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AFM Microscopes in BICEN facility in Physics (UoS)
Atomic Force Microscopy
Atomic Force Microscopy
Our group uses Atomic Force Microscopy (AFM) on a regular basis. We use state-of-the-art machines such as the Fast-Scan from Bruker. The main advantage of this type of microscopy over others is the capability of achieving 1 nm resolution on living cells in their native environment without damaging them or the need of labelling the sample with dyes. We apply AFM to various samples, primarily focusing on the bacterial cell envelope. The type of samples we image are both purified cell wall or live bacteria actively dividing. We can capture dynamics of biological processes at 1 nm resolution!
The bacteria we have previously work on are the following: Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, Pseudomonas aeruginosa and the current focus of the group is Streptococcus pneumoniae. Some of the samples we have imaged are shown below.
Examples of unpublished AFM images
Purified cell wall of S. aureus
B. subtilis live cell before treatment with 3% SDS
Boiled Streptococcus pneumoniae cells
Video-AFM of a B. subtilis cell actively dividing in media
Visualising individual cell wall fibres on B. subtilis live cell
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Correlative approaches (STORMforce)
Correlative approaches (STORMforce)
Our group aims to fully optimise correlative approaches such as AFM + STORM (STORMforce) to obtain more biological relevant information from our samples. For example, we are focused on understanding the peptidoglycan growing process of Streptococcus pneumoniae. The newly inserted peptidoglycan is only placed at the newly formed septa. STORM gives us 20 nm resolution in a cell where peptidoglycan has been labelled for 5 min, we can see where the new material has been inserted versus the old material. Therefore, by combining this temporal framework from the STORM with the AFM on the same cell, we can obtain a direct correlation between structure and age of the cell wall.
STORM Microscope in Wolfson Ligth Microscopy Facility in Biosciences (UoS)
Examples of STORMforce and STORM data
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Open Source Bio-Image Analysis Tools (Coding experience isn't required to join us)
Bio-Image Analysis Tools
Bio-Image Analysis Tools
Previously, microscopy images used to be just qualitative information about a sample, where the researchers could finally visualise those microscopic systems they were studying. However, much like other types of scientific images (e.g. in the Astronomy field), microscopic images of biological samples are a type of data. Therefore, we need Quantitative analysis tools that are unbiased, automatic and reproducible to obtain robust conclusions from our samples. Developing Bio-Image Analysis tools is a key step in our research pipeline because it allow us to extract valuable information from our precious microscopy images.
Examples of unpublished Bio-Image Analysis Tools
S. aureus peptidoglycan fibre map presented in 3D and rotating
Agar plate with bacterial colonies detected by ilastik (machine learning segmentation)
Pores in 3D of the cell wall of S. aureus and coluor coded by depth using visualisation software Avizo. Work from here.
CURRENT RESEARCH PROJECT
Current funding from Wellcome - Early Career Award (5 years)
Title: Revealing cell wall homeostasis mechanisms by interrogating the architecture of Streptococcus pneumoniae peptidoglycan with unprecedented resolution using AFM and STORMforce
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