title: Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells.
abstract: 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.
authors.affiliation: Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, NC 27695, USA. newbhanu@gmail.com.
authors.forename: Bhanu
authors.initials: B
authors.lastname: Neupane
authors.affiliation: Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA. tjin2@ncsu.edu.
authors.forename: Tao
authors.initials: T
authors.lastname: Jin
authors.affiliation: Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, NC 27695, USA. lfmellor@ncsu.edu.
authors.forename: Liliana F
authors.initials: LF
authors.lastname: Mellor
authors.affiliation: Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, NC 27695, USA. egloboa@ncsu.edu.
authors.forename: Elizabeth G
authors.initials: EG
authors.lastname: Loboa
authors.affiliation: Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, NC 27695, USA. fsligler@ncsu.edu.
authors.forename: Frances S
authors.initials: FS
authors.lastname: Ligler
authors.affiliation: Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA. gwang10@ncsu.edu.
authors.forename: Gufeng
authors.initials: G
authors.lastname: Wang
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival; D019902:Chondrocytes / Q000166:cytology; D005453:Fluorescence; D021621:Imaging, Three-Dimensional / Q000295:instrumentation*; D008853:Microscopy / Q000295:instrumentation*; D018613:Microscopy, Confocal; D016716:PC12 Cells; D051381:Rats; D016707:Tissue Fixation
keywords: PC-12 cells; actin cytoskeleton; rat chondrosarcoma cells; stimulated emission depletion (STED) microscopy; super resolution optical microscopy