Cell Imaging Techniques: Methods and Protocols

Cell Imaging Techniques: Methods and Protocols, Second Edition
Preface
Contents
Contributors
1 Digital Images Are Data: And Should Be Treated as Such
	1. Introduction
	2. How Bad Is the Problem of Inappropriate Image Manipulation?
	3. Learning from the Past
		3.1. Manipulated Images Lead to an Inaccurate Historical Record
		3.2. The Failure, to Capture a Representative Sampling of Images of the Subject Being Studied Could Lead Others to Misinterpret the Data
		3.3. “Artistic” Changes to an Image Can Unintentionally Alter the Factual Content and/or a Viewer’s Interpretation of the Image
		3.4. Photo-Illustrations That “Look” Real Are Misleading
	4. Before the Image Is Captured: Appropriate Image Acquisition Strategies
	5. How Digital Image Data Are Stored
		5.1. Scientific Digital Images Are Data That Can Be Compromised by Inappropriate Manipulations
		5.2. Sampling (See Subheadings  6.9 and 6.10)
		5.3. Digital Image Artifacts
		5.4. Data Management
		5.5. Manipulation of Digital Images Should Only Be Performed on a Copy of the Unprocessed Image Data File
	6. Post-processing
		6.1. Simple Adjustments Performed on the Entire Image Are Usually Considered an Acceptable Practice
		6.2. Cropping an Image Is Usually Considered an Acceptable Form of Image Manipulation
		6.3. Digital Images That Will Be Compared to One Another Should Be Acquired Under Identical Conditions, and Any Postacquisition
Image
Processing Should
Also Be Identical
		6.4. Manipulations That Are Specific to One Area of an Image and Are Not Performed on Other Areas Are Questionable
		6.5. Use of Software Filters to Improve Image Quality Is Usually Not Recommended for Biological Images
		6.6. Cloning or Copying Objects into a Digital Image, from Other Parts of the Same Image or from a Different Image, Is Very Questionable
		6.7. Intensity Measurements Should Be Performed on Uniformly Processed Image Data, and the Data Should Be Calibrated to a Known
Standard
		6.8. Avoid the Use of Lossy Compression
		6.9. Magnification and Resolution Are Important
		6.10. Be Careful When Changing the Size (in Pixels) of a Digital Image
		6.11. Reviewing the Processed Image
	7. Going to Press
		7.1. High Bit Depth Images
		7.2. Line Art
		7.3. RGB and CYMK
		7.4. File Compression Issues
	8. Conclusions
	References
2 Epi-Fluorescence Microscopy
	1. Introduction
	2. The Fluorescence Light Path
	3. Components
		3.1. Light Sources
		3.2. Objective Choice
		3.3. Filter Choice
		3.4. Cameras
	4. Fluorescence Probes and Immunofluorescence
		4.1. Fluorescent Proteins (FP)
		4.2. Fluorescent Probes and Biosensors
		4.3. Immunofluorescence
	5. Sample Immuno-Fluorescence Staining Protocol
	6. Image Collection, Corrections, and Display
		6.1. Collecting Images
		6.2. Saving Images
		6.3. Correctingfor Nonuniformity and Background
		6.4. Image Display
		6.5. Correcting for Excitation and Emission Cross-talk
	7. 3D Imaging and Deconvolution
		7.1. Collecting 3D image series
		7.2. Image deconvolution
		7.3. Deblurring or Nearest-Neighbor Non-quantitative Deconvolution
		7.4. Quantitative Restorative Deconvolution
		7.5. Blind Deconvolution
	8. Conclusions
	References
3 Live-Cell Migration and Adhesion Turnover Assays
	1. Introduction
		1.1. A Bit of History
		1.2. Ways to Minimize Light Exposure
		1.3. Microscopy Platforms
	2. Materials
		2.1. Cell Culture
		2.2. Cell Viability Materials
		2.3. Live-Cell Imaging Materials
		2.4. Image Analysis Tools
	3. Methods
		3.1. Before the Microscope: Cell Culture
		3.2. Monitoring Cell Health Before Imaging
		3.3. Control Experiments
		3.4. Plating Cells for Migration and Adhesion Assays
		3.5. Live-Cell Imaging Conditions: Tissue Culture on the Microscope
		3.6. Cell Migration Assay
		3.7. Adhesion Turnover Assay
		3.8. Measuring Cell Migration
		3.9. Measuring Adhesion Turnover
		3.10. Post-acquisition Monitoring of Cell Health
	4. Notes
	References
4 Multifluorescence Confocal Microscopy: Application for a Quantitative Analysis of Hemostatic Proteins in Human Venous Valves
	1. Introduction
	2. Materials
		2.1. Reagents
		2.2. Equipment
		2.3. Antibody Sources
		2.4. Software
	3. Methods
		3.1. Tissue Processing and Sectioning
		3.2. Multifluorescence Labeling Using Anti-Thrombomodulin, -EPCR, and -vWF
			3.2.1. Deparaf fi nization and Rehydration of Tissue Sections
			3.2.2. Antigen Retrieval
			3.2.3. Blocking Step
			3.2.4. Secondary Negative Control
			3.2.5. Primary Antibody Incubation: Each Primary Antibody Must Be Raised in Different Host Species for This Multi fl uorescence Technique
			3.2.6. Secondary Antibody Incubation: Use Secondary Antibodies Raised in the Same Host to Prevent Potential Interspecies Cross-Reactivity
			3.2.7. Labeling of Nuclei
			3.2.8. Application of Cover Slips and Slide Storage
		3.3. Quantitative Fluorescence Imaging via Laser Scanning Confocal Microscopy
			3.3.1. Confocal Settings
			3.3.2. Quantitative Analysis: Detector Gain Settings
		3.4. Semi-Quantitative Computer-Assisted Image Analysis
			3.4.1. Determination of Minimal Threshold Value
			3.4.2. Creation of a Reference Image
			3.4.3. Measurement of Fluorescence Intensity
			3.4.4. Percentage Conversion Technique
	4. Notes
	References
5 Colocalization Analysis in Fluorescence Microscopy
	1. Introduction
	2. Materials
		2.1. Slides
		2.2. Imaging Systems
		2.3. Software
	3. Methods
		3.1. Choice of Fluorophores and Filters
		3.2. Microscope Setup
		3.3. Image Acquisition
		3.4. Display of Colocalization
		3.5. Measurement of Colocalization
			3.5.1. Thresholding and Selection of an ROI
			3.5.2. Co-occurrence Analysis of Colocalization
			3.5.3. Correlation Analysis of Colocalization
			3.5.4. Replicate Based Noise Corrected Correlation Analysis of Colocalization
	4. Notes
	References
6 A Time-Lapse Imaging Assay to Study Nuclear Envelope Breakdown
	1. Introduction
	2. Materials
		2.1. Solutions for Xenopus Egg Extract Preparation
		2.2. Cell Culture and Preparation
		2.3. Preparing Reaction Mix for Nuclear Envelope Breakdown Assay
		2.4. Reagents for Permeabilization
	3. Methods
		3.1. Xenopus Egg Extract Preparation (17, 18)
		3.2. Plating Cells
		3.3. Preparing Soluble Components for Addition to HeLa Cells in Imaging Chamber
		3.4. Digitonin Permeabilization
		3.5. Setting Up and Analyzing Time-Lapse Microscopy
	4. Notes
	References
7 Light Sheet Microscopy in Cell Biology
	1. Introduction
	2. Materials
		2.1. Sample Preparation in Transparent Plastic Cylinders
		2.2. Sample Embedding in Soft Agarose Gels
			2.2.1. Specimen Embedding
			2.2.2. Drosophila Preparation and Live Imaging
			2.2.3. Zebra fish Preparation and Live Imaging
		2.3. Light Sheet-Based Microscopy
		2.4. Basic Image Processing
	3. Methods
		3.1. Sample Preparation Protocol for In Vitro Imaging of Cell Extracts and Soft Gels
			3.1.1. Preparing Cell Extracts and Soft Gels in Plastic Cylinders
		3.2. Sample Preparation Protocol for In Vivo Imaging of Drosophila and Zebra fish Embryos
			3.2.1. Embedding Drosophila Embryos (See Note 2)
			3.2.2. Embedding Zebra fish Embryos (See Note 2)
		3.3. Light Sheet-Based Microscopy Imaging Protocol
		3.4. Basic Image Processing and Data Inspection in Light Sheet-Based Microscopy
			3.4.1. Deconvolution
			3.4.2. Image Registration and Fusion
	4. Notes
	References
8 Image-Based High-Throughput Screening for Inhibitors of Angiogenesis
	1. Introduction
	2. Materials
		2.1. Propagation of Primary Endothelial Cells and Vascular Smooth Muscle Cells
		2.2. Retroviral Vector Transfection (See Note 2)
		2.3. Compounds
		2.4. Microtiter Plates
		2.5. Hoechst and Propidium Iodide Staining
	3. Methods
		3.1. Propagation of Primary Endothelial Cells and Vascular Smooth Muscle Cells (T175 Flasks)
		3.2. Retroviral Vector Transfection
			3.2.1. Transfection of Retroviral Packaging Cells (To Be Conducted in an BL-2 Cell Laboratory)
			3.2.2. Infection of HUVEC
			3.2.3. FACS Sorting of GFP-Expressing HUVEC
		3.3. Co-culture Assay (See Notes 10–12)
		3.4. Hoechst and Propidium Iodide Staining
		3.5. Compound Addition to Co-cultures in 96-Well Plates
			3.5.1. Manual Addition of Compounds (See Notes 17–21)
			3.5.2. Robotic Compound Addition
		3.6. Image Capture
		3.7. Image Analysis (See Fig.  2)
		3.8. Data Analysis
		3.9. Z-Factor Calculation (See Fig.  3)
	4. Notes
	References
9 Intravital Microscopy to Image Membrane Trafficking in Live Rats
	1. Introduction
	2. Materials
		2.1. Animals and Surgical Tools
		2.2. Fluorescent Probes
		2.3. In Vivo Gene Delivery to Submandibular Salivary Gland
		2.4. Animal Holders and Positioning Devices
		2.5. Microscope
	3. Methods
		3.1. Animals and Anesthesia
		3.2. In Vivo Gene Delivery to Submandibular Salivary Gland
		3.3. Insertion of the Catheter in the Tail Artery
		3.4. Animal Surgery and Positioning for Intravital Microscopy
		3.5. Imaging Parameters
	4. Notes
	References
10 Imaging Non-fluorescent Nanoparticles in Living Cells with Wavelength-Dependent Differential Interference Contrast Microscopy and Planar Illumination Microscopy
	1. Introduction
		1.1. Differential Interference Contrast Microscopy
		1.2. Planar Illumination Microscopy
	2. Materials
		2.1. Microscope Systems
		2.2. Detectors
		2.3. Materials and Reagents
		2.4. Software
	3. Methods
		3.1. Cleaning Glass Slides and Coverslips
		3.2. Immobilizing Gold Nanoparticles on Glass Slides or Coverslips
		3.3. Optimization of the DIC Microscope
		3.4. Optical Path Alignment in PIM
		3.5. Live-Cell Imaging
			3.5.1. Treatment of Coverslips with PLL to Aid Cell Attachment
			3.5.2. Growing Cells on Coated Coverslips
			3.5.3. Observing Live Cells with DIC Microscopy
			3.5.4. Observing Live Cells with PIM
	4. Notes
	References
11 Laser Scanning Cytometry: Principles and Applications—An Update
	1. Introduction: Limitations of Flow Cytometry
	2. Features of LSC and Parameters That Can Be Measured
	3. Maximal Pixel of Fluorescence Intensity
	4. Nuclear vs. Cytoplasmic Localization of Fluorescence
	5. The Micronucleus Assay and DNA Damage Signaling
	6. Applications of LSC Utilizing the Software Designed for FISH Analysis
		6.1. FISH Analysis, Cytogenetic Studies
		6.2. Analysis of Nucleoli and Protein Translocations Between Nucleoli and Nucleoplasm
		6.3. Progeny of Individual Cells/Clonogenicity Assay
	7. Cell Immuno-phenotyping
	8. The Relocation Attribute
		8.1. Visual Cell Examination: Imaging
		8.2. Sequential Analysis of the Same Cells with Different Probes
		8.3. Enzyme Kinetics and Other Time-Resolved Events
	9. LSC in Clinical Pathology
	10. Utility of LSC in Other Applications
	References
12 Laser Capture Microdissection for Protein and NanoString RNA Analysis
	1. Introduction
	2. Materials
		2.1. Preparation of Tissue Sections for ArcturusXT Infrared Capture Mode
		2.2. Preparation of Frozen Tissue Sections for UV Cutting Mode (Protein Analysis)
		2.3. Preparation of FFPE Tissue Sections for UV Cutting Mode (NanoString Analysis)
		2.4. Hematoxylin and Eosin Staining for Frozen/FFPE Sections for Protein Analysis
		2.5. One-Step Cresyl Violet Acetate/Eosin Y Staining of FFPE Sections for NanoString Analysis
		2.6. Laser Capture Microdissection Infrared Mode
		2.7. Laser Capture Microdissection UV Mode
		2.8. Protein and RNA Extraction Buffers
		2.9. Adaptation of LCM Workflow for NanoString Platform
		2.10. NanoString Analysis of LCM Samples
	3. Methods
		3.1. Frozen Tissue Sectioning for Protein Analysis
		3.2. FFPE Tissue Sectioning for NanoString Analysis
		3.3. Hematoxylin and Eosin Staining
			3.3.1. H&E Staining Procedure for Frozen Tissue Sections
			3.3.2. H&E Staining Procedure for Formalin-Fixed or Ethanol-Fixed Paraffin-Embedded Tissue Sections
			3.3.3. One-Step Cresyl Violet Acetate/Eosin Y Staining Procedure for FFPE Sections (NanoString Analysis)
		3.4. Laser Capture Microdissection: Infrared (IR) Capture Mode
			3.4.1. ArcturusXT Instrument Setup
			3.4.2. Inspect Image and Locate Cells of Interest
			3.4.3. LCM Cap Placement and Laser Location
			3.4.4. Mark the Cells for Microdissection
			3.4.5. Capturing the Cells
			3.4.6. Unload the Samples and Microdissected Tissue
		3.5. UV Cutting Mode Microdissection Using the ArcturusXT
			3.5.1. UV Laser Location
			3.5.2. Mark the Cells for UV Cutting
			3.5.3. Setting Cut and Capture Properties
			3.5.4. Cutting the Cells
			3.5.5. UV Cutting Mode Microdissection for NanoString Analysis Using the mmi CellCut Plus®
			3.5.6. mmi CellCut Plus® Instrument Setup
			3.5.7. Laser Setup
			3.5.8. Dissection of Serial Slides and Target Collection
		3.6. Lysis and Protein Extraction of Microdissected Material for Downstream Analysis
			3.6.1. Lysis of Microdissected Material for NanoString Analysis
		3.7. Adaptation of LCM Workflow for NanoString Platform Requirements
			3.7.1. Aperio Digital Image of H&E Sections
			3.7.2. Pilot Studies: Amount of Microdissected Material for NanoString Input
			3.7.3. Optimize LCM Sample Lysis
		3.8. NanoString Analysis of LCM Samples
			3.8.1. Assay Validity and Sample Quality Assessment
	4. Notes
	References
13 Viewing Dynamic Interactions of Proteins and a Model Lipid Membrane with Atomic Force Microscopy
	1. Introduction
	2. Materials
		2.1. Buffer Components
		2.2. Buffers
		2.3. Lipid Components: Applicable Apparatus
		2.4. Protein(s) (See Note 1)
		2.5. Drug (See Note 2)
		2.6. Preparation of Mica Substrates and Artificial Membrane Immobilization
		2.7. MFP-3D-BIO AFM and Accessories (See Note 5; Fig.  2)
	3. Methods
		3.1. Preparation of Lipid Vesicles (5 mM 30% PS/70% PC in Bilayer Buffer)
			3.1.1. Preparation of Phospholipid Suspension
			3.1.2. Extrusion of Phospholipid Suspension
		3.2. Preparation of Mica Substrates for Artificial Membrane Immobilization
		3.3. Adsorption of PSPC to Mica (See Note 3)
		3.4. Addition of Protein(s) (See Note 17)
		3.5. Operation of the Asylum Research MFP-3D-BIO AFM (See Note 18)
	4. Notes
	References
14 Mica Functionalization for Imaging of DNA and Protein-DNA Complexes with Atomic Force Microscopy
	1. Introduction
	2. Materials
		2.1. General Equipment and Supplies
		2.2. Reagents and Solutions
			2.2.1. AP-Mica Preparation
			2.2.2. APS-Mica Preparation
	3. Methods
		3.1. Synthesis of APS
		3.2. Mica Functionalization with APS
		3.3. Mica Functionalization with AP (Evaporation Method)
			3.3.1 Vacuum Distillation of APTES
			3.3.2. Preparation of AP-Mica
		3.4. Sample Preparations for AFM Imaging
			3.4.1 Sample Preparation for Imaging in Air
				Droplet Procedure
				The Immersion Procedure
		3.5. AFM Imaging of the Samples
			3.5.1. Imaging in Air
			3.5.2. Imaging in Liquid
	4. Notes
	References
15 Measuring the Elastic Properties of Living Cells with Atomic Force Microscopy Indentation
	1. Introduction
	2. Materials
		2.1. AFM System
		2.2. Additional Microscope Components
		2.3. Additional Supplies
	3. Methods
		3.1. Sample Preparation
		3.2. Cantilever Spring Constant Calibration
		3.3. Deflection Sensitivity Calibration in Water
		3.4. Force Measurements on Cells
		3.5. Data Analysis
	4. Notes
	References
16 Atomic Force Microscopy Functional Imaging on Vascular Endothelial Cells
	1. Introduction
	2. Materials
		2.1. AFM System
		2.2. Coating of Recombinant VE-Cadherin-Fc Molecules to the AFM Tip
		2.3. Components for the Cell Probes
	3. Methods
		3.1. Attachment of VE-Cadherin-Fc Molecules onto the AFM Tip
		3.2. Cell Preparation for AFM Measurements
		3.3 Simultaneous Topography and RECognition Imaging
		3.4. Determination of the Free Oscillation Amplitude
		3.5. Half-Amplitude Imaging (for Picoscan Software)
	4. Notes
	References
17 Porosome: The Secretory NanoMachine in Cells
	1. Introduction
	2. Discovery of the Porosome
	References
18 Stereology and Morphometry of Lung Tissue
	1. Introduction
	2. Materials
		2.1. Basic Microscopy Equipment
			2.1.1. Sample Preparation
			2.1.2. Microscopes
		2.2. Specific Stereology Equipment
			2.2.1. Sample Preparation
	3. Methods
		3.1. Reference Volume
			3.1.1. Fluid Displacement
			3.1.2. Cavalieri Principle
		3.2. Sampling of Tissue Blocks (Location)
		3.3. Sampling of Tissue Blocks (Orientation)
			3.3.1. Isector
			3.3.2. Orientator
		3.4. Processing and Embedding
			3.4.1. Paraffin Embedding
			3.4.2. Glycol Methacrylate Embedding (Here: Technovit 7100)
			3.4.3. Epoxy Resin Embedding (Here Araldite)
		3.5. Measurements
			3.5.1. Light Microscopy
				Alveolar Septal and Airspace Volume, Alveolar Duct Airspace Volume
				Alveolar Epithelial Surface Area
				Alveolar Number
				Alveolar Size: Number-Weighted Mean Volume
				Alveolar Size: Volume-Weighted Mean Volume
			3.5.2. Transmission Electron Microscopy
				Volume of Different Cell Types of Alveolar Septa
			3.5.3. Paraffin
		3.6. A Case Study
	4. Notes
	References
19 A Novel Combined Imaging/Morphometrical Method for the Analysis of Human Sural Nerve Biopsies for Clinical Diagnosis
	1. Introduction
	2. Materials
		2.1. Tissue Procurement
		2.2. Tissue Processing
		2.3. Tissue Block Sectioning
		2.4. Staining
	3. Methods
		3.1. Tissue Procurement
		3.2. Tissue Processing
		3.3. Glass Knife Preparation
		3.4. Block Sectioning
		3.5. Thick Section Staining
		3.6. Confocal Microscopy Imaging
		3.7. MetaMorph Image Analysis
		3.8. Excel Calculations and Display
	4. Notes
	References
20 Correlative Light–Electron Microscopy as a Tool to Study In Vivo Dynamics and Ultrastructure of Intracellular Structures
	1. Introduction
	2. Materials
	3. Methods
		3.1. Cell Transfection, Observation, and Fixation
		3.2. Immunolabeling
		3.3. Embedding
		3.4. Sectioning
		3.5. Serial Section Analysis and 3D Reconstruction
	4. Notes
	References
21 Photooxidation Technology for Correlative Light and Electron Microscopy
	1. Introduction
	2. Materials
		2.1. Minor Equipment
		2.2. Major Equipment
		2.3. Reagents
		2.4. General Processing of Cell Cultures
	3. Methods
		3.1. BODIPY-Ceramide Photooxidation
		3.2. WGA-Alexa Fluor555 Photooxidation
		3.3. HDL-Alexa568 Photooxidation
	4. Notes
	References
22 Electron Microscopy of Endocytic Pathways
	1. Introduction
	2. Materials
		2.1. Reagents
		2.2. Media and Solutions
		2.3. Equipment
	3. Methods
		3.1. General Cell Culture Procedures
		3.2. WGA Internalization
		3.3. DAB-Cytochemistry after Glutaraldehyde Fixation
		3.4. DAB-Cytochemistry In Vivo Prior to Fixation
		3.5. High Pressure-Freezing and Freeze Substitution
			3.5.1. High Pressure-Freezing
			3.5.2. Freeze Substitution
	4. Notes
	References
23 Morphological Analysis of Autophagy
	1. Introduction
		1.1. The Process of Autophagy
		1.2. Strategy to Analyze Autophagy
		1.3. Electron Microscopy
	2. Materials and Reagents
		2.1. Cells
		2.2. Cell Culture
		2.3. Autophagy Inhibitors
		2.4. Transfection
		2.5. Antibody Sources
		2.6. Immunofluorescent Microscopy
		2.7. Electron Microscopy
			2.7.1. Conventional TEM
			2.7.2. Electron Tomography
			2.7.3. Immuno-EM Using Pre-embedding Nanogold Intensification
	3. Methods
		3.1. Cell Culture
		3.2. Autophagy Induction
		3.3. Immunofluorescence
		3.4. Microscopy
		3.5. Monitoring GFP-LC3
			3.5.1. Method of Monitoring GFP-LC3
		3.6. Analysis of Autophagosome Maturation
			3.6.1. Monitoring tfLC3
			3.6.2. Colocalization of GFP-LC3 and LAMP1
		3.7. Electron Microscopy
			3.7.1. Conventional TEM
				Cell Culture and Fixation
				Embedding in Epoxy Resin
				Ultrathin Sectioning
			3.7.2. Electron Tomography
				Preparation of Formvar and Carbon-Coated Grids (see Note 12)
				Ultrathin Sectioning
				3D Tomographic Reconstruction and Modeling
			3.7.3. Immuno-EM Using Pre-embedding Nanogold Intensification
				Fixation and Permeabilization of Cultured Cells
				Immunogold Labeling
				Post-fixation, Embedding, and Ultrathin Sectioning
	4. Notes
	References
24 Cytochemical Detection of Peroxisomes and Mitochondria
	1. Introduction
	2. Materials
		2.1. Culturing of Mammalian Cells
		2.2. Equipment and Reagents
			2.2.1. Labeling of Peroxisomes and Mitochondria by Immunofluorescence
			2.2.2. DAB-Staining of Peroxisomes for Electron Microscopy
		2.3. Controls
		2.4. Antibody Sources and DNA Constructs
	3. Methods
		3.1. Dual Labeling of Peroxisomes and Mitochondria
			3.1.1. Immunofluorescence Labeling of Membrane Proteins
			3.1.2. Immunofluorescence Labeling of Matrix Proteins
			3.1.3. Organelle-Specific Targeting of Expressed Fusion Proteins
			3.1.4. Detection of Mitochondria Using Mitochondrion-Selective Probes
			3.1.5. Cytochemical Detection of Peroxisomes in Cultured Cells
	4. Notes
	References
25 Histochemical Detection of Lipid Droplets in Cultured Cells
	1. Introduction
	2. Materials
		2.1. Solutions
		2.2. Fluorescent Dyes
		2.3. Antibodies
		2.4. Equipment
		2.5. Controls
	3. Methods
		3.1. Staining of LDs in Fixed Cells
			3.1.1. BODIPY 493/503
			3.1.2. Nile Red
			3.1.3. Oil Red O
		3.2. Staining of LDs in Live Cells
			3.2.1. BODIPY 558/568 C12
		3.3. Immunolabeling of ADRP
		3.4. Double Labeling of LDs with Fluorescent Dye and ADRP
			3.4.1. Double Labeling using BODIPY 493/503
			3.4.2. Double Labeling Using BODIPY 558/568 C12
	4. Notes
	References
26 Environmental Scanning Electron Microscopy in Cell Biology
	1. Introduction
		1.1. ESEM Imaging of Plant Tissue
		1.2. ESEM Imaging of Mammalian Cells
		1.3. ESEM–STEM Imaging of Bacteria
	2. Materials
		2.1. ESEM Imaging of Plant Tissue
		2.2. ESEM imaging of Mammalian Cells
		2.3. ESEM–STEM Imaging of Bacteria
	3. Methods
		3.1. ESEM Imaging of Plant Tissue
			3.1.1. Prepare the Microscope and Cooling Stage
			3.1.2. Prepare the Tissue
			3.1.3. Pumpdown and Final Pressure
			3.1.4. Imaging
			3.1.5. Beam Damage Considerations
		3.2. ESEM Imaging of Mammalian Cells
			3.2.1. Cell Culture and Fixation
			3.2.2. Sample Preparation
			3.2.3. Reaching Imaging Pressure
			3.2.4. Imaging Parameters
		3.3. ESEM–STEM of bacteria
			3.3.1. Cell Culture and Resuspension
			3.3.2. Washing Procedure
			3.3.3. Sample Placement
			3.3.4. Reaching Imaging Pressure
			3.3.5. Imaging Parameters
			3.3.6. Beam Damage Considerations
			3.3.7. Microscope Decontamination
	4. Notes
	5. Appendix: Safe Handling of Biological Samples in the Microscopy Lab
		5.1. Handling Category 1 Samples
			5.1.1. Mammalian Cells
			5.1.2. Bacteria
		5.2. Handling Category 2 Samples
	References
27 Environmental Scanning Electron Microscopy Gold Immunolabeling in Cell Biology
	1. Introduction
	2. Materials
	3. Methods
		3.1. Galectin-3 Immunogold Labeling
		3.2. Silver Enhancement Reaction
		3.3. Environmental Scanning Electron Microscopy
		3.4. Image Analysis
	4. Notes
	References
28 High-Pressure Freezing for Scanning Transmission Electron Tomography Analysis of Cellular Organelles
	1. Introduction
	2. Materials
		2.1. Reagents and Disposables
		2.2. Equipment
	3. Methods
		3.1. Cell Cultures on Sapphire Discs
		3.2. High-Pressure Freezing
			3.2.1. High-Pressure Freezing of Adherent Cells
				High-Pressure Freezing of Adherent Cells Using Aluminum Sapphire Sandwiches
				High-Pressure Freezing of Adherent Cells Using Sapphire Gold Sandwiches
			3.2.2. High-Pressure Freezing of Cells in Solution
				High-Pressure Freezing of Cells in Solution Using Capillaries
				High-Pressure Freezing of Cells in Solution Using Aluminum Sapphire Sandwiches
			3.2.3. High-Pressure Freezing of Tissue
			3.2.4. High-Pressure Freezing of Pre fi xed Infectious Material
		3.3. Freeze Substitution, Embedding, and Sectioning
		3.4. STEM Tomography
		3.5. Tomogram Reconstruction and Visualization
	4. Notes
	References
29 MALDI Imaging Mass Spectrometry for Direct Tissue Analysis
	1. Introduction
	2. Materials
		2.1. Tissue Preparation
		2.2. Matrix Application
		2.3. MALDI Imaging Mass Spectrometry Measurement
		2.4. Tissue Staining
		2.5. Data Analysis
	3. Methods
		3.1. Tissue Preparation
			3.1.1. Section Preparation from Native Tissues
			3.1.2. Section Preparation from Alcohol-Fixed and Paraffin-Embedded Tissues
		3.2. Histological Staining of Tissue Sections on Microscopy Slides
			3.2.1. Native Tissues
			3.2.2. Alcohol-Fixed and Paraffin-Embedded Tissues
		3.3. Matrix Application
		3.4. MALDI Imaging Mass Spectrometry Measurement
		3.5. Histological Staining of Tissue Sections After MALDI Imaging Mass Spectrometry
		3.6. Data Analysis
	4. Notes
	References
Index