Halo-Flipper is a fluorescent probe that specifically labels Halo-tag™* and reports membrane tension changes through its fluorescence lifetime changes. It contains a ChloroAlkane Halo-tag™* ligand as well as a tethered Flipper-TR fluorophore which senses changes of the organization of lipid bilayer membranes surrounding the Halo-tag™* protein. Halo-Flipper is cell permeable, spontaneously labels Halotag expressing cells and is only fluorescent when inserted in a lipid membrane. It has a broad absorption and emission spectrum, excitation can be commonly performed with a 488nm laser, while emission is collected between 575 and 625nm. It is the perfect tool to precisely localize the Flipper-TR membrane tension fluorophore within cells. ChloroAlkane (CA) is the substrate of the self labeling tag Halo-tag™*. Upon reaction with a CA derivative, Halo-tag™* forms a covalent bond with the substrate. It allows to permanently attach a fluorescent label to any protein of interest (POI) expressed as Halo-tag™* fusion
Photophysical Properties
λabs 488 nm
λem 600 nm
εmax 1.66x104 mol-1·cm-1 (DMSO)
lifetime 2.8 - 7 ns
QY 30% (AcOEt)
For product Datasheets and MSDSs please click on the PDF links below.
Spirochrome Technical Tips and Ex/Em spectra in graphical form (PDF)
FAQs of the revolutionary Flipper-TR® probe
Q1. What is FLIM microscopy and how does it work for Flipper-TR?
A1. FLIM microscopy stands for Fluorescent Lifetime Imaging Microscopy. The importance for membrane tension studies is that prior to Flipper-TR membrane tension measurements were very labor and equipment intensive, but now relatively straightforward adaptation of your current microscope will enable highly sensitive tension measurements. Nowadays it is a very standard technique with equipment available from many suppliers, it is based on recording the time that emission takes after excitation of the fluorophore, which is usually very rapid, on the order of 1-10 nano-seconds. FLIM can also be combined with other high resolution microscopic techniques such as Total Internal Reflection Fluorescence (TIRF) or Stimulated Emission Depletion (STED) microscopy for high spatial resolution. FLIM microscopy requires time resolved light detectors which many scientific microscope vendors have available, for example PicoQuant's upgrade kit (https://www.picoquant.com/news/item/picoquants-flim-fcs-upgrade-kit-now-supports-zeiss-lsm780-and-leica-sp2). Reference 1 describes more details about the experimental setup for FLIM microscopy. (Flipper-TR is a registered trademark of UNIGEM, Switzerland)
Q2. Does the Flipper-TR probe work to change fluorescence lifetime?
A2. The fluorescent Flipper-TR® probe works by specifically targeting the plasma membrane of cells and reports membrane tension changes through its fluorescence lifetime changes. It is the most advanced member of the Flipper probes family (Ref. 2,3,4,5). Flipper-TR® spontaneously inserts into the plasma membrane of cells and is only fluorescent when inserted into a lipid membrane. The probe senses changes in the organization of lipid bilayer membranes through the twist angle and polarization between two twisted dithienothiophenes of the mechanophore (see low tension/high tension figure). The emission lifetime is short (2-4 ns) when in the tense state (dithienothiophenes aligned), and longer in the relaxed state (4.1-8.0 ns, dithienothiophenes twisted). Variance (cv) is in the order of 0.3 ns (cv = 4-15%) which allows high resolution of subtle changes in membrane tension. Typically the shorter lifetimes are color coded green, medium lifetimes coded yellow, and longer lifetimes are orange and red (see low tension/high tension figure).
Q3. What are the filter sets for these probes?
A3. The Flipper-TR probe is visualized with a long separation filter set because its excitation peak is more than 100 nm shorter than the emission peak. Thus the ideal filter set is an excitation of 488 +/- 20 nm and an emission of 575 to 675 +/- 40 nm. The time resolved measurement method allows very low bacground, which is additive with its low fluorescence in aqueous envirnments, see Q4 below.
Q4. Why does the Flipper-TR probe have a low background compared to other plasma-membrane probes?
A4. The Flipper-TR probes has very low background in aquence enviroments e.g. tissue culture media or fixative buffer, because it is a fully twisted state and tends to form micelles which quenches itself (Ref.3). After insertion into the membrane it becomes less twisted and starts to emit with high fluorescence.
Q5: Is the Flipper-TR probe stable at room temperature?
A5: Yes, the probe is stable in the powder form at room temperature for a few days. After reconstitution in anhydrous DMSO (do not use old-pre-opened bottles of DMSO, but do use ampoules of dry DMSO from Sigma or Spectrum Chemicals. It is stable to freezing and thawing at –20°C, but it is not recommended to divide into small aliquots for storage because it will degrade under these conditions.
Q6: Is the Flipper-TR toxic to cells?
A6: No, under the conditions given in the datasheet the probe is not toxic. Cells will be viable and fluorescent for 2-4 days depending on cell type and culture condition.
Q7: Which organisms and tissues are stained by the Flipper-TR probe?
A7: Currently all known organism have been stained with Flipper-TR, these include tissue culture cells, tissue section both living and fixed, mammalian cells, insect cells, plant cells, yeast and bacteria. .
Q8. Does the Flipper-TR probe work in 3D cell cultures?
A8: Yes, the probe is able to stain cells in a 3D growth environment.
Q9: What is the quantum yield and extinction coefficient in the membrane?
A9: Quantum yield = 0.30 in ethylacetate.
References
1. FLIM microscopy: Lakowicz JR et al. 1994. Emerging biomedical and advanced applications of time-resolved fluorescence spectroscopy. J Fluoresc. 4(1):117-36. doi: 10.1007/BF01876666.
2. Riggi M et al. 2018. Decrease in plasma membrane tension triggers PtdIns(4,5)P2 phase separation to inactivate TORC2. Nat. Cell Biol. 20, 1043–1051.
3. Colom A et al. 2018. A fluorescent membrane tension probe. Nat. Chem. 10, 1118–1125.
4. Dal Molin M. et al. 2015. Fluorescent flippers for mechanosensitive membrane probes. J. Am. Chem. Soc. 137, 568-571.
5. Soleimanpour S. et al. 2016. Headgroup engineering in mechanosensitive membrane probes. Chem. Commun. (Camb). 52, 14450-14453.
