scholarly journals Fluorescence lifetime DNA-PAINT for multiplexed super-resolution imaging of cells

2022 ◽  
Vol 5 (1) ◽  
Author(s):  
Nazar Oleksiievets ◽  
Yelena Sargsyan ◽  
Jan Christoph Thiele ◽  
Nikolaos Mougios ◽  
Shama Sograte-Idrissi ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Laser Scanning ◽  
Super Resolution ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wide Field ◽  
Fluid Exchange ◽  
Multiplexed Imaging ◽  
Bio Imaging ◽  
A Cell

AbstractDNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. Here, we combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT and use the lifetime information as a multiplexing parameter for targets identification. In contrast to Exchange-PAINT, fluorescence lifetime PAINT (FL-PAINT) can image multiple targets simultaneously and does not require any fluid exchange, thus leaving the sample undisturbed and making the use of flow chambers/microfluidic systems unnecessary. We demonstrate the potential of FL-PAINT by simultaneous imaging of up to three targets in a cell using both wide-field FLIM and 3D time-resolved confocal laser scanning microscopy (CLSM). FL-PAINT can be readily combined with other existing techniques of multiplexed imaging and is therefore a perfect candidate for high-throughput multi-target bio-imaging.

scholarly journals Confocal Laser-Scanning Fluorescence-Lifetime Single-Molecule Localisation Microscopy

2020 ◽  
Author(s):  
Jan Christoph Thiele ◽  
Dominic Helmerich ◽  
Nazar Oleksiievets ◽  
Roman Tsukanov ◽  
Eugenia Butkevich ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Spectral Properties ◽  
Laser Scanning ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Localisation Microscopy

AbstractFluorescence lifetime imaging microscopy (FLIM) is an important technique that adds another dimension to the intensity and colour information of conventional microscopy. In particular, it allows for multiplexing fluorescent labels that have otherwise similar spectral properties. Currently, the only super-resolution technique that is capable of recording super-resolved images with lifetime information is STimulated Emission Depletion (STED) microscopy. In contrast, all Single-Molecule Localisation Microscopy (SMLM) techniques that employ wide-field cameras completely lack the lifetime dimension. Here, we combine Fluorescence-Lifetime Confocal Laser-Scanning Microscopy (FL-CLSM) with SMLM for realising single-molecule localisation-based fluorescence-lifetime super-resolution imaging (FL-SMLM). Besides yielding images with a spatial resolution much beyond the diffraction limit, it determines the fluorescence lifetime of all localised molecules. We validate our technique by applying it to direct STochastic Optical Reconstruction Microscopy (dSTORM) and Points Accumulation for Imaging in Nanoscale Topography (PAINT) imaging of fixed cells, and we demonstrate its multiplexing capability on samples with two different labels that differ only by fluorescence lifetime but not by their spectral properties.

scholarly journals Effect of Clove and Thyme Essential Oils on Candida Biofilm Formation and the Oil Distribution in Yeast Cells

Molecules ◽  
2019 ◽  
Vol 24 (10) ◽  
pp. 1954 ◽  
Author(s):  
Katarzyna Rajkowska ◽  
Paulina Nowicka-Krawczyk ◽  
Alina Kunicka-Styczyńska
Keyword(s):  
Essential Oils ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Yeast Cells ◽  
Confocal Laser ◽  
Minimal Inhibitory Concentrations ◽  
Candida Sp ◽  
A Cell ◽  
Candida Biofilm

Candida biofilm structure is particularly difficult to eradicate, since biofilm is much more resistant to antifungal agents than planktonic cells. In this context, a more effective strategy seems to be the prevention of biofilm formation than its eradication. The aim of the study was to examine whether the process of initial colonization of materials (glass, polyethylene terephthalate, polypropylene) by food-borne Candida sp. can be impeded by clove and thyme essential oils, used at their minimal inhibitory concentrations. In the presence of clove oil, 68.4–84.2% of the yeast tested showed a statistically significant reduction in biofilm formation, depending on the material. After treatment with thyme oil, statistically significant decrease in biofilm cell numbers was observed for 63.2–73.7% of yeasts. Confocal laser scanning microscopy showed diverse compounds of clove and thyme oils that were disparately located in C. albicans cell, on a cell wall and a cell membrane, in cytoplasm, and in vacuoles, depicting the multidirectional action of essential oils. However, essential oils that were used in sub-inhibitory concentration were sequestrated in the yeast vacuoles, which indicate the activation of Candida defense mechanisms by cell detoxification. Clove and thyme essential oils due to their anti-biofilm activity can be efficiently used in the prevention of the tested abiotic surfaces colonization by Candida sp.

scholarly journals A Selective Ratiometric Fluorescent Probe for No-Wash Detection of PVC Microplastic

Polymers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1588
Author(s):  
Valeria Caponetti ◽  
Alexandra Mavridi-Printezi ◽  
Matteo Cingolani ◽  
Enrico Rampazzo ◽  
Damiano Genovese ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Direct Detection ◽  
Low Cost ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wide Field ◽  
Fluorescence Intensity Ratio ◽  
Green Fluorescence ◽  
One Pot ◽  
Emerging Pollutant

Microplastics (MP) are micrometric plastic particles present in drinking water, food and the environment that constitute an emerging pollutant and pose a menace to human health. Novel methods for the fast detection of these new contaminants are needed. Fluorescence-based detection exploits the use of specific probes to label the MP particles. This method can be environmentally friendly, low-cost, easily scalable but also very sensitive and specific. Here, we present the synthesis and application of a new probe based on perylene-diimide (PDI), which can be prepared in a few minutes by a one-pot reaction using a conventional microwave oven and can be used for the direct detection of MP in water without any further treatment of the sample. The green fluorescence is strongly quenched in water at neutral pH because of the formation dimers. The ability of the probe to label MP was tested for polyvinyl chloride (PVC), polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), polystyrene (PS), poly methyl methacrylate (PMMA) and polytetrafluoroethylene (PTFE). The probe showed considerable selectivity to PVC MP, which presented an intense red emission after staining. Interestingly, the fluorescence of the MP after labeling could be detected, under excitation with a blue diode, with a conventional CMOS color camera. Good selectivity was achieved analyzing the red to green fluorescence intensity ratio. UV–Vis absorption, steady-state and time-resolved fluorescence spectroscopy, fluorescence anisotropy, fluorescence wide-field and confocal laser scanning microscopy allowed elucidating the mechanism of the staining in detail.

scholarly journals Direct fluorescence imaging of lignocellulosic and suberized cell walls in roots and stems

AoB Plants ◽  
2020 ◽  
Vol 12 (4) ◽  
Author(s):  
Peter Kitin ◽  
Satoshi Nakaba ◽  
Christopher G Hunt ◽  
Sierin Lim ◽  
Ryo Funada
Keyword(s):  
Cell Walls ◽  
Laser Scanning ◽  
Cell Types ◽  
Plant Evolution ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Chemical Information ◽  
Wide Field ◽  
Tissue Preparation ◽  
Plant Structure

Abstract Investigating plant structure is fundamental in botanical science and provides crucial knowledge for the theories of plant evolution, ecophysiology and for the biotechnological practices. Modern plant anatomy often targets the formation, localization and characterization of cellulosic, lignified or suberized cell walls. While classical methods developed in the 1960s are still popular, recent innovations in tissue preparation, fluorescence staining and microscopy equipment offer advantages to the traditional practices for investigation of the complex lignocellulosic walls. Our goal is to enhance the productivity and quality of microscopy work by focusing on quick and cost-effective preparation of thick sections or plant specimen surfaces and efficient use of direct fluorescent stains. We discuss popular histochemical microscopy techniques for visualization of cell walls, such as autofluorescence or staining with calcofluor, Congo red (CR), fluorol yellow (FY) and safranin, and provide detailed descriptions of our own approaches and protocols. Autofluorescence of lignin in combination with CR and FY staining can clearly differentiate between lignified, suberized and unlignified cell walls in root and stem tissues. Glycerol can serve as an effective clearing medium as well as the carrier of FY for staining of suberin and lipids allowing for observation of thick histological preparations. Three-dimensional (3D) imaging of all cell types together with chemical information by wide-field fluorescence or confocal laser scanning microscopy (CLSM) was achieved.

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 Microscope-Based Imaging Platform for Large-Scale Analysis of Oral Biofilms

2012 ◽  
Vol 78 (24) ◽  
pp. 8703-8711 ◽  
Author(s):  
L. Karygianni ◽  
M. Follo ◽  
E. Hellwig ◽  
D. Burghardt ◽  
M. Wolkewitz ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Large Scale ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wide Field ◽  
Bacterial Composition ◽  
Entire Area ◽  
Content Type ◽  
Oral Biofilms

ABSTRACTA microscopic method for noninvasively monitoring oral biofilms at the macroscale was developed to describe the spatial distribution of biofilms of different bacterial composition on bovine enamel surfaces (BES). For this purpose, oral biofilm was grownin situon BES that were fixed at approximal sites of individual upper jaw acrylic devices worn by a volunteer for 3 or 5 days. Eubacteria,Streptococcusspp., andFusobacterium nucleatumwere stained using specific fluorescencein situhybridization (FISH) probes. The resulting fluorescence signals were subsequently tested by confocal laser scanning microscopy (CLSM) and monitored by an automated wide-field microscope-based imaging platform (Scan∧R). Automated image processing and data analysis were conducted by microscope-associated software and followed by statistical evaluation of the results. The full segmentation of biofilm images revealed a random distribution of bacteria across the entire area of the enamel surfaces examined. Significant differences in the composition of the microflora were recorded across individual as well as between different enamel surfaces varying from sparsely colonized (47.26%) after 3 days to almost full surface coverage (84.45%) after 5 days. The enamel plates that were positioned at the back or in the middle of the oral cavity were found to be more suitable for the examination of biofilms up to 3 days old. In conclusion, automated microscopy combined with the use of FISH can enable the efficient visualization and meaningful quantification of bacterial composition over the entire sample surface. Due to the possibility of automation, Scan∧R overcomes the technical limitations of conventional CLSM.

scholarly journals Laser Scanning versus Wide-Field—Choosing the Appropriate Microscope in Life Sciences

2021 ◽  
Vol 11 (2) ◽  
pp. 733
Author(s):  
Herbert Schneckenburger ◽  
Verena Richter
Keyword(s):  
Laser Scanning ◽  
Light Exposure ◽  
Life Sciences ◽  
Super Resolution ◽  
Light Sheet ◽  
Laser Scanning Microscopy ◽  
Wide Field ◽  
Optimal Method ◽  
Recording Time ◽  
Light Sheet Microscopy

Methods and applications of light microscopy in the life sciences are compared with respect to 3D imaging, resolution, light exposure, sensitivity, and recording time. While conventional wide-field or laser scanning microscopy appear appropriate for smaller samples of only a few micrometers in size with a limited number of light exposures, light sheet microscopy appears to be an optimal method for larger 3D cell cultures, biopsies, or small organisms if multiple exposures or long measuring periods are desired. Super-resolution techniques should be considered in the context of high light exposure possibly causing photobleaching and photo-toxicity to living specimens.

Phase modulation nanoscopy: a simple approach to enhanced optical resolution

2015 ◽  
Vol 177 ◽  
pp. 507-515 ◽  
Author(s):  
Robert Pal
Keyword(s):  
Phase Modulation ◽  
Laser Scanning ◽  
Optical Resolution ◽  
Super Resolution ◽  
Laser Scanning Microscopy ◽  
Optical Diffraction ◽  
Optical Modulator ◽  
Confocal Laser ◽  
Electro Optical Modulator ◽  
Experimental Resolution

A new modular super-resolution technique called Phase Modulation Nanoscopy (PhMoNa) has been developed in order to break the optical diffraction barrier in Confocal Laser Scanning Microscopy (LSCM). This technique is based on using spatially modulated illumination intensity, whilst harnessing the fluorophore's non-linear emission response. It allows experimental resolution in both lateral and axial domains to be improved by at least a factor of 2. The work is in its initial phase, but by using a custom built Electro Optical Modulator (EOM) in conjunction with functionalised Ln(iii) complexes as probes, a sub-diffraction resolution of ∼60 nm was achieved of selected cellular organelles in long term live cell imaging experiments.

scholarly journals Fluorescence lifetime imaging of red yeast Cystofilobasidium capitatum during growth

2018 ◽  
Vol 2 (2) ◽  
pp. 114-120 ◽  
Author(s):  
Martin Vanek ◽  
Filip Mravec ◽  
Martin Szotkowski ◽  
Dana Byrtusova ◽  
Andrea Haronikova ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Laser Scanning ◽  
Light Exposure ◽  
Laser Scanning Microscopy ◽  
Model Systems ◽  
Confocal Laser ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Red Yeast

AbstractRed yeast Cystofilobasidium capitatum autofluorescence was studied by means of confocal laser scanning microscopy (CLSM) to reveal distribution of carotenoids inside the cells. Yeasts were cultivated in 2L fermentor on glucose medium at permanent light exposure and aeration. Samples were collected at different times for CLSM, gravimetric determination of biomass and HPLC determination of pigments. To compare FLIM (Fluorescence Lifetime Imaging Microscopy) images and coupled data (obtained by CLSM) with model systems, FLIM analysis was performed on micelles of SDS:ergosterol and SDS:coenzyme Q with different content of ergosterol and coenzyme Q, respectively, and with constant addition of beta-carotene. Liposomes lecithin:ergosterol:beta-carotene were investigated too. Two different intracellular forms of carotenoids were observed during most of cultivations, with third form appeared at the beginning of stationary phase. Observed behavior is probably due to formation of some kind of carotenoid protective system in membranes of different compartments of yeast cell, especially cytoplasmic membrane.

Different patterns of rDNA distribution in Pisum sativum nucleoli correlate with different levels of nucleolar activity

1993 ◽  
Vol 104 (3) ◽  
pp. 843-852 ◽  
Author(s):  
M. I. Highett ◽  
D. J. Rawlins ◽  
P. J. Shaw
Keyword(s):  
Pisum Sativum ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Ccd Camera ◽  
Cell Types ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wide Field ◽  
Root Tips ◽  
Root Cells

We have used in situ hybridization with probes to rDNA, labelled either with digoxygenin or directly with fluorescein, to determine the arrangement of these genes within the nucleoli of Pisum sativum L. root cells. Confocal laser scanning microscopy was used to image the three-dimensional structures revealed, but we have also compared this technique with deconvolution of conventional (wide-field) fluorescence images measured with a cooled CCD camera, and have shown that the results are remarkably similar. When the deconvolution technique was applied to the confocal data it gave clearer images than could be achieved by confocal microscopy alone. We have analysed the distribution of rDNA in the different cell types observable in root tips: the quiescent centre; active meristematic cells; and relatively differentiated root cap, epidermal and cortical cells. In addition to four perinucleolar knobs of condensed, inactive rDNA genes, corresponding to the four nucleolar organizers in P. sativum, which were the most brightly labelled structures, several characteristic patterns of intranucleolar labelling were apparent, including bright foci, large central chromatin masses, and fine, decondensed interconnecting fibres. The larger and more active the nucleolus, the smaller the proportion of condensed perinucleolar rDNA. In some large and active meristematic nucleoli, all the internal rDNA is decondensed, showing that transcription cannot be restricted to the bright foci, and is most likely to occur on the decondensed fibres.

Sign in / Sign up

Export Citation Format

Share Document