Freeze, Zoom, Enhance : Increasing precision and resolution of cryoCLEM

Microscopes are a very important tool to study and visualise living systems. Two popular microscope techniques Fluorescence Microscopy (FM) and Electron Microscopy (EM) can be combined on the same sample, which provides complementary information called correlative light and electron microscopy (CLEM). This thesis describes improvements in CLEM-techniques of cryofixed samples.The discovery of the bright fluorescence of uranyl acetate is reported, after cooling with liquid nitrogen. The fluorescent signal of uranyl-acetate makes it very straightforward to find back regions in the EM. Furthermore, accurately alignment of the FM and EM images could be achieved using this phenomenon.The optical resolution of cryoFM is limited by practical restrictions. This results in a large resolution-gap between cryoFM and cryoEM, which can make it difficult to precisely interpret cryoCLEM data. In this thesis, super-resolution on cryosamples is developed. To do so three major challenges had to be overcome: increased drift, sample damage due to high intensity lasers and the unknown behaviour of fluorescent proteins under cryo-conditions. Overcoming these challenges allowed the performance of super-resolution cryoCLEM (SR-cryoCLEM), with a localisation accuracy of 30 nm and structural resolution of 50-100 nm, an increase of 4-8 times. Thermo Fisher Scientific Scientific Volume ImagingLUMC / Geneeskunde Repositoriu

Structures of the eukaryotic ribosome and its translational states in situ

Ribosomes translate genetic information into primary structure. During translation, various cofactors transiently bind to the ribosome that undergoes prominent conformational and structural changes. Different translational states of ribosomes have been well characterized in vitro. However, to which extent the known translational states are representative of the native situation inside cells has thus far only been addressed in prokaryotes. Here, we apply cryo-electron tomography to cryo-FIB milled Dictyostelium discoideum cells combined with subtomogram averaging and classification. We obtain an in situ structure that is locally resolved up to 3 Angstrom, the distribution of eukaryotic ribosome translational states, and unique arrangement of rRNA expansion segments. Our work demonstrates the use of in situ structural biology techniques for identifying distinct ribosome states within the cellular environment

Correlated Cryo Super‐Resolution Light and Cryo‐Electron Microscopy on Mammalian Cells Expressing the Fluorescent Protein rsEGFP2

Super‐resolution light microscopy (SRM) enables imaging of biomolecules within cells with nanometer precision. Cryo‐fixation by vitrification offers optimal structure preservation of biological specimens and permits sequential cryo electron microscopy (cryoEM) on the same sample, but is rarely used for SRM due to various technical challenges and the lack of fluorophores developed for vitrified conditions. Here, a protocol to perform correlated cryoSRM and cryoEM on intact mammalian cells using fluorescent proteins and commercially available equipment is described. After cell culture and sample preparation by plunge‐freezing, cryoSRM is performed using the reversibly photoswitchable fluorescent protein rsEGFP2. Next, a super‐resolved image is reconstructed to guide cryoEM imaging to the feature of interest. Finally, the cryoSRM and cryoEM images are correlated to combine information from both imaging modalities. Using this protocol, a localization precision of 30 nm for cryoSRM is routinely achieved. No impediments to successive cryoEM imaging are detected, and the protocol is compatible with a variety of cryoEM techniques. When the optical set‐up and analysis pipeline is established, the total duration of the protocol for experienced cryoEM users is 3 days, not including cell culture. Microscopic imaging and technolog

Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins

Sample fixation by vitrification is critical for the optimal structural preservation of biomolecules and subsequent high-resolution imaging by cryo-correlative light and electron microscopy (cryoCLEM). There is a large resolution gap between cryo fluorescence microscopy (cryoFLM), ~400-nm, and the sub-nanometre resolution achievable with cryo-electron microscopy (cryoEM), which hinders interpretation of cryoCLEM data. Here, we present a general approach to increase the resolution of cryoFLM using cryo-super-resolution (cryoSR) microscopy that is compatible with successive cryoEM investigation in the same region. We determined imaging parameters to avoid devitrification of the cryosamples without the necessity for cryoprotectants. Next, we examined the applicability of various fluorescent proteins (FPs) for single-molecule localisation cryoSR microscopy and found that all investigated FPs display reversible photoswitchable behaviour, and demonstrated cryoSR on lipid nanotubes labelled with rsEGFP2 and rsFastLime. Finally, we performed SR-cryoCLEM on mammalian cells expressing microtubule-associated protein-2 fused to rsEGFP2 and performed 3D cryo-electron tomography on the localised areas. The method we describe exclusively uses commercially available equipment to achieve a localisation precision of 30-nm. Furthermore, all investigated FPs displayed behaviour compatible with cryoSR microscopy, making this technique broadly available without requiring specialised equipment and will improve the applicability of this emerging technique for cellular and structural biology. Microscopic imaging and technolog

Selective bacterial separation of critical metals:Towards a sustainable method for recycling lithium ion batteries

The large scale recycling of lithium ion batteries (LIBs) is essential to satisfy global demands for the raw materials required to implement this technology as part of a clean energy strategy. However, despite what is rapidly becoming a critical need, an efficient and sustainable recycling process for LIBs has yet to be developed. Biological reactions occur with great selectivity under mild conditions, offering new avenues for the implementation of more environmentally sustainable processes. Here, we demonstrate a sequential process employing two bacterial species to recover Mn, Co and Ni, from vehicular LIBs through the biosynthesis of metallic nanoparticles, whilst Li remains within the leachate. Moreover the feasibility of Mn recovery from polymetallic solutions was demonstrated at semi-pilot scale in a 30 L bioreactor. Additionally, to provide insight into the biological process occurring, we investigated selectivity between Co and Ni using proteomics to identify the biological response and confirm the potential of a bio-based method to separate these two essential metals. Our approach determines the principles and first steps of a practical bio-separation and recovery system, underlining the relevance of harnessing biological specificity for recycling and up-cycling critical materials

Megafire:An ambiguous and emotive term best avoided by science

Background: As fire regimes are changing and wildfire disasters are becoming more frequent, the term megafire is increasingly used to describe impactful wildfires, under multiple meanings, both in academia and popular media. This has resulted in a highly ambiguous concept.Approach: We analysed the use of the term ‘megafire’ in popular media to determine its origin, its developments over time, and its meaning in the public sphere. We subsequently discuss how relative the term ‘mega’ is, and put this in the context of an analysis of Portuguese and global data on fire size distribution.Results: We found that ‘megafire’ originated in the popular news media over 20 years before it appeared in science. Megafire is used in a diversity of languages, considers landscape fires as well as urban fires, and has a variety of meanings in addition to size. What constitutes ‘mega’ is relative and highly context-dependent in space and time, given variation in landscape, climate, and anthropogenic controls, and as revealed in examples from the Netherlands, Portugal and the Global Fire Atlas. Moreover, fire size does not equate to fire impact.Conclusion: Given the diverse meanings of megafire in the popular media, we argue that redefining megafire in science potentially leads to greater disparity between science and practice. Megafire is widely used as an emotive term that is best left for popular media. For those wanting to use it in science, what constitutes a megafire should be defined by the context in which it is used, not by a metric of one-size-fits-all.</p

Freeze, Zoom, Enhance : Increasing precision and resolution of cryoCLEM

Microscopes are a very important tool to study and visualise living systems. Two popular microscope techniques Fluorescence Microscopy (FM) and Electron Microscopy (EM) can be combined on the same sample, which provides complementary information called correlative light and electron microscopy (CLEM). This thesis describes improvements in CLEM-techniques of cryofixed samples.The discovery of the bright fluorescence of uranyl acetate is reported, after cooling with liquid nitrogen. The fluorescent signal of uranyl-acetate makes it very straightforward to find back regions in the EM. Furthermore, accurately alignment of the FM and EM images could be achieved using this phenomenon.The optical resolution of cryoFM is limited by practical restrictions. This results in a large resolution-gap between cryoFM and cryoEM, which can make it difficult to precisely interpret cryoCLEM data. In this thesis, super-resolution on cryosamples is developed. To do so three major challenges had to be overcome: increased drift, sample damage due to high intensity lasers and the unknown behaviour of fluorescent proteins under cryo-conditions. Overcoming these challenges allowed the performance of super-resolution cryoCLEM (SR-cryoCLEM), with a localisation accuracy of 30 nm and structural resolution of 50-100 nm, an increase of 4-8 times. </p

Freeze, Zoom, Enhance : Increasing precision and resolution of cryoCLEM

Microscopes are a very important tool to study and visualise living systems. Two popular microscope techniques Fluorescence Microscopy (FM) and Electron Microscopy (EM) can be combined on the same sample, which provides complementary information called correlative light and electron microscopy (CLEM). This thesis describes improvements in CLEM-techniques of cryofixed samples.The discovery of the bright fluorescence of uranyl acetate is reported, after cooling with liquid nitrogen. The fluorescent signal of uranyl-acetate makes it very straightforward to find back regions in the EM. Furthermore, accurately alignment of the FM and EM images could be achieved using this phenomenon.The optical resolution of cryoFM is limited by practical restrictions. This results in a large resolution-gap between cryoFM and cryoEM, which can make it difficult to precisely interpret cryoCLEM data. In this thesis, super-resolution on cryosamples is developed. To do so three major challenges had to be overcome: increased drift, sample damage due to high intensity lasers and the unknown behaviour of fluorescent proteins under cryo-conditions. Overcoming these challenges allowed the performance of super-resolution cryoCLEM (SR-cryoCLEM), with a localisation accuracy of 30 nm and structural resolution of 50-100 nm, an increase of 4-8 times. </p