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

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

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

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

Developing probes for cryo-superresolution light and electron microscopy

Microscopic imaging and technolog