Structured illumination microscope (SIM) enables high temporal resolution wide field-of-view super-resolution imaging but typically provides only two-fold resolution improvement over the diffraction limit. We report speckle metamaterial-assisted illumination nanoscopy (Speckle-MAIN) which brings the resolution down to 40nm and beyond. A hyperbolic metamaterial structure is implemented as substrate to generate deep sub-wavelength speckle-like illumination pattern at the near field of the metamaterial. Fluorescent objects are illuminated by such high spatial-frequency near field illuminations and are reconstructed by a Blind-SIM algorithm. Speckle-MAIN provides a new route for low-cost easy-implemented super-resolution imaging with ultra-high resolution and biocompatibility.
In the past two decades, various super-resolution fluorescence microscopic techniques have achieved an axial resolution on the order of tens of nanometers and been applied for a wide range of biological studies. However, these imaging techniques still face technical challenges to reach a resolution below 10 nm. Moreover, the required complex system for these techniques limits their wide applications in practice. In this talk, we present a new cellular fluorescence imaging method with a nanometer-scale axial resolution, based on a distance-dependent photobleaching suppression of fluorophores on hyperbolic metamaterial. We will show that by applying this technology to image HeLa cell membranes tagged with fluorescent proteins, an axial resolution of ~3 nm at multiple colors can be achieved, allowing for a precise determination of the architecture of cell adhesion.
KEYWORDS: Super resolution, Microscopy, Signal to noise ratio, Luminescence, Data processing, Biomedical optics, MATLAB, Image analysis, Biological research, Image processing
We present Localizer, a freely available and open source software package that implements the computational data processing inherent to several types of superresolution fluorescence imaging, such as localization (PALM/STORM/GSDIM) and fluctuation imaging (SOFI/pcSOFI). Localizer delivers high accuracy and performance and comes with a fully featured and easy-to-use graphical user interface but is also designed to be integrated in higher-level analysis environments. Due to its modular design, Localizer can be readily extended with new algorithms as they become available, while maintaining the same interface and performance. We provide front-ends for running Localizer from Igor Pro, Matlab, or as a stand-alone program. We show that Localizer performs favorably when compared with two existing superresolution packages, and to our knowledge is the only freely available implementation of SOFI/pcSOFI microscopy. By dramatically improving the analysis performance and ensuring the easy addition of current and future enhancements, Localizer strongly improves the usability of superresolution imaging in a variety of biomedical studies.
Genetically encodable fluorescent biosensors based on fluorescence resonance energy transfer (FRET) are being
developed for analyzing spatiotemporal dynamics of various signaling events in living cells, as these events are often
dynamically regulated and spatially compartmentalized within specific signaling context. In particular, to investigate the
phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in the cellular context, we have developed a series of such
biosensors that enable dynamic visualization of several key signaling events in this pathway, namely InPAkt for lipid
second messenger dynamics, BAKR for Akt activity, and ReAktion for the action of Akt during its multi-step activation
process. Discussed here are several studies that have been carried out with these novel biosensors. First, we examined
nuclear phosphatidylinositol-3,4,5-triphosphate (PIP3) in living cells using nucleus-targeted InPAkt. Second, we
analyzed signal propagation from the plasma membrane to the nucleus by using plasma membrane-targeted InPAkt and
nucleus-targeted BKAR to simultaneously monitor PIP3 dynamics and Akt activity in the same cell. Of note, results from
these co-imaging experiments suggest that active Akt can dissociate from the plasma membrane and translocate into the
nucleus in the presence of high levels of PIP3 at the plasma membrane. This finding has led to a further study of the
action of Akt during its activation process, particularly focusing on how Akt dissociates from the membrane. In this
regard, a live-cell molecular analysis using ReAktion reveals a conformational change in Akt that is critically dependent
on the existence of a phosphorylatable T308 in the activation loop. Subsequently this has led to the discovery of new
regulatory roles of this critical phosphorylation event of Akt for ensuring its proper activation and function.
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