scholarly journals SPITFIR(e): A supermaneuverable algorithm for restoring 2D-3D fluorescence images and videos, and background subtraction

2022 ◽  
Author(s):  
Sylvain Prigent ◽  
Hoai-Nam Nguyen ◽  
Ludovic Leconte ◽  
Cesar Augusto Valades-Cruz ◽  
Bassam Hajj ◽  
...  
Keyword(s):  
Fluorescent Microscopy ◽  
Direct Consequence ◽  
Light Sheet ◽  
Stimulated Emission ◽  
Wide Field ◽  
Modern Biology ◽  
Microscopy Imaging ◽  
Cell Behaviors ◽  
Primal Dual ◽  
Restoration Algorithms

While fluorescent microscopy imaging has become the spearhead of modern biology as it is able to generate long-term videos depicting 4D nanoscale cell behaviors, it is still limited by the optical aberrations and the photon budget available in the specimen and to some extend to photo-toxicity. A direct consequence is the necessity to develop flexible and "off-road" algorithms in order to recover structural details and improve spatial resolution, which is critical when pushing the illumination to the low levels in order to limit photo-damages. Moreover, as the processing of very large temporal series of images considerably slows down the analysis, special attention must be paid to the feasibility and scalability of the developed restoration algorithms. To address these specifications, we present a very flexible method designed to restore 2D-3D+Time fluorescent images and subtract undesirable out-of-focus background. We assume that the images are sparse and piece-wise smooth, and are corrupted by mixed Poisson-Gaussian noise. To recover the unknown image, we consider a novel convex and non-quadratic regularizer Sparse Hessian Variation) defined as the mixed norms which gathers image intensity and spatial second-order derivatives. This resulting restoration algorithm named SPITFIR(e) (SParse fIT for Fluorescence Image Restoration) utilizes the primal-dual optimization principle for energy minimization and can be used to process large images acquired with varied fluorescence microscopy modalities. It is nearly parameter-free as the practitioner needs only to specify the amount of desired sparsity (weak, moderate, high). Experimental results in lattice light sheet, stimulated emission depletion, multifocus microscopy, spinning disk confocal, and wide-field microscopy demonstrate the generic ability of the SPITFIR(e) algorithm to efficiently reduce noise and blur, and to subtract undesirable fluorescent background, while avoiding the emergence of deconvolution artifacts.

scholarly journals Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

2021 ◽  
Vol 22 (4) ◽  
pp. 1903
Author(s):  
Ivona Kubalová ◽  
Alžběta Němečková ◽  
Klaus Weisshart ◽  
Eva Hřibová ◽  
Veit Schubert
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Chromatin Condensation ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Wide Field ◽  
Structured Illumination Microscopy ◽  
Metaphase Chromosomes ◽  
Localization Microscopy ◽  
Super Resolution Microscopy

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

scholarly journals Light Sheet Microscopy Imaging of Light Absorption and Photosynthesis Distribution in Plant Tissue

2017 ◽  
Vol 175 (2) ◽  
pp. 721-733 ◽  
Author(s):  
Mads Lichtenberg ◽  
Erik C.L. Trampe ◽  
Thomas C. Vogelmann ◽  
Michael Kühl
Keyword(s):  
Light Absorption ◽  
Plant Tissue ◽  
Light Sheet ◽  
Microscopy Imaging ◽  
Light Sheet Microscopy

scholarly journals Three-Dimensional Cross-Sectional Light-Sheet Microscopy Imaging of the Inflamed Mouse Gut

2017 ◽  
Vol 153 (4) ◽  
pp. 898-900 ◽  
Author(s):  
Sebastian Zundler ◽  
Anika Klingberg ◽  
Daniela Schillinger ◽  
Sarah Fischer ◽  
Clemens Neufert ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Light Sheet ◽  
Cross Sectional ◽  
Microscopy Imaging ◽  
Light Sheet Microscopy

scholarly journals Light sheet-excited spontaneous Raman imaging of a living fish by optical sectioning in a wide field Raman microscope

2012 ◽  
Vol 20 (15) ◽  
pp. 16195 ◽  
Author(s):  
Yusuke Oshima ◽  
Hidetoshi Sato ◽  
Hiroko Kajiura-Kobayashi ◽  
Tetsuaki Kimura ◽  
Kiyoshi Naruse ◽  
...  
Keyword(s):  
Light Sheet ◽  
Raman Imaging ◽  
Wide Field ◽  
Optical Sectioning

scholarly journals 3D visualization of macromolecule synthesis

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Timothy J Duerr ◽  
Ester Comellas ◽  
Eun Kyung Jeon ◽  
Johanna E Farkas ◽  
Marylou Joetzjer ◽  
...  
Keyword(s):  
3D Visualization ◽  
Fluorescent Microscopy ◽  
Three Dimensional ◽  
Light Sheet ◽  
Macromolecular Synthesis ◽  
Volumetric Imaging ◽  
Processing Pipeline ◽  
Powerful Means

Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole-mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization.

scholarly journals Long-term, in toto live imaging of the developing mouse heart

2020 ◽  
Author(s):  
Yanzhu Yue ◽  
Xin Li ◽  
Youdong Zhang ◽  
Aibin He
Keyword(s):  
Imaging System ◽  
Light Sheet ◽  
Live Imaging ◽  
Mouse Heart ◽  
Cell Behaviors ◽  
Oriented Cell Division ◽  
Lower Vertebrates ◽  
Imaging Strategy ◽  
Cell Intercalation ◽  
Gated Imaging

Abstract Mapping holistic cell behaviors sculpting mammalian heart has been a goal, but so far only successes in transparent invertebrates and lower vertebrates. Using a live-imaging system comprising a customized vertical light-sheet microscope equipped with a culture module, a heartbeat-gated imaging strategy, and a digital image processing framework, we realized imaging of developing mouse hearts with uninterrupted cell lineages for up to 1.5 days. Four-dimensional landscapes of cell behaviors revealed a blueprint for ventricle chamber formation in which biased outward migration of outermost cardiomyocytes coupled with cell intercalation and horizontal division. The trabeculae, an inner muscle architecture, was developed through early fate segregation and transmural cell arrangement involving both oriented cell division and directional migration. Thus, live-imaging reconstruction affords a transformative means for deciphering mammalian organogenesis.

Near-Surface Velocimetry Using Evanescent Wave Illumination

2003 ◽  
Author(s):  
Songwan Jin ◽  
Peter Huang ◽  
Jinil Park ◽  
Jung Yul Yoo ◽  
Kenneth S. Breuer
Keyword(s):  
Slip Velocity ◽  
Finite Thickness ◽  
Fluorescent Microscopy ◽  
Direct Consequence ◽  
Microfluidic System ◽  
Wall Region ◽  
Near Surface ◽  
Shear Rates ◽  
Seed Particles ◽  
Apparent Slip

Total internal reflection fluorescent microscopy (TIRFM) is used to measure particle motion in the near wall region of a microfluidic system. TIRFM images have minimum background noise and contain only particles that are very close to channel surface, where slip velocities may be present. Submicron sized fluorescent particles suspended in water are used as seed particles and images are analyzed with a PTV algorithm to extract information about apparent slip velocity. At relatively low shear rates (less than 2500 sec−1), an apparent slip velocity, proportional to the shear rate was observed. However, numerical simulations show that this observation is a direct consequence of the small, but finite thickness of the illuminated region, and most likely not due to physical slip at the surface. The statistical difference in apparent slip velocities measured over hydrophilic and hydrophobic surfaces is found to be minimal. Issues associated with the experimental technique and the interpretation of the experimental results are also discussed.

Anti-HER2 VHH Targeted Fluorescent Liposome as Bimodal Nanoparticle for Drug Delivery and Optical Imaging

2021 ◽  
Vol 16 ◽  
Author(s):  
Sepideh Khaleghi ◽  
Fatemeh Rahbarizadeh ◽  
Shahryar Khoshtinat Nikkhoi
Keyword(s):  
Drug Delivery ◽  
Real Time ◽  
Targeted Delivery ◽  
Cell Tracking ◽  
Physicochemical Characterization ◽  
Target Cells ◽  
Wide Field ◽  
Cell Trafficking ◽  
Microscopy Imaging ◽  
Intracellular Drug Delivery

Objective: The aim of this study was to formulate fluorescent-labeled targeted immunoliposome to visualize the delivery and distribution of drugs in real-time. Methods: In this study, fluorescent-labeled liposomes were decorated with anti-HER2 VHH or Herceptin to improve the monitoring of intracellular drug delivery and tumor cell tracking with minimal side effects. The conjugation efficiency of antibodies was analyzed by SDS-PAGE silver staining. In addition, the physicochemical characterization of liposomes was performed using DLS and TEM. Finally, confocal microscopy visualized nanoparticles in the target cells. Results: Quantitative and qualitative methods characterized the intracellular uptake of 110±10 nm particles with near 70% conjugation efficiency. In addition, live-cell trafficking during hours of incubation was monitored by wide-field microscopy imaging. The results show that the fluorescent-labeled nanoparticles can specifically bind to HER2-positive breast cancer with minimal off-target delivery. Conclusion: This kind of nanoparticles can have several applications in personalized medicine, especially drug delivery and real-time visualization of cancer therapy. Moreover, this method also can be applied in the targeted delivery of contrast agents in imaging and thermotherapy.

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