Unmet Need
Differentiating small features, such a membrane fibrils in a cell, or small entities, such as viruses, is made possible by high resolution fluorescence imaging. Stimulated Emission Depletion (STED) Nanoscopy has proven to produce images that can overcome the diffraction limit and provide nanometer resolution of fluorescently labeled samples. The difficulty with this technology is the introduction of noise, and a low signal to background ratio (SBR). New microscopy techniques have made it possible to get closer to seeing biology at the molecular level in real time, but reducing background and increasing signal while keeping high resolution are still challenges in super-resolution microscopy.
Technology Overview
The new technology developed at Johns Hopkins University is an additional hardware control of the incoming light to use polarized switching in combination with a standard STED laser microscope to remove background noise. Current techniques to remove background noise only partially suppress background noise, are not efficient at removing noise due to periphery fluorescent signal, or don’t work well in both high power STED or low power STED. Compared to conventional methods, this new technique has a 20-fold increase in SBR for high power and 10-fold increase in SBR for low power STED. It is a simple adaption of the standard STED system, and is not difficult or costly to incorporate.
Calculated signal to background ratio (SBR) data showed a 10-fold increase in SBR in comparison to standard STED imaging of the fluorescent nanobeads. Simulated SBR of the other background reducing techniques showed that the polarization switching STED, psSTED, has a 20-fold increase in SBR using high power STED and a 10-fold increase using low power STED. In the imaging of biological samples, psSTED outperformed standard STED in the contrast between the fluorescent samples and the background, had an increased SBR, and had a significantly reduced spreading of the fluorescent signal.
Stage of Development
The prototype has been built and used for 2D and 3D imaging of fluorescent nanobeads and biological samples.
Publication
J. C. Lee, Y. Ma, K. Y. Han and T. Ha, “Accurate background subtraction in STED nanoscopy with polarization switching”, ACS Photonics 6, 1789-1797 (2019).
Ma Y, Ha T. Fight against background noise in stimulated emission depletion nanoscopy. Phys Biol. 2019 Jul 5;16(5):051002. doi: 10.1088/1478-3975/ab255c. PMID: 31141791.
