scholarly journals Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

10.3791/60613 ◽  
2019 ◽  
Author(s):  
Alexander Lichius ◽  
Susanne Zeilinger
Keyword(s):  
Cell Wall ◽  
Filamentous Fungi ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Dyes ◽  
Live Cell

Dye selection for live cell imaging of intact siRNA

2012 ◽  
Vol 393 (1-2) ◽  
pp. 23-35 ◽  
Author(s):  
Markus Hirsch ◽  
Dennis Strand ◽  
Mark Helm
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Lapse ◽  
Live Cell ◽  
High Yield ◽  
Ph Variation ◽  
Rnai Efficiency

Abstract Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) efficiency. Because intracellular degradation of RNA may prevent reliable observation of fluorescence-labeled siRNA, new tools for fluorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fluorescence resonance energy transfer (FRET) dye pairs for sensing the integrity of duplex siRNA is reported, which allows an assessment of the degradation status of an siRNA cell population by live cell imaging. A panel of high-yield fluorescent dyes has been investigated for their suitability as FRET pairs for the investigation of RNA inside the cell. Nine dyes in 13 FRET pairs were evaluated based on the performance in assays of photostability, cross-excitation, bleed-through, as well as on quantified changes of fluorescence as a consequence of, e.g., RNA strand hybridization and pH variation. The Atto488/Atto590 FRET pair has been applied to live cell imaging, and has revealed first aspects of unusual trafficking of intact siRNA. A time-lapse study showed highly dynamic movement of siRNA in large perinuclear structures. These and the resulting optimized FRET labeled siRNA are expected to have significant impact on future observations of labeled RNAs in living cells.

scholarly journals Evaluation of Chemical Fluorescent Dyes as a Protein Conjugation Partner for Live Cell Imaging

PLoS ONE ◽  
2014 ◽  
Vol 9 (9) ◽  
pp. e106271 ◽  
Author(s):  
Yoko Hayashi-Takanaka ◽  
Timothy J. Stasevich ◽  
Hitoshi Kurumizaka ◽  
Naohito Nozaki ◽  
Hiroshi Kimura
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Dyes ◽  
Live Cell ◽  
Protein Conjugation

scholarly journals Live-cell imaging with Aspergillus fumigatus-specific fluorescent siderophore conjugates

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Joachim Pfister ◽  
Alexander Lichius ◽  
Dominik Summer ◽  
Hubertus Haas ◽  
Thines Kanagasundaram ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Dyes ◽  
Iron Acquisition ◽  
Live Cell ◽  
Subcellular Localisation ◽  
Gallium 68

Abstract Live-cell imaging allows the in vivo analysis of subcellular localisation dynamics of physiological processes with high spatial–temporal resolution. However, only few fluorescent dyes have been custom-designed to facilitate species-specific live-cell imaging approaches in filamentous fungi to date. Therefore, we developed fluorescent dye conjugates based on the sophisticated iron acquisition system of Aspergillus fumigatus by chemical modification of the siderophore triacetylfusarinine C (TAFC). Various fluorophores (FITC, NBD, Ocean Blue, BODIPY 630/650, SiR, TAMRA and Cy5) were conjugated to diacetylfusarinine C (DAFC). Gallium-68 labelling enabled in vitro and in vivo characterisations. LogD, uptake assays and growth assays were performed and complemented by live-cell imaging in different Aspergillus species. Siderophore conjugates were specifically recognised by the TAFC transporter MirB and utilized as an iron source in growth assays. Fluorescence microscopy revealed uptake dynamics and differential subcellular accumulation patterns of all compounds inside fungal hyphae.[Fe]DAFC-NBD and -Ocean Blue accumulated in vacuoles, whereas [Fe]DAFC-BODIPY, -SiR and -Cy5 localised to mitochondria. [Fe]DAFC -FITC showed a uniform cytoplasmic distribution, whereas [Fe]DAFC-TAMRA was not internalised at all. Co-staining experiments with commercially available fluorescent dyes confirmed these findings. Overall, we developed a new class of fluorescent dyes that vary in intracellular fungal targeting , thereby providing novel tools for live-cell imaging applications for Aspergillus fumigatus.

Deep-Red Emissive Crescent-Shaped Fluorescent Dyes: Substituent Effect on Live Cell Imaging

2015 ◽  
Vol 7 (13) ◽  
pp. 7421-7427 ◽  
Author(s):  
Weimin Liu ◽  
Bingjiang Zhou ◽  
Guangle Niu ◽  
Jiechao Ge ◽  
Jiasheng Wu ◽  
...  
Keyword(s):  
Substituent Effect ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Dyes ◽  
Live Cell
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