میکروسکوپی نوری و الکترونی همبسته IV (جلد ۱۶۲)

Correlative Light and Electron Microscopy IV, Volume 162, a new volume in the Methods in Cell Biology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. Besides the detailed description of protocols for CLEM technologies including time-resolution, Super resolution LM and Volume EM, new chapters cover Workflow (dis)-advantages/spiderweb, Serial section LM + EM, Platinum clusters as CLEM probes, Correlative Light Electron Microscopy with a transition metal complex as a single probe, SEM-TEM-SIMS, HPF-CLEM, A new workflow for high-throughput screening of mitotic mammalian cells for electron microscopy using classic histological dyes, and more. Contains contributions from experts in the field Covers topics using nano-SIMS and EDX for CLEM Presents recent advances and currently applied correlative approaches Gives detailed protocols, allowing for the application of workflows in one's own laboratory setting Covers CLEM approaches in the context of specific applications Aims to stimulate the use of new combinations of imaging modalities Front Cover Correlative Light and Electron Microscopy IV Copyright Contents Contributors Preface to CLEM IV: Broaden the horizon What does it hold for you? Chapter 1: 50 Shades of CLEM: How to choose the right approach for you 1. What is CLEM? 2. The microscopist's dilemma 3. Sample preservation 4. Case studies 5. How to choose your shade of CLEM: Closing remarks Acknowledgments References Chapter 2: The Histo-CLEM Workflow for tissues of model organisms 1. Introduction 2. General design of the Histo-CLEM Workflow 2.1. Histology 2.1.1. Microtome method 2.1.1.1. Tissue processing and embedding 2.1.1.2. Microtome sectioning 2.1.2. Cryostat method 2.1.2.1. Tissue processing and embedding 2.1.2.2. Cryostat sectioning 2.1.3. Vibratome method 2.1.3.1. Tissue processing and embedding 2.1.3.2. Vibratome sectioning 2.2. Light microscopy 2.2.1. Section mounting 2.2.2. Data acquisition 2.2.3. Quality control 2.3. Electron microscopy 2.3.1. Post-fixation, dehydration and resin embedding 2.3.2. Ultramicrotome sectioning 2.3.3. Post-staining 2.3.4. Data acquisition 3. Application example 3.1. Histology 3.1.1. Coverslip coating with 2% APES 3.1.2. Sample processing, embedding and sectioning 3.2. Light microscopy 3.3. Electron microscopy 3.4. Instrumentation and materials 3.4.1. Histology 3.4.2. Light microscopy 3.4.3. Electron microscopy 3.5. Results and discussion 4. Concluding remarks Acknowledgments References Chapter 3: Fluorescent platinum nanoclusters as correlative light electron microscopy probes 1. Introduction 2. CLEM probes 2.1. CLEM probes with an endogenously expressed element 2.2. Affinity-based CLEM probes 2.3. Fluorescent nanoclusters 2.4. Nanocluster scaffold molecules 2.5. Dendrimers and polymers 3. Instrumentation, materials, and reagents 3.1. Platinum nanocluster synthesis 3.2. CLEM workflow 3.2.1. Cell culture 3.2.2. Processing of cells 3.2.3. Laser scanning confocal microscopy 3.2.4. TEM sample processing 4. Methods 4.1. Platinum nanocluster synthesis 4.2. CLEM workflow 4.2.1. Cell culture 4.2.2. Platinum nanocluster uptake in cells 4.2.3. Laser scanning confocal microscopy 4.2.4. TEM sample processing 5. Generation and validation of the probe 5.1. Emission, excitation and quantum yield of PtNCs 5.2. Characterization of PtNCs using HAADF-STEM and EDX 5.3. Silver enhancement of platinum nanoclusters 5.4. UV-vis spectroscopy of silver enhancing PtNCs 6. Use of PtNCs 6.1. Investigation of PtNCs as in vivo probes 6.2. PtNCs as CLEM probes 7. Discussion and outlook Acknowledgments References Chapter 4: Refining a correlative light electron microscopy workflow using luminescent metal complexes 1. Introduction 2. Transition metal Ir complex 1 3. Instrumentation and materials 3.1. Ir complex 1 synthesis 3.2. CLEM workflow 3.2.1. Cell culture 3.2.2. Processing of cells 3.2.3. Laser scanning confocal microscopy 3.2.4. TEM sample processing 3.2.5. CLEM data analysis 4. Methods 4.1. Ir complex 1 Synthesis 4.2. Correlative light and electron microscopy 4.2.1. Cell culture 4.2.2. Choice of finder dishes 4.2.3. CLEM of Ir complex 1 4.2.4. TEM sample processing 5. Results 5.1. Choice of finder dishes 5.2. CLEM of Ir complex 1 6. Discussion Acknowledgments References Chapter 5: Sample preparation for energy dispersive X-ray imaging of biological tissues 1. Introduction 1.1. EDX 1.2. Sample preparation 2. Routine chemical fixation of tissues 2.1. Protocol 2.2. Results and discussion 3. Resin 3.1. Protocol 3.2. Results and discussion 4. Contrasting 4.1. Protocol 4.2. Results and discussion 5. Grids and sample holder 5.1. Protocol 5.2. Results and discussion 6. Other considerations 6.1. Section thickness 6.2. Current 6.3. Acceleration voltage 6.4. Sample tilt 7. Conclusion 8. Materials 8.1. Equipment 8.2. Materials Acknowledgments Author contributions References Chapter 6: HPM live μ for a full CLEM workflow 1. Introduction 2. High pressure freezing principles 2.1. Water phase diagram as a guide for HPF 2.2. The development of high pressure freezing technology 3. Designing the HPM live μ 3.1. Improving the pressure rise speed and cooling rate 3.2. The HPM live μ: A flexible research engine for optimized vitrification efficiency 3.3. User interface for fast assessing and optimization HPF procedures 3.4. A wide range of sample holders for all commercial carriers 3.5. Choosing the right sample preparation method for HPF 4. Integration of the HPM live μ in a CLEM workflow 4.1. Cryo-capturing fast and small biological event 4.2. A flexible design to accommodate CLEM workflows 5. A live imaging, cryo-fluorescence, in-resin fluorescence and EM workflow: Validation at each step 5.1. Sample evaluation under cryogenic conditions after high pressure freezing 5.2. Fluorescent preservation after freeze substitution 5.3. Correlating fluorescence with SEM: Toward 3D CLEM 6. Perspectives Acknowledgments Author contributions References Chapter 7: High-throughput screening of mitotic mammalian cells for electron microscopy using classic histological dyes 1. Introduction 2. Methods 2.1. Preparation of sapphire discs for high-pressure freezing 2.2. Synchronization of cells and enrichment of mitotic stages 2.3. Specimen preparation for electron microscopy 2.4. Selection and staging of mitotic cells using methylene blue 3. Instrumentation and materials 3.1. Preparation of sapphire discs for high-pressure freezing 3.2. Synchronization of cells and enrichment of mitotic stages 3.3. Specimen preparation for electron microscopy 3.4. Selection and staging of mitotic cells using methylene blue 4. Discussion Acknowledgments References Chapter 8: On-section correlative light and electron microscopy of large cellular volumes using STEM tomography 1. Introduction 2. Methods 2.1. Molecular cloning 2.2. Cell lines 2.3. Live-cell nuclear staining with different bisBenzimide dyes 2.4. Sample preparation for CLEM 2.4.1. Cell culture 2.4.2. High-pressure freezing and freeze-substitution 2.4.3. Chemical fixation and progressive lowering of temperature 2.4.4. Ultramicrotomy 2.5. Fluorescence microscopy of resin sections 2.6. Transmission electron microscopy 2.7. Scanning transmission electron microscopy (STEM) and electron tomography 2.8. Data analysis and overlay generation using eC-CLEM 3. Instrumentation and materials 3.1. Molecular cloning 3.2. Cell lines 3.3. Live-cell nuclear staining with different bisBenzimide dyes 3.4. Sample preparation for CLEM 3.4.1. Cell culture 3.4.2. High-pressure freezing and freeze-substitution 3.4.3. Chemical fixation and progressive lowering of temperature 3.4.4. Ultramicrotomy 3.5. Fluorescence microscopy of resin sections 3.6. Transmission electron microscopy 3.7. Scanning transmission electron microscopy (STEM) and electron tomography 3.8. Data analysis and overlay generation using eC-CLEM 4. Results 4.1. bisBenzimide H 33342 is a suitable nuclear fluorescent dye to stain living cells for on-section CLEM 4.2. High-pressure freezing is the fixation method of choice for on-section CLEM 4.3. STEM tomography is a useful tool for investigating the ultrastructure of primary cilia 4.4. On-section CLEM-STEM in combination with fluorescently labeled gold nanoparticles can be used to investigate endosomes 5. Discussion 5.1. Preservation of fluorescence signals and cellular ultrastructure 5.2. Internal landmarks and fluorescent fiducial markers for on-section CLEM 5.3. On-section CLEM-STEM approach to study low-copy and multi-copy organelles 6. Conclusion Acknowledgments References Chapter 9: An accelerated procedure for approaching and imaging of optically branded region of interest in tissue 1. Introduction 2. Rationale 3. Methods 3.1. Dissection of fruit flies 3.2. Light microscopy 3.3. Near-infrared branding 3.4. Sample preparation for EM 3.5. Imaging by SBF-SEM and TEM 4. Instrumentation and materials 4.1. Dissecting of fruit flies 4.2. Light microscopy 4.3. Near-infrared branding 4.4. Sample preparation for EM 4.5. Imaging by SBF-SEM and TEM 5. Discussion and conclusion Acknowledgments References Chapter 10: Cryo-fluorescence microscopy of high-pressure frozen C. elegans enables correlative FIB-SEM 1. Introduction 2. A note on selecting the right cryoprotectant 2.1. Evaluation of toxicity, ease of handling, and degree of cryoprotection 2.2. Measurement of autofluorescence 3. Rationale 4. Methods 4.1. High-pressure freezing of C. elegans hermaphrodites 4.2. Cryo-fluorescence microscopy 4.2.1. Preparation of the cryostage and sample loading 4.2.2. Imaging of samples 4.2.3. Recovery of samples 4.3. Freeze substitution and resin embedding 4.4. Light microscopy of resin-embedded worms 4.5. FIB-SEM sample preparation and imaging 4.5.1. Sample preparation 4.5.2. FIB-SEM data acquisition 4.6. Image processing and analysis 4.7. Observations and results 5. Instrumentation and materials 5.1. High-pressure freezing of C. elegans worms 5.1.1. Cryoprotectants and worm strains 5.1.2. Preparation and freezing of worms 5.2. Cryo super-resolution fluorescence microscopy 5.2.1. Preparation of cryostage and sample loading 5.2.2. Imaging of samples 5.2.3. Recovery of samples 5.3. Freeze substitution and resin embedding 5.4. Light microscopy of resin-embedded worms 5.5. FIB-SEM sample preparation and imaging 5.5.1. Sample preparation 5.5.2. FIB-SEM data acquisition 5.6. Image analysis 6. Discussion Acknowledgments References Chapter 11: Correlative super-resolution fluorescence and electron cryo-microscopy based on cryo-SOFI& 1. Introduction 2. Rationale 3. Materials 3.1. Instrumentation 3.2. Materials 3.3. Reagents 4. Methods 4.1. Grid preparation 4.2. Seeding cells on EM grids 4.3. Plunge-freezing EM grids with cells 4.4. Correlative cryogenic light and electron microscopy 5. Discussion 5.1. Advantages of cryo-SOFI 5.2. Limitations of cryo-SOFI 5.3. Future prospects Acknowledgments References Chapter 12: Cryo-correlative light and electron microscopy workflow for cryo-focused ion beam milled adherent cells 1. Introduction 2. Rationale 3. Materials 3.1. Instruments 3.2. Commercial software 3.3. Materials and reagents 4. Methods 4.1. Sample preparation 4.1.1. Preparation of polydimethylsiloxane (PDMS) coated cell culture dishes 4.1.2. Culturing adherent cells on gold EM grids and subsequent labeling with fluorescent dyes 4.1.3. Plunge freezing cells cultured on EM grids 4.2. Data acquisition and cryo-FIB milling 4.2.1. Cryo-LM of cells before cryo-FIB milling (pre-LM map acquisition) 4.2.1.1. Creating a stitched map of the acquired grid 4.2.2. Cryo-FIB milling 4.2.3. Cryo-TEM 4.2.3.1. Loading FIB autogrids into the cryo-TEM 4.2.3.2. Cryo-TEM mapping and cryo-ET 4.2.3.3. Stitching of lamella cryo-TEM map 4.2.4. Cryo-LM of cells after FIB milling (post-LM map acquisition) 4.3. Correlation analysis 4.3.1. Deconvolution 4.3.2. 3D registration and alignment of the pre- and post-LM maps 4.3.3. Compensate for lamella tilt 4.3.4. Removal of out-of-lamella signal 4.3.5. 2D registration of the cryo-LM and cryo-TEM map using ec-CLEM 4.4. Safety considerations and standards 4.5. Analysis and statistics 4.5.1. Out-of-lamella signal analysis 4.5.2. Correlation precision analysis 4.6. Troubleshooting and optimization 5. Results and discussion 5.1. Limitations and alternative technique 5.2. Pros and cons of this method 5.3. Summary Acknowledgments References Chapter 13: Super-resolution correlative light-electron microscopy using a click-chemistry approach for stu 1. Introduction 1.1. CLEM and super-resolution microscopy 1.2. SMLM-CLEM, advantages over conventional CLEM 1.3. Fixation in SRM-CLEM 1.4. Fluorescence labeling: Advantages of click chemistry 1.5. Case study: Intracellular pathogens 2. Methods 2.1. Bioorthogonal labeling of bacteria and cell infection experiment 2.2. Fixation and preparation of ultrathin cryo-sections 2.3. Click reaction and counterstaining of thawed cryo-sections 2.4. STORM imaging 2.5. STORM analysis 2.6. TEM staining 2.7. TEM imaging and stitching 2.8. Correlation 3. Instrumentation and materials 3.1. Bioorthogonal labeling and cell infection experiments 3.2. Fixation and preparation of ultrathin cryo-sections 3.3. Click reaction and counterstaining of thawed cryo-sections 3.4. Super-resolution microscopy 3.5. Transmission electron microscopy 3.6. Correlation 4. Discussion 4.1. Flexibility offered by click-chemistry 4.2. Choice of grids 4.3. How to correlate 4.4. Added value of STORM 4.5. General applicability of the method Acknowledgments References Chapter 14: Step-by-step guide to post-acquisition correlation of confocal and FIB/SEM volumes using Amira softwar 1. Introduction 2. Precursor techniques 3. Software and hardware 4. Step-by-step guide 4.1. Initial image processing 4.1.1. Light microscopy 4.1.2. FIB/SEM data 4.2. Loading data in Amira 4.3. Rough alignment 4.4. Segmenting internal correlation fiducials 4.4.1. Segmenting FM DAPI signal 4.4.2. Segmenting bacteria from FIB/SEM data 4.5. Fine alignment 5. Summary and discussion Acknowledgments References Chapter 15: Visualization and co-registration of correlative microscopy data with the ImageJ plug-in C 1. Introduction 2. Models for image co-registration 3. Correlia 3.1. Installation of the software 3.2. A quick guide to image registration with Correlia 3.2.1. In preparation of working with Correlia 3.2.2. Starting a Correlia project 3.2.3. Rigid body registration 3.2.4. Deformable registration (de-warping) 3.3. Visualization 4. Two real-life examples 4.1. Distribution of phosphorous in an algal biofilm 4.1.1. Rationale 4.1.2. Sample and microscopy equipment 4.1.3. Methods (for image registration) 4.1.4. Alternatives and troubleshooting 4.1.5. Summary 4.2. Microscopic analysis of a soil sample-An image registration workflow 4.2.1. Rationale 4.2.2. Sample and microscopy equipment 4.2.3. Methods 4.2.4. M1, M2-Acquisition of a treasure map and identification of areas of interest 4.2.5. M3-Start a new Correlia project 4.2.6. M4, M5-Scanning helium-ion microscopy 4.2.7. M6, M7-Measurement of the topography in the vicinity of the plant root 4.2.8. M8, M9-Time-of-flight secondary ion mass spectrometry 4.2.9. M10, M11-Chemical analysis of mineral particles by energy-dispersive X-ray spectroscopy 4.2.10. Summary 5. Summary and conclusions Acknowledgments References Chapter 16: Multimodality imaging beyond CLEM: Showcases of combined in-vivo preclinical imaging and ex-vivo microscopy t ... 1. Introduction 1.1. Correlated multimodality imaging (CMI) 1.2. Vascular mural lesions: Hyperplasia 1.3. Vascular mural lesions: Micro-calcifications 2. Rationale 3. Methods and results 3.1. Mural lesions: Hyperplasia 3.1.1. MicroMRI 3.1.2. MicroCT 3.1.3. HREM and HP 3.2. Mural lesions: Micro-calcifications 3.2.1. MicroPET/MRI and autoradiography 3.2.2. Autoradiography and HP 3.2.3. MicroXRF and HP 3.2.4. Rat protocols 4. Discussion 5. Conclusion and outlook Acknowledgment References Chapter 17: Correlative multimodal imaging: Building a community 1. Setting the scene 2. A brief history 3. Building the community 3.1. COMULIS 3.2. Training 3.3. Dissemination 3.4. Standards 3.5. Public archiving of CLEM and CLXM data 4. Conclusion Acknowledgments References Back Cover Abstract: Correlative light and electron microscopy (CLEM) combines the strengths of light microscopy (LM) and electron microscopy (EM) to pin-point and visualize cellular or macromolecular structures. However, there are many different imaging modalities that can be combined in a CLEM workflow, creating a vast number of combinations that can overwhelm new-comers to the field. Here, we offer a conceptual framework to help guide the decision-making process for choosing the CLEM workflow that can best address your research question, based on the answer to five questions __Correlative Light and Electron Microscopy IV, Volume 162,____Methods in Cell Biology__