A membrane-activatable near-infrared fluorescent probe with ultra-photostability for mitochondrial membrane potentials
TL;DR: A photostable near-infrared (NIR) fluorescent dye for monitoring MMP, named NIMAP, is non-fluorescent in aqueous solution and can be activated by cell membranes, providing high fluorescence contrast and low background fluorescence.
Abstract: Mitochondrial membrane potential (MMP) is a frequently used indicator for mitochondrial function Herein, we report a photostable near-infrared (NIR) fluorescent dye for monitoring MMP This new probe, named NIMAP, is non-fluorescent in aqueous solution and can be activated by cell membranes, providing high fluorescence contrast and low background fluorescence NIMAP has been validated for monitoring MMP in living mammalian cells and in mice Due to the large fluorescence response, low fluorescence background, high photostability, and excellent tissue penetration resulting from red-shifted excitation and emission in the "optical window" above 600 nm, broad applications of this new probe are expected
Summary (2 min read)
- Mitochondrial membrane potential (MMP) is required for cellular respiration and ATP synthesis.
- Measuring absolute MMP in living cells is technically challenging.
- 11 †Electronic supplementary information (ESI) available: Procedures for chemical synthesis, spectroscopic characterization, experimental optimization and comparison, and time-lapse movies.
- NIMAP is highly attractive, because not only does it command the above-mentioned advantages of a NIR probe, but it also shows a large fluorescence response to MMP changes, membrane-activated fluorescence and minimal background signals, ultra-photostability, and excellent tissue penetration in live animals.
Materials and general methods
- All chemicals and reagents were purchased from Fisher Scientific (Pittsburgh, PA) or Sigma-Aldrich (St. Louis, Mo) unless specified elsewhere.
- Plasmid DNA was purified using Syd Laboratories Miniprep columns (Malden, MA).
- The structure and purity of NIMAP were analyzed with ESI-MS, 1H NMR, and 13C NMR.
- ESI-MS was run on an Agilent LC-TOF system by direct fusion.
- NMR spectra were recorded on a Varian Inova 400 instrument with chemical shifts relative to tetramethylsilane.
- A monochromator-based Synergy Mx Microplate Reader (BioTek, Winooski, VT) was used to record all spectra.
- To measure the fluorescence excitation and emission spectra of NIMAP, the authors prepared NIMAP in liposomes as described elswhere.21 L-α-Lecithin egg yolk and cholesterol were purchased from EMD Millipore (Billerica, MA) and Alfa Aesar (Ward Hill, MA), respectively.
- The resulting mixture was then sonicated 5 min on a probe sonicator to yield a slightly hazy and transparent solution.
- To record the excitation spectrum, the emission wavelength was set at 730 nm, and the excitation scanned from 500 nm to 700 nm.
- Mammalian cell culture and dye loading Human embryonic kidney (HEK) 293T cells or cervical cancer HeLa cells were maintained in T25 flasks containing 5 mL Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C with 5% CO2 in humidified air.
Mammalian cell transfection
- The prepared transfection mixture was then added to cells and incubated for 2 h at 37 °C.
- Next, pre-warmed, fresh DMEM containing 10% FBS was used to replace the transfection medium.
- On the following day, cells were stained with NIMAP before imaging.
- Fluorescence microscopy was performed with a Leica SP5 inverted confocal fluorescence microscope with the spectral imaging capability (Leica, Boston, MA), unless otherwise stated.
- A 40× water lens was used for all imaging studies.
- To record the emission spectrum for NIMAP loaded into live-cell mitochondria, the emission bandwidth was set at 10 nm and images were taken from 650 nm to 790 nm.
Designing and preparation of NIMAP
- The authors development of the new NIR MMP probes originated from a Förster resonance energy transfer (FRET) experiment in their laboratory, which used a QSY-21 core structure as a dark quencher.
- The authors prepared a QSY-21 labeled biotin derivative (2), and observed mitochondria-like organellar fluorescence in cultured mammalian cells stained with 100 μM of the dye (ESI Fig. S1A†).
- Optimization of NIMAP loading into mammalian cells.
- By comparison, the background fluorescence of HEK 293T cells stained with a conventional MMP dye, Rhodamine 123, was strong before washing off excess dye molecules (Fig. S3†).
Mitochondrial localization of NIMAP
- To further confirm the mitochondrial localization of NIMAP, the authors loaded the molecule into HEK 293T cells simultaneously expressing a green fluorescent mWasabi protein fused to a mitochondrial localization tag (Mito-mWasabi).
- A previous molecular dynamics (MD) study suggests that QSY-21 fluorescence is quenched by ring rotations and electron transfer in the excited state.
- The authors also measured the absorbance of NIMAP in an aqueous solution, which was similar to the profile of NIMAP fluorescence excitation in liposomes (Fig. 4).
- The authors observed much weaker signals for Rhodamine 123-stained cells, compared to NIMAP-stained cells; and almost no signal for the other two groups.
- In summary, starting from a nonfluorescent dark quencher QSY-21, the authors have developed a new NIR probe, NIMAP, for monitoring MMP in living mammalian cells and in vivo.
- Lipid membranes can activate the fluorescence of NIMAP, and therefore the loading procedures can be simplified to gain low fluorescence background and high fluorescence contrast.
- When NIMAP was loaded into the mitochondria of living mammalian cells, it responded to MMP changes induced by chemicals with high sensitivity.
- Because of its excitation and emission at wavelengths above 600 nm, excellent penetration of mouse skin tissue was also observed.
- In addition, the mitochondrial localization capability of the QSY-21 scaffold may further be exploited to construct other mitochondriatargeting fluorescent probes or selectively deliver inhibitors of mitochondrial enzymes.
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