As part of the Imaging ONEWORLD 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, core facility staff from the University of Cambridge, Gurdon Institute, MRC-LMB and the ICR/Royal Marsden Trust are also able to continue the discussion and provide advice on your imaging projects.
Scientific Organisers
Stefanie Reichelt
Stefanie Reichelt, PhD has been head of the light microscopy facility at the CRUK Cambridge Institute. The core provides state-of-the-art imaging resources, training courses for scientists and students and develop new imaging systems as well as user-friendly analysis and acquisition tools for specific research applications. Stefanie is now Public Engagement Manager for the Biomedical Schools and teaches academically at Cambridge University, in scientific workshops and out-reach events. (http://cargocollective.com/StefanieReichelt)
- Email:
- sr411@cam.ac.uk
Alessandro Esposito
Dr Alessandro Esposito obtained a PhD in Biophysics in 2006 working at the University of Utrecht and the European Neuroscience Institute in Goettingen for which he was awarded the ‘Sergio Ciani’ award by the Italian Society of Pure and Applied Biophysics. At the University of Cambridge, he then developed novel analytical tools contributing to redefining models of red blood cells homeostasis infected by P. falciparum (malaria). In recognition of his early work, in 2009 Alessandro was awarded a Life Science Interface fellowship by the EPSRC to establish foster the development of heavily multiplexed biochemical imaging. Soon after he moved to the MRC Cancer Unit where he lead the ‘Systems Microscopy initiative’ and retrained in cancer biology. During these years, Alessandro’s work developed into two research streams: i) the study of cellular responses to DNA damage and mutations in signalling pathways and ii) the innovation of biochemical imaging technologies. His team contributed to revealing the vast cell-to-cell variability in stress responses of genetically identical cells, a feature of biological systems that hinder the efficacy of disease management and therapeutic efficacy. Since 2019, Alessandro leads a transdisciplinary research programme at the MRC Cancer Unit in Cambridge devoted to understanding how DNA damage and mutations in KRAS derange homeostatic programmes leading to cancer. His group combines multi-omics data with single-cell biochemical imaging techniques aiming to achieve a deeper understanding of cancer phenotypes during the earliest stages of carcinogenesis, with particular attention to cell-to-cell variability of non-genetic origin and cell-to-cell communication.
Nick Barry
An optical physicist and specialist in light microscopy and head of the Light Microscopy facility at the MRC Laboratory of Molecular Biology, University of Cambridge.
Kirti Prakash
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. Formerly at the National Physical Laboratory (NPL), and currently working at the Institute for Cancer Research (ICR) and Royal Marsden Trust, he is working on microscope development and image analysis.
Data fusion in localization based super-resolution microscopy
Single molecule localization microscopy offers in principle resolution down to the molecular level. Ever bright fluorophores, but also the advent of new techniques as MINflux, SIMflux or MNSTED are enabling better localization precision with the same amounts of photons. Complementary to cryo-Electron Microscopy imaging fluorescence at cryogenic temperature also vastly improved precisions. The reported precisions reach the low nano-meter range, even the sub-nanometer range, however, the useful information to the researcher from these recordings is often much poorer than suggested by the reported precision.
The root cause of this is, that in fluorescence microscopy, you image the fluorophores and not the bio-molecule of interest directly. The process of labelling the relevant epitopes with the fluorescent emitters is always incomplete in practise. This deficiency termed, underlabelling, hinders to obtain a complete picture. This missing information can be completed by merging information from many structurally identical particles if these are available.
I will discuss the basis ideas of data fusion or particle averaging in fluorescence microscopy, explain similarities and difference of conceptual and practical nature with cryo-EM particle averaging. I will go into detail of different technicalities and potential issues that could arise when processing data, e.g. data inhomogenitiy, how can we work with the fact that the underlying biological macromolecules are not structural exactly the same; issues from 2D or 3D data and projection; computational aspects; how can we incorporate potential symmetry of the structure; how can we deal with strong underlabelling; can we apply the long existing cryo-EM software or partially incorporate it; what are limiting factors in averaging approaches; can we reach ever higher resolution by imaging 10^5 particles as in EM?
At the end I will show some recent results of our efforts in 3D alignment of particles of the Nuclear Pore Complex.
