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)
Figure 1 - Diagram of FLIM set-up and time resolved color coded cell image.
Figure 1. On the left, standard FLIM microscope layout arranged in simple units that are readily available from microscope vendors. On the right, Flipper-TR® staining of cells. Left side: Image of cells stained with Flipper-TR® Greyscale represents fluorescence intensity, and color codes represent fluorescence lifetime. Images courtesy of Colom et al. 2018 (Ref. 1). Flipper-TR is a registered trademark of UNIGE, 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 Fig. 2). The emission lifetime is long (4.1-8.0 ns) when in the tense state (dithienothiophenes aligned), and shorter in the relaxed state (2.0-4.0 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 Fig. 2).
Figure 2 - Schematic diagram of the structure and mechanism of tension reporting by Flipper-TR
Legend Figure 2. Schematic diagram of the mechanism of Flipper-TR and its interaction with membranes with different tension. On the left, basic molecular structure of Flipper-TR. On the right, low tension green twisted Flipper-TR and on the right high tension planar structure.
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 (Fig. 3). The time resolved measurement method allows very low bacground, which is additive with its low fluorescence in aqueous envirnments, see Q4 below.
Figure 3 - Absortion and emission spectra of Flipper-TR
Legend Figure 3 - Flipper-TR solution in ethyl acetate was subject to absornace and emission scans and the results plotted on the same graph. Absorbance is dotted orange line, and emission is a solid orange line.
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 aqueous enviroments e.g. tissue culture media, because it is a fully twisted state and tends to form micelles and quench itself (Ref.3). After insertion into the membrane it becomes less twisted and starts to emit with high fluorescence (see Fig. 2).
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 sections, 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 ethoxyethanol.
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.