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.
Highlights
In the past few years, with the help of fluorescence microscopy and various biotechnologies, visualization of sub-cellular structures and dynamic processes in fixed and live cells has become a routine procedure, with an associated accumulation of knowledge in biological structures and functions of biomolecules
Our results showed that the continuous wave (CW)-stimulated emission depletion (STED) microscope has improved spatial resolution compared to confocal microscopy and can be used to monitor changes in actin structures over time
Using the full power of a 1-W fiber laser at 592 nm as the depletion source, we were able to achieve a full-width at half maximum (FWHM) spatial resolution of 70 nm routinely for our home-built system
Summary
In the past few years, with the help of fluorescence microscopy and various biotechnologies, visualization of sub-cellular structures and dynamic processes in fixed and live cells has become a routine procedure, with an associated accumulation of knowledge in biological structures and functions of biomolecules. Actin is a globular multi-functional protein that plays important roles in many cellular processes, including maintaining cell shapes [23,24], establishing cell junctions [25], cell movements [26], muscle contraction [27], cell division and cytokinesis [28], cell signaling [29], intracellular vesicle [30] and organelle movements [31], etc Many of these processes involve highly dynamic (milliseconds to seconds time scale) formation and reorganization of fine structures of actin filaments and relevant cell organs [32]. We investigate the feasibility of using our CW-STED microscope to study actin structures and dynamics in fixed and live cells by comparing confocal and STED fluorescence microscopy. It may disclose new information for systems with a proper characteristic length scale
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