| 1. |
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| 2. |
- Dembele, Vamara, et al.
(författare)
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Correlation properties of a spatially quasi-incoherent imaging interferometer
- 2022
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Ingår i: Applied Optics. - : Optical Society of America. - 1559-128X .- 2155-3165. ; 61:19, s. 5806-5812
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Tidskriftsartikel (refereegranskat)abstract
- The depth-gating capacity of a spatially quasi-incoherent imaging interferometer is investigated in relation to the 3D correlation properties of diffraction field laser speckles. The system exploits a phase-stepped imaging Michelson-type interferometer in which spatially quasi-incoherent illumination is generated by passing an unexpanded laser beam through a rotating diffuser. Numerical simulations and optical experiments both verify that the depth-gating capacity of the imaging interferometer scales as ?/2NA2?λ/2NAp2, where ?λ is the wavelength of the laser and NA?NAp is the numerical aperture of the illumination. For a set depth gate of 150 µm, the depth-gating capacity of the interferometer is demonstrated by scanning a standard USAF target through the measurement volume. The results obtained show that an imaging tool of this kind is expected to provide useful capabilities for imaging through disturbing media and where a single wavelength is required.
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| 3. |
- Dembele, Vamara, et al.
(författare)
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Depth-resolved interferometric imaging utilizing a spatially quasi-incoherent light source
- 2022
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Ingår i: Proceedings Digital Holography and 3-D Imaging 2022. - : Optica Publishing Group.
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Konferensbidrag (refereegranskat)abstract
- An interferometric technique that utilize a spatially quasi-incoherent light source to perform interferometric measurements involving diffusely scattering objects is presented. The proposed technique is demonstrated with settings that give a depth gate of 90 µm.
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| 4. |
- Dembele, Vamara, et al.
(författare)
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Depth-resolved speckle correlation using quasi-incoherent imaging interferometry
- 2021
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Ingår i: Proceedings OSA Imaging and Applied Optics Congress 2021 (3D, COSI, DH, ISA, pcAOP). - : Optical Society of America.
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Konferensbidrag (refereegranskat)abstract
- We described a new concept on a depth-resolved investigation based on imaging interferometry. It exploits a quasi-incoherent imaging interferometry scheme and speckle image correlation techniques. We can spatially resolve a depth-gating capacity of the imaging interferometer with a resolution of around 26 µm.
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| 5. |
- Fresard, Laure, et al.
(författare)
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Identification of rare-disease genes using blood transcriptome sequencing and large control cohorts
- 2019
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Ingår i: Nature Medicine. - : NATURE PUBLISHING GROUP. - 1078-8956 .- 1546-170X. ; 25:6, s. 911-919
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Tidskriftsartikel (refereegranskat)abstract
- It is estimated that 350 million individuals worldwide suffer from rare diseases, which are predominantly caused by mutation in a single gene(1). The current molecular diagnostic rate is estimated at 50%, with whole-exome sequencing (WES) among the most successful approaches(2-5). For patients in whom WES is uninformative, RNA sequencing (RNA-seq) has shown diagnostic utility in specific tissues and diseases(6-8). This includes muscle biopsies from patients with undiagnosed rare muscle disorders(6,9), and cultured fibroblasts from patients with mitochondrial disorders(7). However, for many individuals, biopsies are not performed for clinical care, and tissues are difficult to access. We sought to assess the utility of RNA-seq from blood as a diagnostic tool for rare diseases of different pathophysiologies. We generated whole-blood RNA-seq from 94 individuals with undiagnosed rare diseases spanning 16 diverse disease categories. We developed a robust approach to compare data from these individuals with large sets of RNA-seq data for controls (n = 1,594 unrelated controls and n = 49 family members) and demonstrated the impacts of expression, splicing, gene and variant filtering strategies on disease gene identification. Across our cohort, we observed that RNA-seq yields a 7.5% diagnostic rate, and an additional 16.7% with improved candidate gene resolution.
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| 6. |
- Giordano, Luca, et al.
(författare)
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Essential Role of Mitochondrial Cytochrome c Oxidase Subunit 4 Isoform 2 (Cox4i2) for Acute Pulmonary Oxygen Sensing
- 2022
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier. - 0005-2728 .- 1879-2650. ; 1863:Supplement
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondrial Cytochrome c Oxidase Subunit 4 Isoform 2 (Cox4i2) is essential for acute oxygen sensing and signaling in pulmonary arterial smooth muscle cells (PASMCs) by triggering the production of superoxide during acute hypoxia [1]. However, the molecular mechanism underlying Cox4i2-dependent oxygen sensing remains elusive. We analysed oxygen-dependent respiration by high resolution respirometry, redox changes of the electron transport chain (ETC) by RAMAN spectroscopy, and supercomplex formation by blue native gel analysis of PASMCs isolated from wild type (WT) and Cox4i2-/- mice. To understand the role of Cox4i2-specific cysteine residues we determined hypoxia-induced superoxide production and oxygen affinity in a mouse epithelial cell line (CMT167 cells) overexpressing either Cox4i1, or WT Cox4i2, or Cox4i2 mutants (C41S, C55A, C109S). Respiration and supercomplex formation were similar in WT and Cox4i2-/- PASMCs. Interestingly, hypoxia-induced reduction of ETC components (NADH, ubiquinol, and reduced cytochrome c) was prevented in Cox4i2-/- PASMCs. CMT167 cells expressing either Cox4i1, or Cox4i2 mutants lacked hypoxia-induced superoxide release, which was detected only in cells expressing WT Cox4i2. In contrast, overexpression of Cox4i1, or Cox4i2, or Cox4i2 mutants did not affect oxygen affinity. Our findings suggest that Cox4i2 does not alter superoxide production by rearrangement of supercomplexes, whereas its specific cysteines are needed for the superoxide release. In conclusion, Cox4i2 plays a major role in the hypoxia-induced reduction of ETC components, likely mediated through its redox-active cysteine residues.
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| 7. |
- Knoepp, Fenja, et al.
(författare)
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A Microfluidic System for Simultaneous Raman Spectroscopy, Patch-Clamp Electrophysiology, and Live-Cell Imaging to Study Key Cellular Events of Single Living Cells in Response to Acute Hypoxia
- 2021
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Ingår i: Small Methods. - : John Wiley & Sons. - 2366-9608. ; 5:10
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Tidskriftsartikel (refereegranskat)abstract
- The ability to sense changes in oxygen availability is fundamentally important for the survival of all aerobic organisms. However, cellular oxygen sensing mechanisms and pathologies remain incompletely understood and studies of acute oxygen sensing, in particular, have produced inconsistent results. Current methods cannot simultaneously measure the key cellular events in acute hypoxia (i.e., changes in redox state, electrophysiological properties, and mechanical responses) at controlled partial pressures of oxygen (pO2). The lack of such a comprehensive method essentially contributes to the discrepancies in the field. A sealed microfluidic system that combines i) Raman spectroscopy, ii) patch-clamp electrophysiology, and iii) live-cell imaging under precisely controlled pO2 have therefore been developed. Merging these modalities allows label-free and simultaneous observation of oxygen-dependent alterations in multiple cellular redox couples, membrane potential, and cellular contraction. This technique is adaptable to any cell type and allows in-depth insight into acute oxygen sensing processes underlying various physiologic and pathologic conditions.
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| 8. |
- Knoepp, F., et al.
(författare)
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Development of a Gas-Tight Microfluidic System for Raman Sensing of Single Pulmonary Arterial Smooth Muscle Cells Under Normoxic/Hypoxic Conditions
- 2018
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Ingår i: Sensors. - Basel, Switzerland : MDPI. - 1424-8220. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Acute hypoxia changes the redox-state of pulmonary arterial smooth muscle cells (PASMCs). This might influence the activity of redox-sensitive voltage-gated K⁺-channels (Kv-channels) whose inhibition initiates hypoxic pulmonary vasoconstriction (HPV). However, the molecular mechanism of how hypoxia-or the subsequent change in the cellular redox-state-inhibits Kv-channels remains elusive. For this purpose, a new multifunctional gas-tight microfluidic system was developed enabling simultaneous single-cell Raman spectroscopic studies (to sense the redox-state under normoxic/hypoxic conditions) and patch-clamp experiments (to study the Kv-channel activity). The performance of the system was tested by optically recording the O₂-content and taking Raman spectra on murine PASMCs under normoxic/hypoxic conditions or in the presence of H₂O₂. Oxygen sensing showed that hypoxic levels in the gas-tight microfluidic system were achieved faster, more stable and significantly lower compared to a conventional open system (1.6 ± 0.2%, respectively 6.7 ± 0.7%, n = 6, p < 0.001). Raman spectra revealed that the redistribution of biomarkers (cytochromes, FeS, myoglobin and NADH) under hypoxic/normoxic conditions were improved in the gas-tight microfluidic system (p-values from 0.00% to 16.30%) compared to the open system (p-value from 0.01% to 98.42%). In conclusion, the new redox sensor holds promise for future experiments that may elucidate the role of Kv-channels during HPV.
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| 9. |
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| 10. |
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| 11. |
- Sommer, Natascha, et al.
(författare)
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Bypassing mitochondrial complex III using alternative oxidase inhibits acute pulmonary oxygen sensing
- 2020
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Ingår i: Science Advances. - : American Association for the Advancement of Science. - 2375-2548. ; 6:16
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondria play an important role in sensing both acute and chronic hypoxia in the pulmonary vasculature, but their primary oxygen-sensing mechanism and contribution to stabilization of the hypoxia-inducible factor (HIF) remains elusive. Alteration of the mitochondrial electron flux and increased superoxide release from complex III has been proposed as an essential trigger for hypoxic pulmonary vasoconstriction (HPV). We used mice expressing a tunicate alternative oxidase, AOX, which maintains electron flux when respiratory complexes III and/or IV are inhibited. Respiratory restoration by AOX prevented acute HPV and hypoxic responses of pulmonary arterial smooth muscle cells (PASMC), acute hypoxia-induced redox changes of NADH and cytochrome c, and superoxide production. In contrast, AOX did not affect the development of chronic hypoxia-induced pulmonary hypertension and HIF-1α stabilization. These results indicate that distal inhibition of the mitochondrial electron transport chain in PASMC is an essential initial step for acute but not chronic oxygen sensing.
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| 12. |
- Wahl, Joel, et al.
(författare)
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Comparison between process simulation and deformation measured by defocused speckle photography
- 2018
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Ingår i: Speckle 2018. - : SPIE - International Society for Optical Engineering. - 9781510622975
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Konferensbidrag (refereegranskat)abstract
- Defocused laser speckle photography is used as a tool to measure the heat responses in a titanium component during laser heating. The evolution of the response is compared with a set of preprocessed Finite Element Simulations of the corresponding process with the aim to verify the simulation model and to find the simulation settings that best resemble the experimental results. The titanium component consists of a 300 x 100 mm2 substrate of thickness 3.2 mm on which a 200 x 30 x 11 mm3 ridge is built up using the laser metal deposition by wire process. The component is heated on the top of the ridge by a 300 W laser for 10 s and the deformation of the subtrate is followed throughout the heating-cooling cycle. The simulated deformation gradient is shown to resemble the measured response, and the magnitude of the response indicates that about 70 % of the laser power transferres into heat in the metal.
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| 13. |
- Wahl, Joel
(författare)
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Development of Methods to Investigate Pulmonary Arterial Smooth Muscle Cells under Hypoxia
- 2019
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to localized alveolarhypoxia that is intrinsic to the pulmonary circulation. By hypoxia-induced contractionof pulmonary arterial smooth muscle cells (PASMCs), the pulmonary capillary bloodflow is redirected to alveolar areas of high oxygen partial pressure, thus maintaining theventilation-perfusion ratio. Although the principle of HPV was recognized decades agothe underlying pathway remains elusive. The patch clamp technique, imaging and Ramanspectroscopy are methods that can be used to investigate parts of the mechanisms. Toenable measurements at controlled oxygen concentrations a gas-tight microfluidic systemwas developed. In this thesis preparatory experiments to couple the gas-tight systemto a microscope that enabled simultaneous measurements with patch clamp, imagingand Raman spectroscopy are discussed. The patch clamp technique is to be used formeasurements on the dynamics of the ion-channels in the cellular membrane as well aschanges in membrane potential as a response to hypoxia. Imaging of PASMCs is requiredto successfully apply the patch clamp technique. Further, imaging will also reveal whetherthe mechanical response of HPV has been triggered, for this purpose image analysis forestimation of optical flow can be used. Raman spectroscopy enables measurements ofbiochemical changes in redox biomarkers, cytochrome c and NADH, of the mitochondrialelectron transport chain. This thesis shows that the gas-tight microfluidic system providesoptimal control of the oxygen content, in an experimantal setting where the patch clamptechnique can be applied. Raman measurements showed significantly larger variationsin spectra compared to an open fluidic system, which is the conventional approach.However, the results showed a need for improved Raman preprocessing. For this purposea Convolutional Neural Network (CNN) was trained using synthetic spectra that providedoptimal reconstruction of the Raman signal. Finally, simultaneous imaging and Ramanspectroscopy of red blood cells were performed in a home built microscope. The resultspave the way for measurements on PASMCs.
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| 14. |
- Wahl, Joel, et al.
(författare)
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Impact of preprocessing methods on the Raman spectra of brain tissue
- 2022
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Ingår i: Biomedical Optics Express. - : Optica Publishing Group (formerly OSA). - 2156-7085. ; 13:12, s. 6763-6777
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Tidskriftsartikel (refereegranskat)abstract
- Delineating cancer tissue while leaving functional tissue intact is crucial in brain tumor resection. Despite several available aids, surgeons are limited by preoperative or subjective tools. Raman spectroscopy is a label-free optical technique with promising indications for tumor tissue identification. To allow direct comparisons between measurements preprocessing of the Raman signal is required. There are many recognized methods for preprocessing Raman spectra; however, there is no universal standard. In this paper, six different preprocessing methods were tested on Raman spectra (n > 900) from fresh brain tissue samples (n = 34). The sample cohort included both primary brain tumors, such as adult-type diffuse gliomas and meningiomas, as well as metastases of breast cancer. Each tissue sample was classified according to the CNS WHO 2021 guidelines. The six methods include both direct and iterative polynomial fitting, mathematical morphology, signal derivative, commercial software, and a neural network. Data exploration was performed using principal component analysis, t-distributed stochastic neighbor embedding, and k-means clustering. For each of the six methods, the parameter combination that explained the most variance in the data, i.e., resulting in the highest Gap-statistic, was chosen and compared to the other five methods. Depending on the preprocessing method, the resulting clusters varied in number, size, and associated spectral features. The detected features were associated with hemoglobin, neuroglobin, carotenoid, water, and protoporphyrin, as well as proteins and lipids. However, the spectral features seen in the Raman spectra could not be unambiguously assigned to tissue labels, regardless of preprocessing method. We have illustrated that depending on the chosen preprocessing method, the spectral appearance of Raman features from brain tumor tissue can change. Therefore, we argue both for caution in comparing spectral features from different Raman studies, as well as the importance of transparency of methodology and implementation of the preprocessing. As discussed in this study, Raman spectroscopy for in vivo guidance in neurosurgery requires fast and adaptive preprocessing. On this basis, a pre-trained neural network appears to be a promising approach for the operating room.
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| 15. |
- Wahl, Joel
(författare)
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Multimodal applications in medical technology that utilize Raman spectroscopy
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biology can often be explained by structures and interactions between molecules. It is therefore of importance for medical sciences that the chemical reactions and molecular compositions of biological samples can be measured. The purpose of this thesis is twofold. Firstly, to develop methods for measuring the underlying mechanisms of the lung disorder pulmonary hypertension. Secondly, to investigating the possibility of classifying brain tissue for safer resection of brain tumors. A multimodal approach is often motivated, as there exist many different measurement techniques. In this thesis Raman spectroscopy has been applied both as the main measurement modality and in cooperation with other methods.Raman spectroscopy is a label free optical measurement technique that measure inelastic scattering from materials that are illuminated by a monochromatic light source. Raman scattering results in a weak signal that is uniquely proportional to the chemical structure of the sample. When measuring biological samples, Raman scattering is accompanied by a strong intrinsic fluorescent signature that can overshadow the signal. To elucidate the underlying Raman spectrum, it is generally preprocessed to allow further analysis. There are many methods available for preprocessing; a selection of commonly used methodologies has been included here, as well as methods of my own design. The most novel approach being a neural network that was trained on synthetic spectra to perform preprocessing without relying on user defined variables, which is common for other methods (Paper A). The neural network resulted in improved preprocessing when compared to a control predictor, with test data from paraffin, ethanol, and polyethylene, as well as spectra based on simulations.Hypoxic pulmonary hypertension (PH) is a condition where the arteries in the lung-walls are blocked due to prolonged oxygen deprivation or lung disease. People who suffer from PH, often experience shortness of breath and fatigue. If the condition persists the added strain upon the heart from the increased resistance in the arteries will result in right-heart failure. Hypoxic PH is the result of permanently constricted pulmonary arterial smooth muscle cells (PASMCs). PASMCs reside in the arterial walls and react locally to reduced oxygen content by constricting. This effect is called pulmonary vasoconstriction (HPV) and results in the regulation of deoxygenated blood to areas of the lung that have more oxygen available. Full understanding of the mechanisms of oxygen sensing in PASMCs has importance for the development of new treatments against PH. To this end a sealed microfluidic system was designed with the purpose to enable multimodal measurements of the response of cultivated PASMCs to acute hypoxia including Raman spectroscopy, patch clamp, and imaging (Paper B). The microfluidic system was initially tested using Raman spectroscopy and oxygen sensing to investigate the reaction of PASMCs to hypoxia. The results were compared to an open flow system that showed a higher variation of the desired oxygen content (21% or 4%) compared to the designed closed microfluidic system. The system was later reworked and tested with simultaneous measurements using Raman spectroscopy, oxygen sensing, imaging, and patch-clamp (Paper C). With this setup it was possible to track the molecular response in the mitochondria in correlation with the activity of the calcium-ion channels and the mechanical response of the PASMCs.The main modality used in clinics for brain tumor imaging is magnetic resonance imaging (MRI). Structural MRI gives neurosurgeons information regarding the size and mass effects of tumors. However, during surgery it can be difficult to assess the marginal zone of tumors. Improvements have been made by incorporating fluorescence guided resection (FGR) in the standard practice of many operating rooms in Europe. FGR relies on measuring the emission from metabolized precursor molecules. However, the drawback of FGR is that it is not tumor specific and has reduced sensitivity in low-grade tumors and children. In this thesis the option of incorporating a Raman probe setup, to fill in the gaps of other methods has been discussed. During this preliminary discussion it was noted that there is no standard approach for preprocessing and many different methodologies have been employed by various researcher. Therefore, measurement on fresh brain tumor tissue from a Raman microscopy setup was preprocessed using commonly applied methods, in addition to a pretrained neural network, to investigate the variations of the outcome (Paper D). It became apparent that different methods and variable choices can alter the distinctive spectral features. The conclusion being that it is important to be both transparent and specific when explaining how data has been prepared prior to analysis to enable reproducible results.
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| 16. |
- Wahl, Joel, et al.
(författare)
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Raman spectroscopic analysis of fresh tissue samples from brain tumors
- 2021
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Ingår i: Diffuse Optical Spectroscopy and Imaging VIII. - : SPIE - International Society for Optical Engineering. - 9781510647077 - 9781510647060
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Konferensbidrag (refereegranskat)abstract
- We have applied a CNN to preprocess Raman spectra from fresh tissue samples from brain tumors. The neural network can handle the variations that occur naturally, which enables explorative data analysis methods such as PCA.
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| 17. |
- Wahl, Joel, et al.
(författare)
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Single-Step Preprocessing of Raman Spectra Using Convolutional Neural Networks
- 2020
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Ingår i: Applied Spectroscopy. - : Sage Publications. - 0003-7028 .- 1943-3530. ; 74:4, s. 427-438
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Tidskriftsartikel (refereegranskat)abstract
- Preprocessing of Raman spectra is generally done in three separate steps: (1) cosmic ray removal, (2) signal smoothing, and (3) baseline subtraction. We show that a convolutional neural network (CNN) can be trained using simulated data to handle all steps in one operation. First, synthetic spectra are created by randomly adding peaks, baseline, mixing of peaks and baseline with background noise, and cosmic rays. Second, a CNN is trained on synthetic spectra and known peaks. The results from preprocessing were generally of higher quality than what was achieved using a reference based on standardized methods (second-difference, asymmetric least squares, cross-validation). From 105 simulated observations, 91.4% predictions had smaller absolute error (RMSE), 90.3% had improved quality (SSIM), and 94.5% had reduced signal-to-noise (SNR) power. The CNN preprocessing generated reliable results on measured Raman spectra from polyethylene, paraffin and ethanol with background contamination from polystyrene. The result shows a promising proof of concept for the automated preprocessing of Raman spectra.
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