scholarly journals Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging

2014 ◽  
Vol 22 (9) ◽  
pp. 10221 ◽  
Author(s):  
Ming Zhao ◽  
Yu Li ◽  
Leilei Peng
Keyword(s):  
Confocal Microscopy ◽  
Fluorescence Lifetime ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Parallel Excitation

scholarly journals Optimization of Advanced Live-Cell Imaging through Red/Near-Infrared Dye Labeling and Fluorescence Lifetime-Based Strategies

2021 ◽  
Vol 22 (20) ◽  
pp. 11092
Author(s):  
Magalie Bénard ◽  
Damien Schapman ◽  
Christophe Chamot ◽  
Fatéméh Dubois ◽  
Guénaëlle Levallet ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Live Cell Imaging ◽  
Near Infrared ◽  
Cell Imaging ◽  
Light Exposure ◽  
Photon Counting ◽  
Live Cell ◽  
Stimulated Emission ◽  
Plot Analysis ◽  
Phasor Plot

Fluorescence microscopy is essential for a detailed understanding of cellular processes; however, live-cell preservation during imaging is a matter of debate. In this study, we proposed a guide to optimize advanced light microscopy approaches by reducing light exposure through fluorescence lifetime (τ) exploitation of red/near-infrared dyes. Firstly, we characterized key instrumental elements which revealed that red/near-infrared laser lines with an 86x (Numerical Aperture (NA) = 1.2, water immersion) objective allowed high transmission of fluorescence signals, low irradiance and super-resolution. As a combination of two technologies, i.e., vacuum tubes (e.g., photomultiplier) and semiconductor microelectronics (e.g., avalanche photodiode), type S, X and R of hybrid detectors (HyD-S, HyD-X and HyD-R) were particularly adapted for red/near-infrared photon counting and τ separation. Secondly, we tested and compared lifetime-based imaging including coarse τ separation for confocal microscopy, fitting and phasor plot analysis for fluorescence lifetime microscopy (FLIM), and lifetimes weighting for enhanced stimulated emission depletion (STED) nanoscopy, in light of red/near-infrared multiplexing. Mainly, we showed that the choice of appropriate imaging approach may depend on fluorochrome number, together with their spectral/lifetime characteristics and STED compatibility. Photon-counting mode and sensitivity of HyDs together with phasor plot analysis of fluorescence lifetimes enabled the flexible and fast imaging of multi-labeled living H28 cells. Therefore, a combination of red/near-infrared dyes labeling with lifetime-based strategies offers new perspectives for live-cell imaging by enhancing sample preservation through acquisition time and light exposure reduction.

scholarly journals HaloTag8: an engineered protein tag to improve fluorophore performance

2021 ◽  
Author(s):  
Michelle S. Frei ◽  
Miroslaw Tarnawski ◽  
Julia Roberti ◽  
Birgit Koch ◽  
Julien Hiblot ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescent Probes ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Protein Tag

HaloTag8 is an engineered variant of HaloTag7 with up to 40% higher brightness and increased fluorescence lifetime when labeled with fluorogenic rhodamines. Moreover, combining HaloTag8 with HaloTag7 and other fluorescent probes enabled live-cell multiplexing using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. The increased brightness of HaloTag8 and its use in fluorescence lifetime multiplexing makes it a powerful tool for live-cell imaging.

Continuous live cell imaging of cellulose attachment by microbes under anaerobic and thermophilic conditions using confocal microscopy

2013 ◽  
Vol 25 (5) ◽  
pp. 849-856 ◽  
Author(s):  
Zhi-Wu Wang ◽  
Seung-Hwan Lee ◽  
James G. Elkins ◽  
Yongchao Li ◽  
Scott Hamilton-Brehm ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Thermophilic Conditions

scholarly journals KV1.5–KVβ1.3 Recycling Is PKC-Dependent

2021 ◽  
Vol 22 (3) ◽  
pp. 1336
Author(s):  
Alvaro Macias ◽  
Alicia de la Cruz ◽  
Diego A. Peraza ◽  
Angela de Benito-Bueno ◽  
Teresa Gonzalez ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Hek293 Cells ◽  
Dependent Manner ◽  
Fast Inactivation ◽  
Patch Clamp Technique ◽  
Relevant Issue ◽  
Regulatory Subunits

KV1.5 channel function is modified by different regulatory subunits. KVβ1.3 subunits assemble with KV1.5 channels and induce a fast and incomplete inactivation. Inhibition of PKC abolishes the KVβ1.3-induced fast inactivation, decreases the amplitude of the current KV1.5–KVβ1.3 and modifies their pharmacology likely due to changes in the traffic of KV1.5–KVβ1.3 channels in a PKC-dependent manner. In order to analyze this hypothesis, HEK293 cells were transfected with KV1.5–KVβ1.3 channels, and currents were recorded by whole-cell configuration of the patch-clamp technique. The presence of KV1.5 in the membrane was analyzed by biotinylation techniques, live cell imaging and confocal microscopy approaches. PKC inhibition resulted in a decrease of 33 ± 7% of channels in the cell surface due to reduced recycling to the plasma membrane, as was confirmed by confocal microscopy. Live cell imaging indicated that PKC inhibition almost abolished the recycling of the KV1.5–KVβ1.3 channels, generating an accumulation of channels into the cytoplasm. All these results suggest that the trafficking regulation of KV1.5–KVβ1.3 channels is dependent on phosphorylation by PKC and, therefore, they could represent a clinically relevant issue, mainly in those diseases that exhibit modifications in PKC activity.

scholarly journals Engineered HaloTag variants for fluorescence lifetime multiplexing

2021 ◽  
Author(s):  
Michelle S. Frei ◽  
Miroslaw Tarnawski ◽  
M. Julia Roberti ◽  
Birgit Koch ◽  
Julien Hiblot ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescence Lifetime ◽  
Fusion Proteins ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Cellular Targets ◽  
Label Fusion ◽  
Spectral Channel ◽  
Protein Tags

AbstractSelf-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications.

Live Cell Imaging and Confocal Microscopy

2018 ◽  
pp. 117-130 ◽  
Author(s):  
Luciana Renna ◽  
Giovanni Stefano ◽  
Federica Brandizzi
Keyword(s):  
Confocal Microscopy ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell
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