3 fluorescent probes that will count in 2017

Published on December 2, 2016

The detection of molecular events in living cells is booming. In this post, we look at 3 fluorescent probes that will undoubtedly count in the live-cell imaging landscape in 2017.

The use of fluorescent probes for detecting and monitoring cellular and molecular events in living cells is becoming more and more popular.

Interactive live cell imaging tool bow by tebu-bio — Interactive live cell imaging tool box by tebu-bio.

This year was marked by the launch of numerous innovative fluorescent probes covering various areas, such as:

mitophagy
mitochondrial Singlet Oxygen Imaging (Si-DMA)
lipohilic peroxides (MitoPEDPP)…

These new tools complement an already comprehensive offer of well-accepted dyes such as SiR fluorogenic probes for studying cytoskeletal dynamics, DNA or monitoring lysosomal activities, ROSFluor™ Series for the measurement of reactive oxgen species, and NOFluor™ Series for NO monitoring, Fluo-8 and other probes for Calcium concentrations….

To illustrate the dynamism of this offer, an interactive cellular map has been designed for the convenient selection of fluorescent dyes according to the cellular parameter to analyse in live cell imaging (LCI) or flow cytometry (FCM).

What does the future hold?

fluorescence-spectra-of-acidifluor-orange-as-a-function-of-ph-ph-5-0-vs-ph-7-4 — Fluorescence spectra of AcidiFluor Orange was measured in phosphate buffer pH 5.0 (the condition in the acidic organelles) or pH 7.4 ( physiological cytoplasmic conditions). Fluorescent intensity of AcidiFluor Orange at pH 5.0 increased 50 fold to that in the pH 7.4 buffer. ( λex 532 nm / λem 568 nm )

2017 will also see the emergence of promising and innovative fluorescent probes that will bring Life Scientists reliable tools to further analyze events in living cells. Here are 4 dyes that will count.

#1 – AcidiFluor Orange: probe acidic organelles

AcidiFluor™ stains are photostable Orange fluorescent dyes with high S/N ratio to selectively stain acidic cellular organelles:

AcidiFluor™ ORANGE (cat. nr GC301): pH probe for acidic organelle imaging,
AcidiFluor™ ORANGE-NHS (cat. nr GC302): pH probe for labeling protein and nucleic acid,
AcidiFluor™ ORANGE-Zymosan A (cat. nr GC305): For analyzing phagocytosis,
AcidiFluor™ ORANGE-Dextran 10K (cat. nr GC306) and -transferin (cat. nr GC309) for endocytosis detection.

AcidiFluor™ stains enable thus the monitoring of exocytosis, endocytosis, pH dynamics in vesicule in living cells, and are compatible with multicolor imaging.

acidifluor-orange-multicolor-fluorescence-in-hela-cells — AcidiFluor Orange Multicolor fluorescence in HeLa cells HeLa cell expressed Mitochondria-GFP was stained multiplicity with AcidiFluor™ ORANGE and Hoechst33342. Lysosomes were stained orange with AcidiFluor™ ORANGE, nucleuses were stained blue with Hoechst33342 and Mitochondria were stained green with GFP.

acidifluor-orange-ph-probe-for-acidic-organelle — Samples were continuously illuminated for 180 seconds and viewed on a confocal microscopy. Although LysoTracker® Green DND-26 and LysoTracker® Red DND-99 discolored dramatically, fluorescence of AcidiFluor™ ORANGE remained. LysoSensor™ Green DND-189 was not suitable for continuous observation because fluorescence leaked to cytoplasm by excitation light irradiation.

#2 – GlycoYELLOW™-βGal to screening of β-galactosidase activity in live cells

β-galactosidase (β-Gal) activity is widely used as a senescent marker (SA-βGal) or reporter gene such as lacZ (transfection efficiency, gene reporter assays, transcriptional regulation…).

For more biologically relevant lacZ analysis by fluorescent imaging, the GlycoYELLOW™-βGal (cat. nr GC601) has been recently released. This dye probes for β-Gal activity at single-cell level detection with high S/N ratio for both live and fixed cells.

Therefore, it is suitable for pharmacological screening tests, including cell imaging-based screening system as well as detection of cellular senescence.

Live cell imaging of 293/LacZ (b-galactosidase expressing cell line) and HEK293 cell by using GlycoYELLOW™–βGal. Comparing these cell lines, strong fluorescence derived from GlycoYELLOW™–βGal was observed inside 293/LacZ cell. Mean fluorescence intensity in cells of GlycoYELLOW™–βGal is 5.6 times increased.

live-cell-imaging-of-293-lacz-and-hek293-cells — Time-lapse observation of 293/LacZ cell (β-galactosidase expressing cell) treated by GlycoYELLOW™–βGal result indicated that GlycoYELLOW™–βGal possesses excellent retention ability inside the cell.

Live cell imaging of senescence-associated beta-galactosidase (SA-β-gal) in senescent cell by GlycoYellow™-βGal (Yellow color shows SA-b-Gal activity in living cell after irradiation of radial ray).

Representing image (Input image) of GlycoYELLOW™-βGal stained cells acquired on Operetta™ High Contents Imaging System (PerkinElmer). The obtained images were analyzed with Harmony™ software (PerkinElmer). In brief, the individual cells in each field of view (FOV) were recognized by using prebuild software functions as follows: DAPI stained nuclei were recognized by “Find Nuclei” function, and then cytoplasmic region were subsequently detected by “Find Cytoplasm” function. The fluorescence intensity derived from GlycoYELLOWTM-βGal in cytoplasm region was quantitated in every cell in FOV. Find Nuclei: Nuclei recognized in input image with “Find Nuclei” function. Find Cytoplasm: Cytoplasm regions recognized in input image with “Find Cytoplasm” function. Quantitation: GlycoYELLOW™-βGal positive signal detected in the FOV.

#3- MAR – the hypoxia detection probe

fluorescent-intensity-in-a549-cell-under-hypoxia-stained-by-mar-or-pimonidazole — Fluorescent intensity in A549 cell under hypoxia, stained by MAR or pimonidazole. A549 cells were observed after stained by MAR or pimonidazole under the various concentrations of oxygen. While pimonidazole responded to the concentration of oxygen under the 1%, MAR responded to the oxygen concentration of about 5%.

Hypoxia occurs in living tissue and is closely related to various diseases such as cancers and ischemia.
Current hypoxia detection probes (e.g. pimonidazole) require immunostaining of fixed cells. HypoFluor™ MAR fluorogenic probe (cat. nr A101-01) is a new hypoxia detection probe easily applicable for live-cell imaging and flow cytometry with an high level of sensitivity.

MAR is becoming fluorescent (Ex. 498 nm / Em. 520 nm) while hypoxic conditions are appearing (pO2 of about 5%) in cell culture or in tissues