Abstract
We show that far-field fluorescence nanoscopy by stimulated emission depletion (STED) can be realized with compact off-the-shelf laser diodes, such as those used in laser pointers and DVDs. A spatial resolution of 40-50 nm is attained by pulsing a 660 nm DVD-diode. The efficacy of these low-cost STED microscopes in biological imaging is demonstrated by differentiating between clusters of the synaptic protein bassoon and transport vesicles in hippocampal neurons, based on the feature diameter. Our results facilitate the implementation of this all-molecular-transition based superresolution method in many applications ranging from nanoscale fluorescence imaging to nanoscale fluorescence sensing.
Highlights
Due to its sensitivity and ability to image the interior of cells, far-field fluorescence microscopy has become one of the most widely applied methods in modern life sciences
We show that far-field fluorescence nanoscopy by stimulated emission depletion (STED) can be realized with compact off-the-shelf laser diodes, such as those used in laser pointers and DVDs
STED microscopy has become a powerful tool for subdiffraction fluorescence imaging [4,5,6,7] that has been increasingly applied in the biological as well as in the material sciences [8,9,10,11,12]
Summary
Due to its sensitivity and ability to image the interior of cells, far-field fluorescence microscopy has become one of the most widely applied methods in modern life sciences. STED microscopy overcomes the diffraction barrier by using stimulated emission to transiently turn off the capability of fluorophores to emit spontaneously This transient fluorescence silencing is utilized to make features, that are closer than the diffraction barrier, fluoresce sequentially in time. Because stimulated emission has to act on the relatively short-lived fluorescent state (τ 1-5 ns), for efficient fluorescence silencing, the rate for stimulated emission has to be higher than the relatively fast spontaneous decay rate 1/τ This results in values of I s of the order of a few MW/cm making a strong laser source essential for high resolution STED imaging. The ideal STED light source features an excellent beam quality paired with high intensity, preferably operated in a pulsed mode with a repetition rate of several megahertz (for fast recording times) and pulse durations of 0.3-1 ns (for efficient fluorophore silencing). While for a long time it seemed that this demand can be met only with mode-locked Ti:sapphire or mode-locked gas laser based systems, here we show that competitive STED performances can be achieved using compact, off-the-shelf laser diode modules, by laser pointers and lasers used in DVDs
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