Abstract
Superresolution fluorescence microscopy possesses an important role for the study of processes in biological cells with subdiffraction resolution. Recently, superresolution methods employing the emission properties of fluorophores have rapidly evolved due to their technical simplicity and direct applicability to existing microscopes. However, the application of these methods has been limited to samples labeled with fluorophores that can exhibit intrinsic emission properties at a restricted timescale, especially stochastic blinking. Here, we present a superresolution method that can be performed using general fluorophores, regardless of this intrinsic property. Utilizing speckle patterns illumination, temporal emission fluctuation of fluorophores is induced and controlled, from which a superresolution image can be obtained exploiting its statistical property. Using this method, we demonstrate, theoretically and experimentally, the capability to produce subdiffraction resolution images. A spatial resolution of 500 nm, 300 nm and 140 nm with 0.4, 0.5 and 1.4 NA objective lenses respectively was achieved in various samples with an enhancement factor of 1.6 compared to conventional fluorescence microscopy.
Highlights
Analysis which is a form of statistical analysis related to correlation
Since superresolution optical fluctuation imaging (SOFI) requires two important properties known as blinking and correlation, to obtain superresolution images, SOFI has been only applicable for samples labeled with quantum dots and organic and protein fluorophores[18] that have intrinsic blinking characteristic within a timescale limited by the imaging system
To overcome the limitations of SOFI completely, it is important that the optical fluctuation caused by the intrinsic blinking of fluorophores in SOFI is replaced by controllable fluctuation that is directly and induced by an external method, such as random patterns illumination, and that is unaffected by the type of fluorophore
Summary
Simulated images of a virtual sample containing two fluorophores with interdistance a was reconstructed via an analytic model and experimental simulation, under the assumption that the numerical aperture (NA) of an objective lens for illumination is 0.5, and that the wavelength of light is 532 nm. Since D(τ) can be neglected in the calculation when it is rescaled according to optical magnification, and the step size of the motorized stage is small enough, we can get a simple equation, such as in equation (3) (details of D(τ) in Supplementary Information) Based on this equation, the S-SOFI image of an analytic model is calculated (detailed description in Supplementary Information). These results mean that analytic model is in good agreement with the real measurement of S-SOFI. The second order S-SOFI (2nd S-SOFI) showed 1.3× enhancement (Fig. 2d) and the second order S-SOFI with FRW (2nd S-SOFI with FRW) showed 1.6× enhancement (Fig. 2e)
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