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20 pages, 4318 KiB

Open AccessArticle

NeuroActivityToolkit—Toolbox for Quantitative Analysis of Miniature Fluorescent Microscopy Data

by Evgenii Gerasimov, Alexander Mitenev, Ekaterina Pchitskaya, Viacheslav Chukanov and Ilya Bezprozvanny

J. Imaging 2023, 9(11), 243; https://doi.org/10.3390/jimaging9110243 - 06 Nov 2023

Viewed by 1814

Abstract

The visualization of neuronal activity in vivo is an urgent task in modern neuroscience. It allows neurobiologists to obtain a large amount of information about neuronal network architecture and connections between neurons. The miniscope technique might help to determine changes that occurred in [...] Read more.

The visualization of neuronal activity in vivo is an urgent task in modern neuroscience. It allows neurobiologists to obtain a large amount of information about neuronal network architecture and connections between neurons. The miniscope technique might help to determine changes that occurred in the network due to external stimuli and various conditions: processes of learning, stress, epileptic seizures and neurodegenerative diseases. Furthermore, using the miniscope method, functional changes in the early stages of such disorders could be detected. The miniscope has become a modern approach for recording hundreds to thousands of neurons simultaneously in a certain brain area of a freely behaving animal. Nevertheless, the analysis and interpretation of the large recorded data is still a nontrivial task. There are a few well-working algorithms for miniscope data preprocessing and calcium trace extraction. However, software for further high-level quantitative analysis of neuronal calcium signals is not publicly available. NeuroActivityToolkit is a toolbox that provides diverse statistical metrics calculation, reflecting the neuronal network properties such as the number of neuronal activations per minute, amount of simultaneously co-active neurons, etc. In addition, the module for analyzing neuronal pairwise correlations is implemented. Moreover, one can visualize and characterize neuronal network states and detect changes in 2D coordinates using PCA analysis. This toolbox, which is deposited in a public software repository, is accompanied by a detailed tutorial and is highly valuable for the statistical interpretation of miniscope data in a wide range of experimental tasks. Full article

(This article belongs to the Special Issue Fluorescence Imaging and Analysis of Cellular System)

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12 pages, 5869 KiB

Open AccessArticle

Near-Infrared Fluorescence Imaging in Preclinical Models of Glioblastoma

by Monserrat Llaguno-Munive, Wilberto Villalba-Abascal, Alejandro Avilés-Salas and Patricia Garcia-Lopez

J. Imaging 2023, 9(10), 212; https://doi.org/10.3390/jimaging9100212 - 06 Oct 2023

Viewed by 1298

Abstract

Cancer is a public health problem requiring ongoing research to improve current treatments and discover novel therapies. More accurate imaging would facilitate such research. Near-infrared fluorescence has been developed as a non-invasive imaging technique capable of visualizing and measuring biological processes at the [...] Read more.

Cancer is a public health problem requiring ongoing research to improve current treatments and discover novel therapies. More accurate imaging would facilitate such research. Near-infrared fluorescence has been developed as a non-invasive imaging technique capable of visualizing and measuring biological processes at the molecular level in living subjects. In this work, we evaluate the tumor activity in two preclinical glioblastoma models by using fluorochrome (IRDye 800CW) coupled to different molecules: tripeptide Arg-Gly-Asp (RGD), 2-amino-2-deoxy-D-glucose (2-DG), and polyethylene glycol (PEG). These molecules interact with pathological conditions of tumors, including their overexpression of αvβ3 integrins (RGD), elevated glucose uptake (2-DG), and enhanced permeability and retention effect (PEG). IRDye 800CW RGD gave the best in vivo fluorescence signal from the tumor area, which contrasted well with the low fluorescence intensity of healthy tissue. In the ex vivo imaging (dissected tumor), the accumulation of IRDye 800CW RGD could be appreciated at the tumor site. Glioblastoma tumors were presently detected with specificity and sensitivity by utilizing IRDye 800CW RGD, a near-infrared fluorophore combined with a marker of αvβ3 integrin expression. Further research is needed on its capacity to monitor tumor growth in glioblastoma after chemotherapy. Full article

(This article belongs to the Special Issue Fluorescence Imaging and Analysis of Cellular System)

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11 pages, 2013 KiB

Open AccessArticle

Hybrid Autofluorescence and Optoacoustic Microscopy for the Label-Free, Early and Rapid Detection of Pathogenic Infections in Vegetative Tissues

by George J. Tserevelakis, Andreas Theocharis, Stavroula Spyropoulou, Emmanouil Trantas, Dimitrios Goumas, Filippos Ververidis and Giannis Zacharakis

J. Imaging 2023, 9(9), 176; https://doi.org/10.3390/jimaging9090176 - 29 Aug 2023

Viewed by 1316

Abstract

Agriculture plays a pivotal role in food security and food security is challenged by pests and pathogens. Due to these challenges, the yields and quality of agricultural production are reduced and, in response, restrictions in the trade of plant products are applied. Governments [...] Read more.

Agriculture plays a pivotal role in food security and food security is challenged by pests and pathogens. Due to these challenges, the yields and quality of agricultural production are reduced and, in response, restrictions in the trade of plant products are applied. Governments have collaborated to establish robust phytosanitary measures, promote disease surveillance, and invest in research and development to mitigate the impact on food security. Classic as well as modernized tools for disease diagnosis and pathogen surveillance do exist, but most of these are time-consuming, laborious, or are less sensitive. To that end, we propose the innovative application of a hybrid imaging approach through the combination of confocal fluorescence and optoacoustic imaging microscopy. This has allowed us to non-destructively detect the physiological changes that occur in plant tissues as a result of a pathogen-induced interaction well before visual symptoms occur. When broccoli leaves were artificially infected with Xanthomonas campestris pv. campestris (Xcc), eventually causing an economically important bacterial disease, the induced optical absorption alterations could be detected at very early stages of infection. Therefore, this innovative microscopy approach was positively utilized to detect the disease caused by a plant pathogen, showing that it can also be employed to detect quarantine pathogens such as Xylella fastidiosa. Full article

(This article belongs to the Special Issue Fluorescence Imaging and Analysis of Cellular System)

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13 pages, 1800 KiB

Open AccessArticle

Live Cell Light Sheet Imaging with Low- and High-Spatial-Coherence Detection Approaches Reveals Spatiotemporal Aspects of Neuronal Signaling

by Mariana Potcoava, Donatella Contini, Zachary Zurawski, Spencer Huynh, Christopher Mann, Jonathan Art and Simon Alford

J. Imaging 2023, 9(6), 121; https://doi.org/10.3390/jimaging9060121 - 16 Jun 2023

Viewed by 1230

Abstract

Light sheet microscopy in live cells requires minimal excitation intensity and resolves three-dimensional (3D) information rapidly. Lattice light sheet microscopy (LLSM) works similarly but uses a lattice configuration of Bessel beams to generate a flatter, diffraction-limited z-axis sheet suitable for investigating subcellular compartments, [...] Read more.

Light sheet microscopy in live cells requires minimal excitation intensity and resolves three-dimensional (3D) information rapidly. Lattice light sheet microscopy (LLSM) works similarly but uses a lattice configuration of Bessel beams to generate a flatter, diffraction-limited z-axis sheet suitable for investigating subcellular compartments, with better tissue penetration. We developed a LLSM method for investigating cellular properties of tissue in situ. Neural structures provide an important target. Neurons are complex 3D structures, and signaling between cells and subcellular structures requires high resolution imaging. We developed an LLSM configuration based on the Janelia Research Campus design or in situ recording that allows simultaneous electrophysiological recording. We give examples of using LLSM to assess synaptic function in situ. In presynapses, evoked Ca²⁺ entry causes vesicle fusion and neurotransmitter release. We demonstrate the use of LLSM to measure stimulus-evoked localized presynaptic Ca²⁺ entry and track synaptic vesicle recycling. We also demonstrate the resolution of postsynaptic Ca²⁺ signaling in single synapses. A challenge in 3D imaging is the need to move the emission objective to maintain focus. We have developed an incoherent holographic lattice light-sheet (IHLLS) technique to replace the LLS tube lens with a dual diffractive lens to obtain 3D images of spatially incoherent light diffracted from an object as incoherent holograms. The 3D structure is reproduced within the scanned volume without moving the emission objective. This eliminates mechanical artifacts and improves temporal resolution. We focus on LLS and IHLLS applications and data obtained in neuroscience and emphasize increases in temporal and spatial resolution using these approaches. Full article

(This article belongs to the Special Issue Fluorescence Imaging and Analysis of Cellular System)

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13 pages, 4931 KiB

Open AccessArticle

Picomolar Detection of Lead Ions (Pb²⁺) by Functionally Modified Fluorescent Carbon Quantum Dots from Watermelon Juice and Their Imaging in Cancer Cells

by Kundan Singh Rawat, Vikram Singh, Chandra Prakash Sharma, Akanksha Vyas, Priyanka Pandey, Jagriti Singh, Neeraj Mohan Gupta, Monika Sachdev and Atul Goel

J. Imaging 2023, 9(1), 19; https://doi.org/10.3390/jimaging9010019 - 16 Jan 2023

Cited by 6 | Viewed by 2690

Abstract

Water contamination due to the presence of lead is one of the leading causes of environmental and health hazards because of poor soil and groundwater waste management. Herein we report the synthesis of functionally modified luminescent carbon quantum dots (CQDs) obtained from watermelon [...] Read more.

Water contamination due to the presence of lead is one of the leading causes of environmental and health hazards because of poor soil and groundwater waste management. Herein we report the synthesis of functionally modified luminescent carbon quantum dots (CQDs) obtained from watermelon juice as potential nanomaterials for the detection of toxic Pb²⁺ ions in polluted water and cancer cells. By introducing surface passivating ligands such as ethanolamine (EA) and ethylenediamine (ED) in watermelon juice, watermelon-ethanolamine (WMEA)-CQDs and watermelon-ethylenediamine (WMED)-CQDs exhibited a remarkable ~10-fold and ~6-fold increase in fluorescence intensity with respect to non-doped WM-CQDs. The relative fluorescence quantum yields of WMEA-CQDs and WMED-CQDs were found to be 8% and 7%, respectively, in an aqueous medium. Among various functionally-modified CQDs, only WMED-CQDs showed high selectivity towards Pb²⁺ ions with a remarkably good limit of detection (LoD) of 190 pM, which is less than that of the permissible limit (72 nM) in drinking water. The functionally altered WMED-CQDs detected Pb²⁺ metal ions in polluted water and in a human cervical cancer cell line (HeLa), thus advocating new vistas for eco-friendly nanomaterials for their use as diagnostic tools in the environment and biomedical research areas. Full article

(This article belongs to the Special Issue Fluorescence Imaging and Analysis of Cellular System)

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10 pages, 2267 KiB

Open AccessArticle

Gadolinium and Bio-Metal Association: A Concentration Dependency Tested in a Renal Allograft and Investigated by Micro-Synchrotron XRF

by Wolf Osterode, Gerald Falkenberg and Heinz Regele

J. Imaging 2022, 8(10), 254; https://doi.org/10.3390/jimaging8100254 - 21 Sep 2022

Viewed by 1316

Abstract

Aims: This study aimed to investigate gadolinium (Gd) and bio-metals in a renal allograft of a patient who was shortly after transplantation repeatedly exposed to a Gd-based contrast agent (GBCA), with the purpose of determining whether Gd can be proven and spatially and [...] Read more.

Aims: This study aimed to investigate gadolinium (Gd) and bio-metals in a renal allograft of a patient who was shortly after transplantation repeatedly exposed to a Gd-based contrast agent (GBCA), with the purpose of determining whether Gd can be proven and spatially and quantitatively imaged. Further elemental associations between Gd and bio-metals were also investigated. Materials and Methods: Archival paraffin-embedded kidney tissue (eight weeks after transplantation) was investigated by microscopic synchrotron X-ray fluorescence (µSRXRF) at the DORIS III storage ring, beamline L, at HASYLAB/DESY (Hamburg, Germany). For the quantification of elements, X-ray spectra were peak-fitted, and the net peak intensities were normalized to the intensity of the incoming monochromatic beam intensity. Concentrations were calculated by fundamental parameter-based program quant and external standardization. Results: Analysis of about 15,000 µSRXRF spectra (comprising allograft tissue of four cm²) Gd distribution could be quantitatively demonstrated in a near histological resolution. Mean Gd resulted in 24 ± 55 ppm with a maximum of 2363 ppm. The standard deviation of ±55 ppm characterized the huge differences in Gd and not in detection accuracy. Gd was heterogeneously but not randomly distributed and was mostly found in areas with interstitial fibrosis and tubular atrophy. Concentrations of all other investigated elements in the allograft resembled those found in normal kidney tissue. No correlations between Gd and bio-metals such as calcium, strontium or zinc below ~40 ppm Gd existed. In areas with extremely high Gd, Gd was associated with iron and zinc. Conclusions: We could show that no dose-dependent association between Gd and bio-metals exists—least in renal tissue—at Gd concentrations below ~40 ppm Gd. This was proven compared with a GBCA-exposed end-stage renal failure in which the mean Gd was ten-fold higher. Our results could shed additional light on Gd metabolism. Full article

(This article belongs to the Special Issue Fluorescence Imaging and Analysis of Cellular System)

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17 pages, 6390 KiB

Open AccessArticle

upU-Net Approaches for Background Emission Removal in Fluorescence Microscopy

by Alessandro Benfenati

J. Imaging 2022, 8(5), 142; https://doi.org/10.3390/jimaging8050142 - 20 May 2022

Cited by 3 | Viewed by 2577

Abstract

The physical process underlying microscopy imaging suffers from several issues: some of them include the blurring effect due to the Point Spread Function, the presence of Gaussian or Poisson noise, or even a mixture of these two types of perturbation. Among them, auto–fluorescence [...] Read more.

The physical process underlying microscopy imaging suffers from several issues: some of them include the blurring effect due to the Point Spread Function, the presence of Gaussian or Poisson noise, or even a mixture of these two types of perturbation. Among them, auto–fluorescence presents other artifacts in the registered image, and such fluorescence may be an important obstacle in correctly recognizing objects and organisms in the image. For example, particle tracking may suffer from the presence of this kind of perturbation. The objective of this work is to employ Deep Learning techniques, in the form of U-Nets like architectures, for background emission removal. Such fluorescence is modeled by Perlin noise, which reveals to be a suitable candidate for simulating such a phenomenon. The proposed architecture succeeds in removing the fluorescence, and at the same time, it acts as a denoiser for both Gaussian and Poisson noise. The performance of this approach is furthermore assessed on actual microscopy images and by employing the restored images for particle recognition. Full article

(This article belongs to the Special Issue Fluorescence Imaging and Analysis of Cellular System)

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Review

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25 pages, 3360 KiB

Open AccessReview

Unravelling the Mystery inside Cells by Using Single-Molecule Fluorescence Imaging

by Julian Zalejski, Jiachen Sun and Ashutosh Sharma

J. Imaging 2023, 9(9), 192; https://doi.org/10.3390/jimaging9090192 - 19 Sep 2023

Cited by 1 | Viewed by 1810

Abstract

Live-cell imaging is a powerful technique to study the dynamics and mechanics of various biological molecules like proteins, organelles, DNA, and RNA. With the rapid evolution of optical microscopy, our understanding of how these molecules are implicated in the cells’ most critical physiological [...] Read more.

Live-cell imaging is a powerful technique to study the dynamics and mechanics of various biological molecules like proteins, organelles, DNA, and RNA. With the rapid evolution of optical microscopy, our understanding of how these molecules are implicated in the cells’ most critical physiological roles deepens. In this review, we focus on how spatiotemporal nanoscale live-cell imaging at the single molecule level allows for profound contributions towards new discoveries in life science. This review will start by summarizing how single-molecule tracking has been used to analyze membrane dynamics, receptor–ligand interactions, protein–protein interactions, inner- and extra-cellular transport, gene expression/transcription, and whole organelle tracking. We then move on to how current authors are trying to improve single-molecule tracking and overcome current limitations by offering new ways of labeling proteins of interest, multi-channel/color detection, improvements in time-lapse imaging, and new methods and programs to analyze the colocalization and movement of targets. We later discuss how single-molecule tracking can be a beneficial tool used for medical diagnosis. Finally, we wrap up with the limitations and future perspectives of single-molecule tracking and total internal reflection microscopy. Full article

(This article belongs to the Special Issue Fluorescence Imaging and Analysis of Cellular System)

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