scholarly journals An adaptive microscope for the imaging of biological surfaces

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Faris Abouakil ◽  
Huicheng Meng ◽  
Marie-Anne Burcklen ◽  
Hervé Rigneault ◽  
Frédéric Galland ◽  
...  
Keyword(s):  
Light Exposure ◽  
Imaging Modality ◽  
Light Dose ◽  
Fluorescent Imaging ◽  
Small Subset ◽  
Cell Physiology ◽  
Cell Periphery ◽  
Cell Sheets ◽  
Scanning Scheme ◽  
Scanning Strategies

AbstractScanning fluorescence microscopes are now able to image large biological samples at high spatial and temporal resolution. This comes at the expense of an increased light dose which is detrimental to fluorophore stability and cell physiology. To highly reduce the light dose, we designed an adaptive scanning fluorescence microscope with a scanning scheme optimized for the unsupervised imaging of cell sheets, which underly the shape of many embryos and organs. The surface of the tissue is first delineated from the acquisition of a very small subset (~0.1%) of sample space, using a robust estimation strategy. Two alternative scanning strategies are then proposed to image the tissue with an improved photon budget, without loss in resolution. The first strategy consists in scanning only a thin shell around the estimated surface of interest, allowing high reduction of light dose when the tissue is curved. The second strategy applies when structures of interest lie at the cell periphery (e.g. adherens junctions). An iterative approach is then used to propagate scanning along cell contours. We demonstrate the benefit of our approach imaging live epithelia from Drosophila melanogaster. On the examples shown, both approaches yield more than a 20-fold reduction in light dose -and up to more than 80-fold- compared to a full scan of the volume. These smart-scanning strategies can be easily implemented on most scanning fluorescent imaging modality. The dramatic reduction in light exposure of the sample should allow prolonged imaging of the live processes under investigation.

scholarly journals The α Isoform of Protein Kinase C Is Involved in Signaling the Response of Desmosomes to Wounding in Cultured Epithelial Cells

2000 ◽  
Vol 11 (3) ◽  
pp. 1077-1092 ◽  
Author(s):  
Sarah Wallis ◽  
Susan Lloyd ◽  
Irene Wise ◽  
Grenham Ireland ◽  
Tom P. Fleming ◽  
...  
Keyword(s):  
Cell Periphery ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Cell Sheets ◽  
Wound Edge ◽  
Calcium Dependence ◽  
Confluent Cell ◽  
Rapid Transition ◽  
Darby Canine Kidney ◽  
Canine Kidney

Initiation of reepithelialization upon wounding is still poorly understood. To enhance this understanding, we focus here on changes in the adhesive state of desmosomes of cultured Madin-Darby canine kidney cells in response to wounding of confluent cell sheets. Previous results show that desmosomal adhesion in Madin-Darby canine kidney cells changes from a calcium-dependent state to calcium independence in confluent cell sheets. We show that this change, which requires culture confluence to develop, is rapidly reversed upon wounding of confluent cell sheets. Moreover, the change to calcium dependence in wound edge cells is propagated to cells hundreds of micrometers away from the wound edge. Rapid transition from calcium independence to calcium dependence also occurs when cells are treated with phorbol esters that activate PKC. PKC inhibitors, including the conventional isoform inhibitor Gö6976, cause rapid transition from calcium dependence to calcium independence, even in subconfluent cells. The cellular location of the α isoform of PKC correlates with the calcium dependence of desmosomes. Upon monolayer wounding, PKCα translocates rapidly to the cell periphery, becomes Triton X-100 insoluble, and also becomes concentrated in lamellipodia. The PKCα translocation upon wounding precedes both the increase in PKC activity in the membrane fraction and the reversion of desmosomes to calcium dependence. Specific depletion of PKCα with an antisense oligonucleotide increases the number of cells with calcium-independent desmosomes. These results show that PKCα participates in a novel signaling pathway that modulates desmosomal adhesion in response to wounding.

scholarly journals The lamellipodium is a myosin independent mechanosensor

2017 ◽  
Author(s):  
Patrick W. Oakes ◽  
Tamara C. Bidone ◽  
Yvonne Beckham ◽  
Austin V. Skeeters ◽  
Guillermina R. Ramirez-San Juan ◽  
...  
Keyword(s):  
Cell Attachment ◽  
Myosin Ii ◽  
Adhesion Formation ◽  
Leading Edge ◽  
Cell Spreading ◽  
Substrate Stiffness ◽  
Cell Physiology ◽  
Cell Periphery ◽  
Initial Adhesion ◽  
Kinetics Of

AbstractThe ability of adherent cells to sense changes in the mechanical properties of their extracellular environments is critical to numerous aspects of their physiology. It has been well documented that cell attachment and spreading are sensitive to substrate stiffness. Here we demonstrate that this behavior is actually biphasic, with a transition that occurs around a Young’s modulus of ∼7 kPa. Furthermore, we demonstrate that, contrary to established assumptions, this property is independent of myosin II activity. Rather, we find that cell spreading on soft substrates is inhibited due to reduced nascent adhesion formation within the lamellipodium. Cells on soft substrates display normal leading edge protrusion activity, but these protrusions are not stabilized due to impaired adhesion assembly. Enhancing integrin-ECM affinity through addition of Mn2+ recovers nascent adhesion assembly and cell spreading on soft substrates. Using a computational model to simulate nascent adhesion assembly, we find that biophysical properties of the integrin-ECM bond are optimized to stabilize interactions above a threshold matrix stiffness that is consistent with the experimentally observations. Together these results suggest that myosin II-independent forces in the lamellipodium are responsible for mechanosensation by regulating new adhesion assembly, which in turn, directly controls cell spreading. This myosin II-independent mechanism of substrate stiffness sensing could potentially regulate a number of other stiffness sensitive processes.Significance StatementCell physiology can be regulated by the mechanics of the extracellular environment. Here, we demonstrate that cell spreading is a mechanosensitive process regulated by weak forces generated at the cell periphery and independent of motor activity. We show that stiffness sensing depends on the kinetics of the initial adhesion bonds that are subjected to forces driven by protein polymerization. This work demonstrates how the binding kinetics of adhesion molecules are sensitively tuned to a range of forces that enable mechanosensation.

Lensless On-Chip Fluorescent Imaging Over an Ultra Wide Field-of-View

2010 ◽  
Author(s):  
Ahmet F. Coskun ◽  
Ting-Wei Su ◽  
Aydogan Ozcan
Keyword(s):  
High Throughput ◽  
Imaging Modality ◽  
Scattered Light ◽  
Fluorescent Imaging ◽  
Field Of View ◽  
Fluorescent Detection ◽  
Fluorescent Light ◽  
Wide Field ◽  
Excitation Light ◽  
Wide Field Of View

We introduce a lensless high-throughput fluorescent detection modality that can simultaneously image micro-objects and labeled cells over an ultra-wide field-of-view (FOV) of ∼8cm2 without the use of any lenses, thin-film filters and mechanical scanners. This lensfree platform utilizes total-internal-reflection (TIR) to block the excitation light, and an inexpensive absorption filter to remove the weakly scattered light that does not obey TIR. The emitted fluorescent light from the objects is then detected on the same chip without the use of any lenses. A digital deconvolution algorithm is used to resolve overlapping fluorescent spots, enabling a resolution of ∼40–50 μm over the entire field-of-view. Such an ultra wide field-of-view lensfree fluorescent imaging modality might be very valuable for high-throughput screening applications as well as quantification of rare cells such as circulating tumor cells using ultra-large microfluidic devices.

scholarly journals Near-Infrared Fluorescent Imaging for Monitoring of Treatment Response in Endometrial Carcinoma Patient-Derived Xenograft Models

Cancers ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 370
Author(s):  
Tina Fonnes ◽  
Elin Strand ◽  
Kristine E. Fasmer ◽  
Hege F. Berg ◽  
Heidi Espedal ◽  
...  
Keyword(s):  
Endometrial Carcinoma ◽  
Near Infrared ◽  
Imaging Modality ◽  
Therapy Monitoring ◽  
Fluorescent Imaging ◽  
Therapeutic Development ◽  
Spatio Temporal ◽  
Preclinical Animal Models ◽  
Pdx Models

Imaging of clinically relevant preclinical animal models is critical to the development of personalized therapeutic strategies for endometrial carcinoma. Although orthotopic patient-derived xenografts (PDXs) reflecting heterogeneous molecular subtypes are considered the most relevant preclinical models, their use in therapeutic development is limited by the lack of appropriate imaging modalities. Here, we describe molecular imaging of a near-infrared fluorescently labeled monoclonal antibody targeting epithelial cell adhesion molecule (EpCAM) as an in vivo imaging modality for visualization of orthotopic endometrial carcinoma PDX. Application of this near-infrared probe (EpCAM-AF680) enabled both spatio-temporal visualization of development and longitudinal therapy monitoring of orthotopic PDX. Notably, EpCAM-AF680 facilitated imaging of multiple PDX models representing different subtypes of the disease. Thus, the combined implementation of EpCAM-AF680 and orthotopic PDX models creates a state-of-the-art preclinical platform for identification and validation of new targeted therapies and corresponding response predicting markers for endometrial carcinoma.

scholarly journals HIV-1 capsid exploitation of the host microtubule cytoskeleton during early infection

Retrovirology ◽  
2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Mojgan H. Naghavi
Keyword(s):  
Critical Role ◽  
Cell Polarization ◽  
Small Subset ◽  
Cell Periphery ◽  
Virus Transport ◽  
Structural Support ◽  
Wide Range ◽  
Range Transport ◽  
Coordinate Changes ◽  
Hiv 1

AbstractMicrotubules (MTs) form a filamentous array that provide both structural support and a coordinated system for the movement and organization of macromolecular cargos within the cell. As such, they play a critical role in regulating a wide range of cellular processes, from cell shape and motility to cell polarization and division. The array is radial with filament minus-ends anchored at perinuclear MT-organizing centers and filament plus-ends continuously growing and shrinking to explore and adapt to the intracellular environment. In response to environmental cues, a small subset of these highly dynamic MTs can become stabilized, acquire post-translational modifications and act as specialized tracks for cargo trafficking. MT dynamics and stability are regulated by a subset of highly specialized MT plus-end tracking proteins, known as +TIPs. Central to this is the end-binding (EB) family of proteins which specifically recognize and track growing MT plus-ends to both regulate MT polymerization directly and to mediate the accumulation of a diverse array of other +TIPs at MT ends. Moreover, interaction of EB1 and +TIPs with actin-MT cross-linking factors coordinate changes in actin and MT dynamics at the cell periphery, as well as during the transition of cargos from one network to the other. The inherent structural polarity of MTs is sensed by specialized motor proteins. In general, dynein directs trafficking of cargos towards the minus-end while most kinesins direct movement toward the plus-end. As a pathogenic cargo, HIV-1 uses the actin cytoskeleton for short-range transport most frequently at the cell periphery during entry before transiting to MTs for long-range transport to reach the nucleus. While the fundamental importance of MT networks to HIV-1 replication has long been known, recent work has begun to reveal the underlying mechanistic details by which HIV-1 engages MTs after entry into the cell. This includes mimicry of EB1 by capsid (CA) and adaptor-mediated engagement of dynein and kinesin motors to elegantly coordinate early steps in infection that include MT stabilization, uncoating (conical CA disassembly) and virus transport toward the nucleus. This review discusses recent advances in our understanding of how MT regulators and their associated motors are exploited by incoming HIV-1 capsid during early stages of infection.

scholarly journals Modeling the Dynamic Concentration Profiles and Efficacy of Type-I and Type-II Photopolymerization

2018 ◽  
Author(s):  
Jui-Teng Lin ◽  
Da-Chuan Cheng ◽  
Kuo-Ti Chen ◽  
Hsia-Wei Liu
Keyword(s):  
Steady State ◽  
Light Intensity ◽  
Light Exposure ◽  
Light Dose ◽  
Type I ◽  
Concentration Profiles ◽  
Type Ii ◽  
Photoinitiated Polymerization ◽  
Lower Light ◽  
Dynamic Concentration

The kinetics and efficacy profiles of photoinitiated polymerization are theoretically presented. For the same dose, lower light intensity achieves a higher steady-state-efficacy (SSE) in type-I; in contrast, type-II has an equal SSE. Higher light intensity has a faster rising efficacy, due to faster depletion of photoinitiator (PS) concentration. However, type-II process is also affected by the available oxygen. Higher light intensity produces more efficient singlet oxygen, resulting a higher transient efficacy, in which all intensities reach the same SSE when oxygen is completely depleted. With external oxygen, type-II efficacy increases with time, otherwise, it is governed only by the light dose, i.e., same dose achieves same efficacy. Moreover, type-II has an efficacy follows Bunsen Roscoe law (BRL), whereas type-I follows non-BRL. The measured type-I efficacy and gelation profile are analyzed by our analytic formulas. Schematics of the photocrosslinking stage defined by the availability of oxygen is developed, where both type-I and –II coexist until the oxygen is depleted. The overall efficacy may be enhanced by resupply of PS or oxygen during the light exposure. The roles of light dose and PS concentration on the efficacy of photoinitiated polymerization should be are governed a new concept of a volume efficacy (Ve), defined by the product of the crosslink (or gelation) depth (CD) and local [efficacy].

Augmentation of Chicken Thigh Model with Fluorescence Imaging Allows for Real-Time, High Fidelity Assessment in Supermicrosurgery Training

2020 ◽  
Author(s):  
Nicholas J. Albano ◽  
Weifeng Zeng ◽  
Christie Lin ◽  
Adam J. Uselmann ◽  
Kevin W. Eliceiri ◽  
...  
Keyword(s):  
Real Time ◽  
Fluorescence Imaging ◽  
White Light ◽  
Imaging Modality ◽  
Fluorescent Imaging ◽  
Rater Agreement ◽  
Training Tool ◽  
University Of Wisconsin ◽  
Fidelity Assessment ◽  
The University

Abstract Background The skills required for supermicrosurgery are hard-earned and difficult to master. The University of Wisconsin “blue-blood” chicken thigh model incorporates perfusion of the thigh vessels with a blue liquid solution, allowing users to visualize flow across their anastomoses. This model has proven to be an excellent source of small vessels (down to 0.3 mm) but assessing the quality of anastomoses at this spatial scale has proven difficult. We evaluated whether fluorescent imaging with indocyanine green (ICG) in this realistic training model would enhance the assessment of supermicrosurgical anastomoses, and therefore improve real-time feedback to trainees. Methods Anastomoses of vessels ranging from 0.35 to 0.55mm in diameter were performed followed by the capture of white light with and without fluorescence imaging overlay during infusion of “blue-blood” and ICG. Videos were randomized and shown to seven fellowship-trained microsurgeons at the University of Wisconsin-Madison who rated each anastomosis as “patent,” “not patent,” or “unsure.” Surgeon accuracy, uncertainty, and inter-rater agreement were measured for each imaging modality. Results Use of fluorescence significantly increased surgeon accuracy to 91% compared with 47% with white light alone (p = 0.015), decreased surgeon uncertainty to 4% compared with 41% with white light alone (p = 0.011), and improved inter-rater agreement from 53.1% with white light alone to 91.8% (p = 0.016). Conclusion Augmentation of the University of Wisconsin “blue-blood” chicken thigh model with ICG fluorescence improves accuracy, decreases uncertainty, and improves inter-rater agreement when assessing supermicrosurgical anastomoses in a training setting. This improved, real-time feedback enhances this model's value as a supermicrosurgical training tool.

scholarly journals Glucose 6-Phosphate Dehydrogenase from Trypanosomes: Selectivity for Steroids and Chemical Validation in Bloodstream Trypanosoma brucei

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 358
Author(s):  
Cecilia Ortíz ◽  
Francesca Moraca ◽  
Marc Laverriere ◽  
Allan Jordan ◽  
Niall Hamilton ◽  
...  
Keyword(s):  
De Novo ◽  
Pentose Phosphate ◽  
Small Subset ◽  
Cell Physiology ◽  
Single Digit ◽  
In Vitro Proliferation ◽  
African Trypanosomes ◽  
Thiol Redox ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose 6-phosphate dehydrogenase (G6PDH) fulfills an essential role in cell physiology by catalyzing the production of NADPH+ and of a precursor for the de novo synthesis of ribose 5-phosphate. In trypanosomatids, G6PDH is essential for in vitro proliferation, antioxidant defense and, thereby, drug resistance mechanisms. So far, 16α-brominated epiandrosterone represents the most potent hit targeting trypanosomal G6PDH. Here, we extended the investigations on this important drug target and its inhibition by using a small subset of androstane derivatives. In Trypanosoma cruzi, immunofluorescence revealed a cytoplasmic distribution of G6PDH and the absence of signal in major organelles. Cytochemical assays confirmed parasitic G6PDH as the molecular target of epiandrosterone. Structure-activity analysis for a set of new (dehydro)epiandrosterone derivatives revealed that bromination at position 16α of the cyclopentane moiety yielded more potent T. cruzi G6PDH inhibitors than the corresponding β-substituted analogues. For the 16α brominated compounds, the inclusion of an acetoxy group at position 3 either proved detrimental or enhanced the activity of the epiandrosterone or the dehydroepiandrosterone derivatives, respectively. Most derivatives presented single digit μM EC50 against infective T. brucei and the killing mechanism involved an early thiol-redox unbalance. This data suggests that infective African trypanosomes lack efficient NADPH+-synthesizing pathways, beyond the Pentose Phosphate, to maintain thiol-redox homeostasis.

scholarly journals Focused X-ray Luminescence Computed Tomography using a Continuous Scanning Scheme

2021 ◽  
Author(s):  
Michael C. Lun ◽  
Yile Fang ◽  
Changqing Li
Keyword(s):  
Computed Tomography ◽  
Imaging System ◽  
Imaging Modality ◽  
Experimental Studies ◽  
Acquisition Time ◽  
Step Size ◽  
Excitation Scheme ◽  
X Ray ◽  
Continuous Scanning ◽  
Scanning Scheme

AbstractX-ray luminescence computed tomography (XLCT) imaging is a hybrid molecular imaging modality combining the merits of both conventional x-ray imaging (high spatial resolution) and optical imaging (high measurement sensitivity). The narrow x-ray beam based XLCT imaging has been shown to be promising. However due to the selective excitation scheme, the imaging speed is slow thus limiting its practical applications for in vivo imaging. In this work, we have introduced a continuous scanning scheme to acquire data for each angular projection in one motion, eliminating the previous stepping scheme and reducing the data acquisition time, which makes it feasible for multiple transverse scans for three-dimensional (3D) imaging. We have introduced a high accuracy vertical stage to our focused x-ray beam based XLCT imaging system to perform high-resolution and 3D XLCT imaging. We have also included a scintillator crystal coupled to a PMT to act as a single-pixel detector for boundary detection purposes to replace our previous flat panel x-ray detector. We have verified the feasibility of our proposed scanning scheme and imaging system by performing phantom experimental studies. A phantom was embedded with a set of cylindrical targets with 200 µm edge-to-edge distance and was scanned in our imaging system with the proposed method. To test the feasibility for 3D scanning, we took measurements from 4 transverse slices with a vertical step size of 1 mm. The results of the experiments verified the feasibility of our proposed method to perform 3D XLCT imaging using a narrow x-ray beam in a reasonable time.

scholarly journals Transmission Structured Illumination Microscopy for Quantitative Phase and Scattering Imaging

2021 ◽  
Vol 8 ◽  
Author(s):  
Kai Wen ◽  
Ying Ma ◽  
Min Liu ◽  
Jianlang Li ◽  
Zeev Zalevsky ◽  
...  
Keyword(s):  
Imaging Modality ◽  
Ccd Camera ◽  
Fluorescent Labeling ◽  
Fluorescent Imaging ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Quantitative Phase ◽  
Phase Images ◽  
Intensity Images ◽  
Fringe Patterns

In this paper, we demonstrate a digital micromirror device (DMD) based optical microscopic apparatus for quantitative differential phase contrast (qDIC) imaging, coherent structured illumination microscopy (SIM), and dual-modality (scattering/fluorescent) imaging. For both the qDIC imaging and the coherent SIM, two sets of fringe patterns with orthogonal orientations and five phase-shifts for each orientation, are generated by a DMD and projected on a sample. A CCD camera records the generated images in a defocusing manner for qDIC and an in-focus manner for coherent SIM. Both quantitative phase images and super-resolved scattering/fluorescence images can be reconstructed from the recorded intensity images. Moreover, fluorescent imaging modality is integrated, providing specific biochemical structures of the sample once using fluorescent labeling.

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