Concurrent Scientific Session (Imaging): Analysis of Super-Resolution Microscopy Image Data
SIM data acquisition and processing for fast live-cell imaging
Structured illumination microscopy (SIM) allows for fast super-resolution imaging with low photo-toxicity, which makes it a popular fluorescent imaging technique, especially for live-cell imaging. SIM relies on both a microscope to provide precisely structured excitation light, and algorithms to merge its raw data frames into a super-resolved image in post-processing.
This talk will provide and overview of the current research into SIM image acquisition and reconstruction:
- The interplay of SIM instrumentation and post-processing, for both commercially available microscopes and bespoke custom solutions. For the latter, today some well documented systems exists that have been successfully implemented and replicated by various optics laboratories.
- Examples of typical SIM imaging artifacts, how they arise and how they can be avoided during acquisition or mitigated in post-processing.
- Advanced SIM processing algorithms, employing iterative deconvolution approaches. These are the subject of on-going research, and are set out to solve various problems and performance issues that arise in SIM imaging.
- The development of high speed and low-latency SIM processing, which enables real-time super-resolution feedback in live-cell and high-throughput imaging.
In summary, both a collection of processing tools currently at our disposal, and the developments to become available within the next years, will be presented.
Single molecule localization microscopy for quantitative biology
Single-molecule localization microscopy (SMLM) generates super-resolution images by serially detecting individual fluorescent molecules. The power of SMLM, however, goes beyond images: biologically relevant information can be extracted from the mathematical relationships between the positions of the fluorophores in space and time. These analytical methods allow new routes to quantify spatial patterning, clustering, and colocalization at nanometer length scales. Given their separation from typical image-based approaches, these methods come with their own caveats and controls to keep in perspective on both the analysis and experimental side. This talk will review some of these new SMLM pointillist analysis methods, including important considerations for parameterization and control analyses, and highlight where they can be applicable as a core facility offering.