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Open AccessArticle

Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells

1
Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, NC 27695, USA
2
Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA
*
Author to whom correspondence should be addressed.
Current Affiliation: Kathmandu Institute of Applied Sciences, Kathmandu Metropolitan City-15, Banasthali, Kathmandu, Nepal
Academic Editor: W. Rudolf Seitz
Sensors 2015, 15(9), 24178-24190; https://doi.org/10.3390/s150924178
Received: 29 May 2015 / Revised: 12 September 2015 / Accepted: 16 September 2015 / Published: 18 September 2015
Stimulated emission depletion (STED) microscopy provides a new opportunity to study fine sub-cellular structures and highly dynamic cellular processes, which are challenging to observe using conventional optical microscopy. Using actin as an example, we explored the feasibility of using a continuous wave (CW)-STED microscope to study the fine structure and dynamics in fixed and live cells. Actin plays an important role in cellular processes, whose functioning involves dynamic formation and reorganization of fine structures of actin filaments. Frequently used confocal fluorescence and STED microscopy dyes were employed to image fixed PC-12 cells (dyed with phalloidin- fluorescein isothiocyante) and live rat chondrosarcoma cells (RCS) transfected with actin-green fluorescent protein (GFP). Compared to conventional confocal fluorescence microscopy, CW-STED microscopy shows improved spatial resolution in both fixed and live cells. We were able to monitor cell morphology changes continuously; however, the number of repetitive analyses were limited primarily by the dyes used in these experiments and could be improved with the use of dyes less susceptible to photobleaching. In conclusion, CW-STED may disclose new information for biological systems with a proper characteristic length scale. The challenges of using CW-STED microscopy to study cell structures are discussed. View Full-Text
Keywords: super resolution optical microscopy; stimulated emission depletion (STED) microscopy; actin cytoskeleton; PC-12 cells; rat chondrosarcoma cells super resolution optical microscopy; stimulated emission depletion (STED) microscopy; actin cytoskeleton; PC-12 cells; rat chondrosarcoma cells
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Neupane, B.; Jin, T.; Mellor, L.F.; Loboa, E.G.; Ligler, F.S.; Wang, G. Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells. Sensors 2015, 15, 24178-24190.

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