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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: However, it is often necessary that researchers insert such materials into cells for various dynamical and/or drug delivery studies.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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Images of HEK and rat cortical neurons before (bright field, then fluorescence) and after (fluorescence, then bright field) optoporation and injection with rhodamine phalloidin.(a–d) HEK, 126 nM RP concentration. (e–h) HEK, 168 nM RP concentration. (i–l, m–p) Rat cortical neuron, 120 nM RP concentration.
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f2: Images of HEK and rat cortical neurons before (bright field, then fluorescence) and after (fluorescence, then bright field) optoporation and injection with rhodamine phalloidin.(a–d) HEK, 126 nM RP concentration. (e–h) HEK, 168 nM RP concentration. (i–l, m–p) Rat cortical neuron, 120 nM RP concentration.

Mentions: All experiments and optimization of dye and laser parameters were carried out on the same experimental setup (Fig. 1 (a)). In order to optoporate efficiently, the focused laser beam should be symmetric and share a focal plane with the sample's surface. This can be verified by beam symmetry as well as circular rings around the spot which is shown in Fig. 1 (b–c). Optimization of the laser parameters was undertaken by varying the laser power and exposure time (macro-pulse duration, 20–50 ms), while keeping the wavelength constant (800 nm). At higher average laser powers (at or above 130 mW) and macro-pulse durations (above 30 ms), HEK cells were observed to die (quick rise in RP-fluorescence and significant change in morphology) as seen in Suppl. Fig. 1. It is known that the Phalloidin family of dyes are toxic to living cells, and can cause or contribute to cell death22 at high concentrations. Therefore, imaging is typically performed on fixed cells to visualize the cytoskeletal filamentous actin network. Here, we have determined a window of optimized RP dye concentration by monitoring the cell viability (by morphology and calcein exclusion assay) and RP-fluorescence following optoporation. In Suppl. Fig. 2, we show Calcein-AM images of the cells following optoporation. Optical parameters were kept constant while the RP concentration was varied from 40 to 200 nM. At lower concentrations (42 nM), we did not observe any change in fluorescence signal. At higher RP concentration (168 nM), optoporated cells showed a sharp increase in fluorescence immediately following optoporation which was not attributed to RP dye binding, along with notable changes in morphology (Fig. 2 (e–h)), indicating cell death. Since the cell viability was retained following optoporation and injection of 126 nM concentration of RP, live cell imaging of the cytoskeletal actin network could be performed (Fig. 1 (d)).


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

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

Images of HEK and rat cortical neurons before (bright field, then fluorescence) and after (fluorescence, then bright field) optoporation and injection with rhodamine phalloidin.(a–d) HEK, 126 nM RP concentration. (e–h) HEK, 168 nM RP concentration. (i–l, m–p) Rat cortical neuron, 120 nM RP concentration.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Images of HEK and rat cortical neurons before (bright field, then fluorescence) and after (fluorescence, then bright field) optoporation and injection with rhodamine phalloidin.(a–d) HEK, 126 nM RP concentration. (e–h) HEK, 168 nM RP concentration. (i–l, m–p) Rat cortical neuron, 120 nM RP concentration.
Mentions: All experiments and optimization of dye and laser parameters were carried out on the same experimental setup (Fig. 1 (a)). In order to optoporate efficiently, the focused laser beam should be symmetric and share a focal plane with the sample's surface. This can be verified by beam symmetry as well as circular rings around the spot which is shown in Fig. 1 (b–c). Optimization of the laser parameters was undertaken by varying the laser power and exposure time (macro-pulse duration, 20–50 ms), while keeping the wavelength constant (800 nm). At higher average laser powers (at or above 130 mW) and macro-pulse durations (above 30 ms), HEK cells were observed to die (quick rise in RP-fluorescence and significant change in morphology) as seen in Suppl. Fig. 1. It is known that the Phalloidin family of dyes are toxic to living cells, and can cause or contribute to cell death22 at high concentrations. Therefore, imaging is typically performed on fixed cells to visualize the cytoskeletal filamentous actin network. Here, we have determined a window of optimized RP dye concentration by monitoring the cell viability (by morphology and calcein exclusion assay) and RP-fluorescence following optoporation. In Suppl. Fig. 2, we show Calcein-AM images of the cells following optoporation. Optical parameters were kept constant while the RP concentration was varied from 40 to 200 nM. At lower concentrations (42 nM), we did not observe any change in fluorescence signal. At higher RP concentration (168 nM), optoporated cells showed a sharp increase in fluorescence immediately following optoporation which was not attributed to RP dye binding, along with notable changes in morphology (Fig. 2 (e–h)), indicating cell death. Since the cell viability was retained following optoporation and injection of 126 nM concentration of RP, live cell imaging of the cytoskeletal actin network could be performed (Fig. 1 (d)).

Bottom Line: However, it is often necessary that researchers insert such materials into cells for various dynamical and/or drug delivery studies.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