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Introduction of impermeable actin-staining molecules to mammalian cells by optoporation.

Dhakal K, Black B, Mohanty S - Sci Rep (2014)

Bottom Line: Here, we have used a focused femtosecond laser beam to create a small transient hole in the cellular membrane (optoporation) in order to inject nanomolar concentrations of rhodamine phalloidin (an impermeable dye molecule for staining filamentous actin) into targeted living mammalian cells (both HEK and primary cortical neurons).Following optoporation, the dye bound to the intracellular actin network and rise in fluorescence intensity was observed.Theoretical dynamics of the dye's diffusion is discussed, and numerical simulations of diffusion time constants are found to match well with experimental values.

View Article: PubMed Central - PubMed

Affiliation: Biophysics and Physiology Lab, Department of Physics, University of Texas at Arlington, Texas, USA.

ABSTRACT
The selective insertion of foreign materials, such as fluorescent markers or plasmids, into living cells has been a challenging problem in cell biology due to the cell membrane's selective permeability. However, it is often necessary that researchers insert such materials into cells for various dynamical and/or drug delivery studies. This problem becomes even more challenging if the study is to be limited to specific cells within a larger population, since other transfection methods, such as viral transfection and lipofection, are not realizable with a high degree of spatial selectivity. Here, we have used a focused femtosecond laser beam to create a small transient hole in the cellular membrane (optoporation) in order to inject nanomolar concentrations of rhodamine phalloidin (an impermeable dye molecule for staining filamentous actin) into targeted living mammalian cells (both HEK and primary cortical neurons). Following optoporation, the dye bound to the intracellular actin network and rise in fluorescence intensity was observed. Theoretical dynamics of the dye's diffusion is discussed, and numerical simulations of diffusion time constants are found to match well with experimental values.

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Dynamics of dye diffusion.Increase in normalized fluorescence intensity following optoporation in the case of (a) HEK cells (84 nM) and (b) rat cortical neuron (120 nM). (c) Normalized fluorescence intensity vs RP concentration (molarity) in the case of HEK cells. Cells exhibit negative reactions at or above 168 nM concentration and are observed to die near 200 nM concentrations. (d) Theoretical (red) and experimental (blue) variation of diffusion time constant as a function of molarity.
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f5: Dynamics of dye diffusion.Increase in normalized fluorescence intensity following optoporation in the case of (a) HEK cells (84 nM) and (b) rat cortical neuron (120 nM). (c) Normalized fluorescence intensity vs RP concentration (molarity) in the case of HEK cells. Cells exhibit negative reactions at or above 168 nM concentration and are observed to die near 200 nM concentrations. (d) Theoretical (red) and experimental (blue) variation of diffusion time constant as a function of molarity.

Mentions: Figs. 5 (a) and (b) respectively show the increase in normalized fluorescence intensity following optoporation in the case of HEK cells and rat cortical neuron. The normalized fluorescence intensity as a function of RP concentration is shown in Fig. 5 (c). Fig. 5 (d) shows the comparison between these experimental values (blue line) and numerical simulations (red line) based on equation (14), where the time (t) was held fixed (40 sec). We have assumed diffusion of dye inside the cell following optoporation, so we have used the previously measured31 intracellular viscosity coefficient, with the diffusion coefficient calculated by equation (13) and was found to be D = 2.13 × 10−8 cm2/s, which matches with experimentally determined values of the diffusion coefficient of RP = 1.38 × 10−8 cm2/s.


Introduction of impermeable actin-staining molecules to mammalian cells by optoporation.

Dhakal K, Black B, Mohanty S - Sci Rep (2014)

Dynamics of dye diffusion.Increase in normalized fluorescence intensity following optoporation in the case of (a) HEK cells (84 nM) and (b) rat cortical neuron (120 nM). (c) Normalized fluorescence intensity vs RP concentration (molarity) in the case of HEK cells. Cells exhibit negative reactions at or above 168 nM concentration and are observed to die near 200 nM concentrations. (d) Theoretical (red) and experimental (blue) variation of diffusion time constant as a function of molarity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4197434&req=5

f5: Dynamics of dye diffusion.Increase in normalized fluorescence intensity following optoporation in the case of (a) HEK cells (84 nM) and (b) rat cortical neuron (120 nM). (c) Normalized fluorescence intensity vs RP concentration (molarity) in the case of HEK cells. Cells exhibit negative reactions at or above 168 nM concentration and are observed to die near 200 nM concentrations. (d) Theoretical (red) and experimental (blue) variation of diffusion time constant as a function of molarity.
Mentions: Figs. 5 (a) and (b) respectively show the increase in normalized fluorescence intensity following optoporation in the case of HEK cells and rat cortical neuron. The normalized fluorescence intensity as a function of RP concentration is shown in Fig. 5 (c). Fig. 5 (d) shows the comparison between these experimental values (blue line) and numerical simulations (red line) based on equation (14), where the time (t) was held fixed (40 sec). We have assumed diffusion of dye inside the cell following optoporation, so we have used the previously measured31 intracellular viscosity coefficient, with the diffusion coefficient calculated by equation (13) and was found to be D = 2.13 × 10−8 cm2/s, which matches with experimentally determined values of the diffusion coefficient of RP = 1.38 × 10−8 cm2/s.

Bottom Line: Here, we have used a focused femtosecond laser beam to create a small transient hole in the cellular membrane (optoporation) in order to inject nanomolar concentrations of rhodamine phalloidin (an impermeable dye molecule for staining filamentous actin) into targeted living mammalian cells (both HEK and primary cortical neurons).Following optoporation, the dye bound to the intracellular actin network and rise in fluorescence intensity was observed.Theoretical dynamics of the dye's diffusion is discussed, and numerical simulations of diffusion time constants are found to match well with experimental values.

View Article: PubMed Central - PubMed

Affiliation: Biophysics and Physiology Lab, Department of Physics, University of Texas at Arlington, Texas, USA.

ABSTRACT
The selective insertion of foreign materials, such as fluorescent markers or plasmids, into living cells has been a challenging problem in cell biology due to the cell membrane's selective permeability. However, it is often necessary that researchers insert such materials into cells for various dynamical and/or drug delivery studies. This problem becomes even more challenging if the study is to be limited to specific cells within a larger population, since other transfection methods, such as viral transfection and lipofection, are not realizable with a high degree of spatial selectivity. Here, we have used a focused femtosecond laser beam to create a small transient hole in the cellular membrane (optoporation) in order to inject nanomolar concentrations of rhodamine phalloidin (an impermeable dye molecule for staining filamentous actin) into targeted living mammalian cells (both HEK and primary cortical neurons). Following optoporation, the dye bound to the intracellular actin network and rise in fluorescence intensity was observed. Theoretical dynamics of the dye's diffusion is discussed, and numerical simulations of diffusion time constants are found to match well with experimental values.

Show MeSH
Related in: MedlinePlus