
Mass Spectrometry-Based Proteomics Workflows in Cancer Research: The Relevance of Choosing the Right Steps
The qualitative and quantitative evaluation of proteome changes that condition cancer
development can be achieved with liquid chromatography–mass spectrometry (LC-MS). LC-MSbased
proteomics strategies are carried out according to predesigned workflows that comprise
several steps such as sample selection, sample processing including labeling, MS acquisition methods,
statistical treatment, and bioinformatics to understand the biological meaning of the findings and set
predictive classifiers. As the choice of best options might not be straightforward, we herein review
and assess past and current proteomics approaches for the discovery of new cancer biomarkers.
Moreover, we review major bioinformatics tools for interpreting and visualizing proteomics results
and suggest the most popular machine learning techniques for the selection of predictive biomarkers.
Finally, we consider the approximation of proteomics strategies for clinical diagnosis and prognosis
by discussing current barriers and proposals to circumvent them.Research Council of Norway INFRASTRUKTUR-program
(project number: 295910
Lung Disease Classification using Dense Alex Net Framework with Contrast Normalisation and Five-Fold Geometric Transformation
lung disease is one of the leading causes of death worldwide. Most cases of lung diseases are found when the disease is in an advanced stage. Therefore, the development of systems and methods that begin to diagnose quickly and prematurely plays a vital role in today's world. Currently, in detecting differences in lung cancer, an accurate diagnosis of cancer types is needed. However, improving the accuracy and reducing training time of the diagnosis remains a challenge. In this study, we have developed an automated classification scheme for lung cancer presented in histopathological images using a dense Alex Net framework. The proposed methodology carries out several phases includes pre-processing, contrast normalization, data augmentation and classification. Initially, the pre-processing step is accompanied to diminish the noisy contents present in the image. Contrast normalization has been explored to maintain the same illumination factor among histopathological lung images next to pre-processing. Afterwards, data augmentation phase has been carried out to enhance the dataset further to avoid over-fitting problems. Finally, the Dense Alex Net is utilized for classification that comprises five convolutional layers, one multi-scale convolution layer, and three fully connected layers. In evaluation experiments, the proposed approach was trained using our original database to provide rich and meaningful features. The accuracy attained by the proposed methodology is93%, which is maximum compared with the existing algorithm
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