Abstract
Pixel reassignment image scanning microscopy (PRISM) is a useful tool to improve the resolution of confocal laser scanning microscopy (CLSM) only equipped with a detector array. However, while it can improve the lateral resolution, it has little effect on the axial resolution. Here, new microscopy has been proposed which combines three-dimension fluorescence emission difference microscopy (3D FED) with PRISM to further improve three-dimension resolution. We call this method three-dimension pixel reassignment fluorescence emission difference microscopy (3D-PRFED). Detailed theoretical analysis and simulation are presented in this paper. Additionally, the performance of lateral and axial resolution improvement of this method has been demonstrated by imaging 100 nm fluorescent beads and nuclear pore complexes samples. Experiment results show that this method in our system can improve lateral resolution by a factor of 1.85 and axial resolution by a factor of 1.48 compared with CLSM.
Highlights
Over the past several decades, confocal laser scanning microscopy (CLSM) has become a basic tool for most fluorescence microscopy applications, such as observing morphologies and dynamics in living cells, which has contributed to its ability to produce high-contrast, optical sectioned images while providing enough versatility to satisfy plentiful samples and application demands [1]
It is significantly noted that the resolution of CLSM, including lateral resolution and axial resolution, can even be improved by a factor of 2 with infinitely small pinhole compared to wide-field fluorescence microscopy, which cannot be achieved in practice for the awful signal-to-noise (SNR) and the inexistence of infinitely small pinhole [2,3]
To further dig out the potential of CLSM, for the past thirty years, lots of super-resolution microscopy methods based on CLSM have been proposed, such as stimulated emission depletion microscopy (STED) [6], fluorescence emission difference microscopy (FED) [7], and image scanning microscopy (ISM) [8]
Summary
Over the past several decades, confocal laser scanning microscopy (CLSM) has become a basic tool for most fluorescence microscopy applications, such as observing morphologies and dynamics in living cells, which has contributed to its ability to produce high-contrast, optical sectioned images while providing enough versatility to satisfy plentiful samples and application demands [1]. Compared with wide-field fluorescence microscopy, CLSM can supply little lateral resolution improvement but superior sectioned imaging with a finitesize pinhole [4]. Fluorescence emission difference microscopy (FED), breaking the resolution limit by nonlinearly decreasing the peripheral fluorescence signal of the focus through digital subtraction between positive confocal image and negative confocal image, is versatile to all kinds of fluorophores as same as CLSM [7]. Over the past several years, several three-dimension fluorescence emission difference microscopy (3D FED) methods have been proposed which utilize three-dimension negative focus modulated by spatial light modulator (SLM) to realize 3D resolution improvement [9,10]. Using SLM, a compact system can be designed to generate a three-dimension doughnut focus [10]
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