Imaging ONE WORLD - "Decrypting MINFLUX and laser-free super-resolution microscopy"
7 December 2020
This week will feature Kirti Prakash from the National Physical Laboratory.
Scientific Organisers: Stefanie Reichelt, Alex Sossick, Nick Barry, Alessandro Esposito and Kirti Prakash
The meeting will begin at 1pm UK Time.
As part of the 'Imaging ONE WORLD' series, the focus of these lectures is on microscopy and image analysis methods and how to apply these to your research. Almost all aspects of imaging such as sample preparation, labelling strategies, experimental workflows, ‘how-to’ image and analyse, as well as facilitating collaborations and inspiring new scientific ideas will be covered. Speakers will be available for questions and answers. The organisers, CRUK CI core facility staff, Gurdon Institute, MRC-LMB, MRC Cancer Unit and NPL will be able to continue the discussion and provide advice on your imaging projects.
Dr Kirti Prakash
National Physical Laboratory
Kirti Prakash is a computer scientist by training (Bachelors and Masters degree) but a biologist at heart (PhD degree). Kirti aspires to be an inventor and develop new imaging tools for cell biology and neuroscience. Kirti did his Masters in Computer Science from Aalto University (Finland) and PhD in Biology from Heidelberg University (Germany). During his PhD, he developed a new method to image DNA which led to the first high-resolution images of the epigenetic landscape of meiotic chromosomes and mechanisms behind chromosome condensation. The doctoral research earned him several awards including Springer Best PhD Thesis Prize. After his PhD, he did a couple of postdocs at Carnegie Institution for Science (USA) and University of Cambridge (UK). The primary highlights of his research here were laser-free superresolution microscopy and development of a high-content imaging pipeline to quantify single-cell gene expression. Presently at National Physical Laboratory (NPL), he is working on microscope development and image analysis.
The talk will be divided into two sections. In the first part, I compare the performance of MINFLUX with other super-resolution methods, focusing particularly on spatial resolution claims, visualisation enhancement, labelling efficiency, linker length (localisation precision vs resolution), filtering of localizations and the possible limitations when imaging samples with dense structures. I hope the analysis and evaluation parameters presented here are not only useful for the future research directions but also for the microscope users, developers and core facility managers’ when deciding on an investment for the next “state-of-the-art” instrument.
In the second part of the talk, I show how low cost, laser-free, single-molecule super-resolution microscopy can be achieved using a Mercury arc-discharge lamp, with blue/UV emission lines to reactivate fluorophores. A lateral spatial resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). I further demonstrate that STED and SMLM can be performed on the same biological sample using a simple imaging medium. I hope that this type of accessible correlative imaging approach will provide a basis for structure validation and resolution enhancement.