scholarly journals Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Eli Slenders ◽  
Marco Castello ◽  
Mauro Buttafava ◽  
Federica Villa ◽  
Alberto Tosi ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Single Photon ◽  
Function Analysis ◽  
Pair Correlation ◽  
Correlation Spectroscopy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Array Detector ◽  
Fluorescence Lifetime Imaging ◽  
Fluorescence Fluctuation Spectroscopy

AbstractThe combination of confocal laser-scanning microscopy (CLSM) and fluorescence fluctuation spectroscopy (FFS) is a powerful tool in studying fast, sub-resolution biomolecular processes in living cells. A detector array can further enhance CLSM-based FFS techniques, as it allows the simultaneous acquisition of several samples–essentially images—of the CLSM detection volume. However, the detector arrays that have previously been proposed for this purpose require tedious data corrections and preclude the combination of FFS with single-photon techniques, such as fluorescence lifetime imaging. Here, we solve these limitations by integrating a novel single-photon-avalanche-diode (SPAD) array detector in a CLSM system. We validate this new implementation on a series of FFS analyses: spot-variation fluorescence correlation spectroscopy, pair-correlation function analysis, and image-derived mean squared displacement analysis. We predict that the unique combination of spatial and temporal information provided by our detector will make the proposed architecture the method of choice for CLSM-based FFS.

scholarly journals Degradation of Drug Delivery Nanocarriers and Payload Release: A Review of Physical Methods for Tracing Nanocarrier Biological Fate

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 770
Author(s):  
Patrick M. Perrigue ◽  
Richard A. Murray ◽  
Angelika Mielcarek ◽  
Agata Henschke ◽  
Sergio E. Moya
Keyword(s):  
Drug Delivery ◽  
Laser Scanning ◽  
Single Photon ◽  
Photon Emission ◽  
Correlation Spectroscopy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Emission Computed Tomography ◽  
Systemic Delivery

Nanoformulations offer multiple advantages over conventional drug delivery, enhancing solubility, biocompatibility, and bioavailability of drugs. Nanocarriers can be engineered with targeting ligands for reaching specific tissue or cells, thus reducing the side effects of payloads. Following systemic delivery, nanocarriers must deliver encapsulated drugs, usually through nanocarrier degradation. A premature degradation, or the loss of the nanocarrier coating, may prevent the drug’s delivery to the targeted tissue. Despite their importance, stability and degradation of nanocarriers in biological environments are largely not studied in the literature. Here we review techniques for tracing the fate of nanocarriers, focusing on nanocarrier degradation and drug release both intracellularly and in vivo. Intracellularly, we will discuss different fluorescence techniques: confocal laser scanning microscopy, fluorescence correlation spectroscopy, lifetime imaging, flow cytometry, etc. We also consider confocal Raman microscopy as a label-free technique to trace colocalization of nanocarriers and drugs. In vivo we will consider fluorescence and nuclear imaging for tracing nanocarriers. Positron emission tomography and single-photon emission computed tomography are used for a quantitative assessment of nanocarrier and payload biodistribution. Strategies for dual radiolabelling of the nanocarriers and the payload for tracing carrier degradation, as well as the efficacy of the payload delivery in vivo, are also discussed.

scholarly journals From Macro to Mesoporous ZnO Inverse Opals: Synthesis, Characterization and Tracer Diffusion Properties

Nanomaterials ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 196
Author(s):  
Shravan Kousik ◽  
Diane Sipp ◽  
Karina Abitaev ◽  
Yawen Li ◽  
Thomas Sottmann ◽  
...  
Keyword(s):  
Open Porosity ◽  
Laser Scanning ◽  
High Surface ◽  
Stop Band ◽  
Correlation Spectroscopy ◽  
Laser Scanning Microscopy ◽  
Added Value ◽  
Confocal Laser ◽  
Inverse Opals ◽  
Mesoporous Zno

Oxide inverse opals (IOs) with their high surface area and open porosity are promising candidates for catalyst support applications. Supports with confined mesoporous domains are of added value to heterogeneous catalysis. However, the fabrication of IOs with mesoporous or sub-macroporous voids (<100 nm) continues to be a challenge, and the diffusion of tracers in quasi-mesoporous IOs is yet to be adequately studied. In order to address these two problems, we synthesized ZnO IOs films with tunable pore sizes using chemical bath deposition and template-based approach. By decreasing the size of polystyrene (PS) template particles towards the mesoporous range, ZnO IOs with 50 nm-sized pores and open porosity were synthesized. The effect of the template-removal method on the pore geometry (spherical vs. gyroidal) was studied. The infiltration depth in the template was determined, and the factors influencing infiltration were assessed. The crystallinity and photonic stop-band of the IOs were studied using X-Ray diffraction and UV-Vis, respectively. The infiltration of tracer molecules (Alexa Fluor 488) in multilayered quasi-mesoporous ZnO IOs was confirmed via confocal laser scanning microscopy, while fluorescence correlation spectroscopy analysis revealed two distinct diffusion times in IOs assigned to diffusion through the pores (fast) and adsorption on the pore walls (slow).

scholarly journals Functionalized Solid-Sphere PEG-b-PCL Nanoparticles to Target Brain Capillary Endothelial CellsIn Vitro

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Philip Grossen ◽  
Gabriela Québatte ◽  
Dominik Witzigmann ◽  
Cristina Prescianotto-Baschong ◽  
Le-Ha Dieu ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Laser Scanning ◽  
Solid Sphere ◽  
Correlation Spectroscopy ◽  
Laser Scanning Microscopy ◽  
Brain Barrier ◽  
Critical Aggregation Concentration ◽  
Confocal Laser ◽  
Blood Brain ◽  
Human Liver Cell

Nanoparticles are increasingly used to implement drug targeting strategies. In the present study, solid-sphere nanoparticles (SNPs) made of poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-b-PCL) were covalently linked to a monoclonal antibody (83-14 mAb) targeted against the human insulin receptor that is highly expressed on human brain microvascular endothelial cells. Resulting targeted SNPs were characterized using transmission electron microscopy (TEM), cryo-TEM, dynamic light scattering, and fluorescence correlation spectroscopy. The critical aggregation concentration was determined using a fluorescence approach. Interaction with a well-characterized humanin vitromodel of the blood-brain barrier (hCMEC/D3) was analysed using an array of methods (flow cytometry, confocal laser scanning microscopy, and TEM). The toxicity on hCMEC/D3 cells and in addition on the human liver cell line HepG2 was assessed using the MTT assay. SNPs with a diameter of 80 nm and a homogeneous size distribution were obtained. Successful conjugation of 83-14 mAb using a heterobifunctional linker resulted in 5-6 molecules of fluorescently labeled 83-14 mAb per SNP. Functionalized SNPs were taken up by hCMEC/D3 cells efficiently without showing a significant toxic effect on cells of the blood-brain barrier and HepG2 cells. These results indicate that functionalized PEG-b-PCL SNPs are a promising candidate to deliver drugs to the CNS.

Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy

2016 ◽  
Vol 22 (2) ◽  
pp. 290-299 ◽  
Author(s):  
Martina Laňková ◽  
Jana Humpolíčková ◽  
Stanislav Vosolsobě ◽  
Zdeněk Cit ◽  
Jozef Lacek ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Laser Scanning ◽  
Correlation Spectroscopy ◽  
Laser Scanning Microscopy ◽  
Image Correlation ◽  
Fluorescence Recovery ◽  
Confocal Laser ◽  
Raster Image ◽  
Image Correlation Spectroscopy ◽  
Plant Plasma Membrane

AbstractA number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

scholarly journals Twin-Chain Polymer Networks Loaded With Nanostructured Fluids for the Selective Removal of A Non-Original Varnish From A 20th Century Oil Painting At The Peggy Guggenheim Collection, Venice

2020 ◽  
Author(s):  
Luciano Pensabene Buemi ◽  
Maria Laura Petruzzellis ◽  
David Chelazzi ◽  
Michele Baglioni ◽  
Rosangela Mastrangelo ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Polymer Network ◽  
Polymer Networks ◽  
Mechanical Action ◽  
Correlation Spectroscopy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Oil Painting ◽  
Chain Polymer ◽  
Osmotic Balance

Abstract This paper reports on the evaluation of a polyvinyl alcohol (PVA) “twin-chain” polymer network (TC-PN) combined with an oil-in-water nanostructured fluid (NSF) for the removal of a polyvinyl acetate (PVAc) varnish. Small Angle X-ray Scattering, Confocal Laser Scanning Microscopy, and Fluorescence Correlation Spectroscopy showed that the structure of the gel and the NSF are only minimally altered by loading the fluid into the gel. The NSF is partially free to diffuse through the network, but also interacts with the gel walls. During the cleaning, the dynamics of the fluid at the gel-substrate interface are controlled by the osmotic balance taking place among the interconnected pores. These features grant effective and controlled cleaning performances. The case study identified for this research is Pablo Picasso’s The Studio (L’Atelier, 1928), one of the masterpieces in the Peggy Guggenheim Collection, Venice (PGC). In 1969 the oil painting, originally unprotected, was wax-lined and then varnished using a PVAc varnish. Over the years, the white shades of the painting have been compromised by the yellowing of the varnish and soiling of deposits. On painting mock-ups, the NSF-loaded hydrogels allowed the swelling and softening of PVAc varnish and wax layers, which were then removed with gentle mechanical action. Effective varnish and wax removal at the micron scale, and the absence of residues from the cleaning system (gel and NSF), were confirmed by Fourier Transform Infrared Spectroscopy (FTIR) 2D imaging. The effective and safe removal of the aged PVAc varnish and wax layer from the surface of the painting was then carried out using the same cleaning protocol successfully tested on the mock-ups, setting the NSF-loaded PVA TC-PNs as robust and reliable tools for the cleaning of sensitive works of art.

scholarly journals Imaging molecular interactions in cells by dynamic and static fluorescence anisotropy (rFLIM and emFRET)

2003 ◽  
Vol 31 (5) ◽  
pp. 1020-1027 ◽  
Author(s):  
D.S. Lidke ◽  
P. Nagy ◽  
B.G. Barisas ◽  
R. Heintzmann ◽  
J.N. Post ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Laser Scanning ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging

We report the implementation and exploitation of fluorescence polarization measurements, in the form of anisotropy fluorescence lifetime imaging microscopy (rFLIM) and energy migration Förster resonance energy transfer (emFRET) modalities, for wide-field, confocal laser-scanning microscopy and flow cytometry of cells. These methods permit the assessment of rotational motion, association and proximity of cellular proteins in vivo. They are particularly applicable to probes generated by fusions of visible fluorescence proteins, as exemplified by studies of the erbB receptor tyrosine kinases involved in growth-factor-mediated signal transduction.

scholarly journals QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY

2011 ◽  
Vol 25 (3) ◽  
pp. 111 ◽  
Author(s):  
Merete Krog Raarup ◽  
Jens Randel Nyengaard
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
3D Structure ◽  
Correlation Spectroscopy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Two Photon ◽  
Photon Excitation

This paper discusses recent advances in confocal laser scanning microscopy (CLSM) for imaging of 3D structure as well as quantitative characterization of biomolecular interactions and diffusion behaviour by means of one- and two-photon excitation. The use of CLSM for improved stereological length estimation in thick (up to 0.5 mm) tissue is proposed. The techniques of FRET (Fluorescence Resonance Energy Transfer), FLIM (Fluorescence Lifetime Imaging Microscopy), FCS (Fluorescence Correlation Spectroscopy) and FRAP (Fluorescence Recovery After Photobleaching) are introduced and their applicability for quantitative imaging of biomolecular (co-)localization and trafficking in live cells described. The advantage of two-photon versus one-photon excitation in relation to these techniques is discussed.

scholarly journals Twin-chain polymer networks loaded with nanostructured fluids for the selective removal of a non-original varnish from the Picasso “L’Atelier” at the Peggy Guggenheim Collection, Venice

2020 ◽  
Author(s):  
Luciano Pensabene Buemi ◽  
Maria Laura Petruzzellis ◽  
David Chelazzi ◽  
Michele Baglioni ◽  
Rosangela Mastrangelo ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Polymer Network ◽  
Polymer Networks ◽  
Mechanical Action ◽  
Correlation Spectroscopy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Oil Painting ◽  
Chain Polymer ◽  
Osmotic Balance

Abstract This paper reports on the evaluation of a polyvinyl alcohol (PVA) “twin-chain” polymer network (TC-PN) combined with an oil-in-water nanostructured fluid (NSF) for the removal of a polyvinyl acetate (PVAc) varnish. Small Angle X-ray Scattering, Confocal Laser Scanning Microscopy, and Fluorescence Correlation Spectroscopy showed that the structure of the gel and the NSF are only minimally altered by loading the fluid into the gel. The NSF is partially free to diffuse through the network, but also interacts with the gel walls. During the cleaning, the dynamics of the fluid at the gel-substrate interface are controlled by the osmotic balance taking place among the interconnected pores. These features grant effective and controlled cleaning performances. The case study identified for this research is Pablo Picasso’s The Studio (L’Atelier, 1928), one of the masterpieces in the Peggy Guggenheim Collection, Venice (PGC). In 1969 the oil painting, originally unprotected, was wax-lined and then varnished using a PVAc varnish. Over the years, the white shades of the painting have been compromised by the yellowing of the varnish and soiling of deposits. On painting mock-ups, the NSF-loaded hydrogels allowed the swelling and softening of PVAc varnish and wax layers, which were then removed with gentle mechanical action. Effective varnish and wax removal at the micron scale, and the absence of residues from the cleaning system (gel and NSF), were confirmed by Fourier Transform Infrared Spectroscopy (FTIR) 2D imaging. The effective and safe removal of the aged PVAc varnish and wax layer from the surface of the painting was then carried out using the same cleaning protocol successfully tested on the mock-ups, setting the NSF-loaded PVA TC-PNs as robust and reliable tools for the cleaning of sensitive works of art.

scholarly journals Antimicrobial efficacy and biocompatibility of extracts from Cryptocarya species

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0261884
Author(s):  
Jacqueline de Oliveira Zoccolotti ◽  
Alberto José Cavalheiro ◽  
Camilla Olga Tasso ◽  
Beatriz Ribeiro Ribas ◽  
Túlio Morandin Ferrisse ◽  
...  
Keyword(s):  
High Performance ◽  
Laser Scanning ◽  
Acrylic Resin ◽  
Laser Scanning Microscopy ◽  
Live Cells ◽  
Confocal Laser ◽  
Array Detector ◽  
Control Group ◽  
Oral Keratinocytes ◽  
Denture Base

This study evaluated the efficacy of Cryptocarya spp extracts on biofilm of Candida albicans and its biocompatibility. Mature biofilm of C. albicans was formed on denture base acrylic resin samples and the fungicidal effect of the extracts was evaluated by Alamar Blue® assay, counting colony-forming units (CFU/mL) and confocal laser scanning microscopy (CLSM). Cytotoxicity of extracts from Cryptocarya species was evaluated by AlamarBlue® assay, using normal oral keratinocytes (NOK) cells. In additional, Analysis of plant extracts by ultra-high-performance liquid chromatography–diode array detector–tandem mass spectrometry (UPLC-DAD-MS) was performed. The results showed significant reduction in the cellular metabolism and in the number of CFU/mL of C. albicans (p<0.05). The concentration of 0.045 g/mL completely inhibited the number of CFU/mL. Regarding cytotoxicity, all extracts decreased cell viability compared to the control group. CLSM analysis showed predominance of live cells, but with a great difference between the groups. Antimicrobial activity of extracts from Cryptocarya on C. albicans biofilm was confirmed. However, all extracts showed toxicity on NOK cells.

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