Monitoring Sol-to-Gel Transitions via Fluorescence Lifetime Determination Using Viscosity Sensitive Fluorescent Probes

Hungerford, Graham; Allison, Archie; McLoskey, David; Kuimova, Marina K.; Yahioglu, Gökhan; Suhling, Klaus

doi:10.1021/jp902727y

Cited by 71 publications

(61 citation statements)

References 48 publications

(104 reference statements)

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“…The need to detect microscopic viscosity is widely recognised in biochemical and biological research, 1 and new applications are emerging in material and atmospheric sciences. [2][3][4] Among several methods that allow the detection of viscosity on a microscopic scale, such as fluorescence correlation spectroscopy (FCS), 5,6 fluorescence recovery after photobleaching (FRAP), 7,8 single particle tracking (SPT), 9,10 steady state and time-resolved fluorescence anisotropy, [11][12][13] detection fluorescence of molecular rotors has secured its place as a versatile technique that allows non-destructive imaging of viscosity in heterogeneous samples and on-line detection of changing viscosity. These two advantages are not accessible simultaneously with any other existing technique.…”

Section: Introductionmentioning

confidence: 99%

Dual mode quantitative imaging of microscopic viscosity using a conjugated porphyrin dimer

Vyšniauskas

Baláž

Anderson

et al. 2015

Phys. Chem. Chem. Phys.

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Microviscosity is of paramount importance in materials and bio-sciences. Fluorescence imaging using molecular rotors has emerged as a versatile tool to measure microviscosity, either using a fluorescence lifetime or a ratiometric signal of the rotor; however, only a limited number of blue -to-green-emitting fluorophores with both the lifetime and the ratiometric signal sensitivity to viscosity have been reported to date. Here we report a deep red emitting dual viscosity sensor, which allows both the ratiometric and the lifetime imaging of viscosity. We study viscosity in a range of lipid-based systems and conclude that in complex dynamic systems dual detection is preferable in order to independently verify the results of the measurements as well as perform rapid detection of changing viscosity .

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Section: Introductionmentioning

confidence: 99%

Dual mode quantitative imaging of microscopic viscosity using a conjugated porphyrin dimer

Vyšniauskas

Baláž

Anderson

et al. 2015

Phys. Chem. Chem. Phys.

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“…[36] Styryl dyes, and in particular 4-[4-(dimethylamino)styryl]pyridine (DMASP) derivatives are also molecular rotors [37] and have also been used to probe microviscosity in cells based on ratiometric fluorescence measurements [38,39] and fluorescence lifetime measurements of sol to gel transitions. [40,41] The fluorescence lifetimes of malononitriles in non-viscous media are short and can be difficult to measure, restricting their use somewhat. More recently there has been interest in a class of molecular rotors based on the boron dipyrromethene (BODIPY) fluorophore.…”

Section: Introductionmentioning

confidence: 99%

“…More recently there has been interest in a class of molecular rotors based on the boron dipyrromethene (BODIPY) fluorophore. [40,[42][43][44][45][46][47][48] Rotation of a phenyl group attached to the meso-position of the BODIPY core unit leads to variation in the non-radiative decay constant. The orientationdependent potential energy surfaces can be significantly different for malononitriles and specific BODIPY-based molecular rotors.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence Anisotropy of Molecular Rotors

et al. 2011

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We present polarization-resolved fluorescence measurements of fluorescent molecular rotors 9-(2-carboxy-2-cyanovinyl)julolidine (CCVJ), 9-(2,2-dicyanovinyl)julolidine (DCVJ), and a meso-substituted boron dipyrromethene (BODIPY-C(12)). The photophysical properties of these molecules are highly dependent on the viscosity of the surrounding solvent. The relationship between their quantum yields and the viscosity of the surrounding medium is given by an equation first described and presented by Förster and Hoffmann and can be used to determine the microviscosity of the environment around a fluorophore. Herein we evaluate the applicability of molecular rotors as probes of apparent viscosity on a microscopic scale based on their viscosity dependent fluorescence depolarization. We develop a theoretical framework, combining the Förster-Hoffmann equation with the Perrin equation and compare the dynamic ranges and usable working regimes for these dyes in terms of utilising fluorescence anisotropy as a measure of viscosity. We present polarization-resolved fluorescence spectra and steady-state fluorescence anisotropy imaging data for measurements of intracellular viscosity. We find that the dynamic range for fluorescence anisotropy for CCVJ and DCVJ is significantly lower than that of BODIPY-C(12) in the viscosity range 0.6<η<600 cP. Moreover, using steady-state anisotropy measurements to probe microviscosity in the low (<3 cP) viscosity regime, the molecular rotors can offer a better dynamic range in anisotropy compared with a rigid dye as a probe of microviscosity, and a higher total working dynamic range in terms of viscosity.

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“…Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment. [6][7][8] A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the Förster Hoffman equation. 9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.…”

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Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Suhling

Levitt

Chung

et al. 2012

JoVE

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Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues. Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm. 6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects.In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors. Video LinkThe video component of this article can be found at https://www.jove.com/video/2925/ ProtocolThe protocols for FLIM sample preparation do not differ from those for confocal or wide-field intensity-based fluorescence microscopy. The data acquisition is followed by the main task of data analysis, i.e. extracting the fluorescence lifetimes from the raw data. Once these have been obtained, data interpretation helps to verify or falsify hypotheses. Staining cells with molecular rotors1. Prepare a stock solution (10 ml) by dissolving approximately 1 mg/ml of the dye in an appropriate solvent (e.g. methanol for BODIPY-C 12 ) 6,7 using an accurate balance and a pipette. 2. The cells (a model cancer cell line, HeLa in our case) to be stained are grown on in a multiwell plate with a coverslide underside for microscopy, in an incubator at 37 °C with a 5 % CO 2 atmosphere until ~ 80% confluent. 3. Add 10 -20 μl of the stock solution to the living cells growing in a multi-well plate (SmartSlide 50 micro-incubation system, Wafergen) in 4 ml of Opti-MEM medium (GIBCO) per well for a 6-well plate. This yields a micro-molar dye concentration in the well. 4. Return the multi-well plate to an incubator at 37 °C with a 5 % CO 2 atmosphere for 10-45 mins for staining. 5. Remove the multiwell plate from the incubator and wash the cells 3-4 times with 4 ml optically clear cell culture medium (e.g. Opti-MEM) to remove excess dye.

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Monitoring Sol-to-Gel Transitions via Fluorescence Lifetime Determination Using Viscosity Sensitive Fluorescent Probes

Cited by 71 publications

References 48 publications

Dual mode quantitative imaging of microscopic viscosity using a conjugated porphyrin dimer

Dual mode quantitative imaging of microscopic viscosity using a conjugated porphyrin dimer

Fluorescence Anisotropy of Molecular Rotors

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

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