Abstract
BackgroundOptical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample.Methodology/Principal FindingsWe show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to <1%. Using astigmatism an axial resolution of ∼65 nm (std. dev.) was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule events. With the typical photon yields from Alexa 680 of ∼2000 up to 5 markers may in principle be resolved with <2% crosstalk.Conclusions/SignificanceOur approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories.
Highlights
IntroductionSingle molecule localisation microscopy (known as PALM, STORM, fPALM, etc.) allows far field optical imaging with a resolution of ,20 nm [1,2,3], or ,1/25th of the wavelength of light
Single molecule localisation microscopy allows far field optical imaging with a resolution of,20 nm [1,2,3], or,1/25th of the wavelength of light
Extending localisation microscopy to multiple labels greatly enhances the utility of this high-resolution method [9,10,11]
Summary
Single molecule localisation microscopy (known as PALM, STORM, fPALM, etc.) allows far field optical imaging with a resolution of ,20 nm [1,2,3], or ,1/25th of the wavelength of light. This remarkable increase in resolution was obtained using special switchable dyes and multiple laser excitation sources to limit the number of fluorescent molecules active at any one time to enable the position of each molecule to be determined. It is important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and can visualize multiple protein species in the same sample
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