Abbelight
Caterina Severi
Workshop Room 7
Biological imaging has successfully pierced the nanoscale in the recent years. Resolving the intricate interaction of multiple targets in a system, naturally represents the next step in the evolution of fluorescence nanoscopy. Multicolor imaging is a powerful approach which allows to elucidate the interaction between different components in a biological environment. However, multicolor imaging comes with a host of technical challenges, mainly chromatic aberration, crosstalk and longer imaging times. Hurdles that are amplified when performing nanoscopy.
For single molecule localization microscopy (SMLM), one of the most promising innovations to overcome the aforementioned obstacles is spectral demixing, which employs dyes that have very similar emission properties. In this workshop spectral demixing will be showcased in a stochastic optical reconstruction microscopy (STORM) experiment
Meanwhile, DNA-PAINT (DNA-Point accumulation for imaging in nanoscale topography) inherently holds great potential for multicolor imaging due to its different approach to attaining the “blinking” required for SMLM. DNA-PAINT relies on the rapid hybridization and dehybridization of short complementary DNA strands, retaining a dye long enough close to the target to collect a few thousand photons and ‘localize’ it before the dye diffuses off again.
With this workshop, we would like to demonstrate the versatility of our SAFe imaging platform in acquiring simultaneous multi-color SMLM images using STORM and DNA-PAINT methods. We will image several types of sample: COS7 cells, hippocampal rat neurons and others.
Schedule
10 min Introduction: SMLM (STORM and DNA-PAINT) and Abbelight setup (Presentation)
5 min Acquisition on a one-color image, explanation of the imaging parameters
15 min Simultaneous two color DNA-PAINT imaging using Cy3b/Atto655 dye combination
15 min Simultaneous two color STORM imaging using AF647/CF680 dye combination
15 min Summary of the workshop & Questions
