Preface
Contents
Contributors
Part I: Imaging with Passive Optical Readouts
Chapter 1: Fluorescent Labeling of the Nuclear Envelope Without Relying on Inner Nuclear Membrane Proteins
1 Introduction
2 Materials
2.1 Preparation of Cells for FL Imaging
2.2 Instruments for FL Imaging
3 Methods
3.1 FL Imaging of the NE
3.2 Simultaneous Imaging of Two Nuclear Components and 3D Reconstruction
3.3 Time-Lapse Imaging of the Cells
4 Notes
References
Chapter 2: Quantitative Analysis of Membrane Receptor Trafficking Manipulated by Optogenetic Tools
1 Introduction
2 Materials
2.1 Cell Culture and Transfection
2.2 Plasmids
2.3 Antibodies
2.4 Live Imaging of ADRB2CIB
2.5 Immunostaining
2.6 Western Blotting
2.7 Immunoprecipitation
3 Methods
3.1 Cell Cultivation and Transfection
3.2 Transfection
3.3 Live Imaging of ADRB2CIB and ArrestinCRY Trafficking Using Confocal Microscopy
3.4 ELISA Assay to Quantify the Amount of ADRB2CIB on the Cell Surface
3.5 Immunostaining of ADRB2CIB and Endosomes
3.6 Western Blotting
3.7 Detection of Ubiquitinated ADRB2CIB by Immunoprecipitation
4 Notes
References
Chapter 3: Simultaneous Detection of Four Cell Cycle Phases with Live Fluorescence Imaging
1 Introduction
2 Materials
2.1 Plasmids (See Fig. 1)
2.2 Imaging Equipment
2.3 Tissue Culture Reagents
2.4 Tissue Culture Equipment
3 Methods
3.1 Lentiviral Packaging
3.2 Lentiviral Transduction
3.3 Cell Seeding
3.4 Imaging
4 Notes
References
Chapter 4: A Murine Bone Metastasis Model Using Caudal Artery Injection and Bioluminescence Imaging
1 Introduction
2 Materials
2.1 Cells and Animals
2.2 Equipment
2.3 Disposables
2.4 Reagents and Media
2.5 Formulation
3 Methods
3.1 Cell Preparation
3.2 Caudal Artery Injection
3.3 Postinjection Analysis
4 Notes
References
Chapter 5: A New Lineage of Artificial Luciferases for Mammalian Cell Imaging
1 Introduction
2 Materials
2.1 Reagents and Lab Equipment
2.2 Instrumentation
2.3 Software
3 Methods
3.1 Design of ALucs by Extracting Frequently Occurring Amino Acids from the Alignment of Copepod Luciferases
3.2 Determination of the Sequential Identity Ranking and Phylogenetic Trees of the New Lineage of Artificial Luciferases (See ...
3.3 Synthesis of cDNA Constructs Encoding the New Lineage of Artificial Luciferases
3.4 Determination of the Relative Optical Intensities of the New ALucs and Comparison with Conventional Luciferases (See Fig. ...
3.5 Substrate Specificity of the New Lineage of ALucs (See Fig. 3)
3.6 Live-Cell BL Imaging and Substrate Selectivity of ALuc49 and Selected Marine Luciferases (See Fig. 4)
4 Notes
References
Chapter 6: Use of Bacterial Luciferase as a Reporter Gene in Eukaryotic Systems
1 Introduction
2 Materials
2.1 Cell Culture Medium
2.2 Cell Plating Reagents
2.3 Transfection Reagents
2.4 Reagents for Measuring Transfected Cells
2.5 Cell Culture Equipment
2.6 BL Equipment
3 Methods
3.1 Seeding Cells for Transient Transfection
3.2 Transient Transfection
3.3 Activator Screening
3.4 Inhibitor Screening
3.5 Lux Assay Activity
3.6 RLuc Assay Activity
3.7 Data Analysis
4 Notes
References
Part II: Imaging with Activatable Bioluminescent Probes
Chapter 7: Quantitative Determination and Imaging of GΞ±q Signaling in Live Cells via Split-Luciferase Complementation
1 Introduction
2 Materials
3 Methods
3.1 Generation of the pIRESpuro3 CBRN-PLC-Ξ²3-2A-GΞ±q(123) Vector
3.2 Generation of Stably Transfected Cells
3.3 Using the GΞ±q Probe for Ligand Characterization
3.4 Employing the GΞ±q Probe in Bioluminescence Imaging
4 Notes
References
Chapter 8: A Split-Luciferase-Based Cell Fusion Assay for Evaluating the Myogenesis-Promoting Effects of Bioactive Molecules
1 Introduction
2 Materials
2.1 Preparation of Retroviruses
2.2 Preparation of N-Cell and C-Cell
2.3 96-Well Microplate-Based Assay
2.3.1 Day 0: Proliferation
2.3.2 Days 2 and 4: Differentiation and Compound Addition
2.3.3 Day 6: BL Measurement
3 Methods
3.1 Preparation of Retroviruses Harboring the N- or C-Probe RNAs
3.2 Preparation of N- and C-Cells
3.3 The 96-Well Microplate Assay
4 Notes
References
Chapter 9: Development of a Single Fluorescent Protein-Based Green Glucose Indicator by Semirational Molecular Design and Mole...
1 Introduction
2 Materials
2.1 Linker Length Optimization
2.2 Random Mutagenesis to Optimize the Linker Amino Acid Sequence
2.3 Cell Culture and Fluorescence Imaging
3 Methods
3.1 Linker Length Optimization Screen
3.2 Site-Directed Random Mutagenesis to the Linker Amino Acid Sequence
3.3 Fluorescence Live-Cell Imaging
4 Notes
References
Part III: Imaging with Functional Substrates and Luciferases
Chapter 10: Near-Infrared Bioluminescence Imaging of Animal Cells with Through-Bond Energy Transfer Cassette
1 Introduction
2 Materials
2.1 Reagents and Lab Equipments
2.2 Instrumentation
2.3 Software
3 Methods
3.1 UV-Vis Absorption Measurements (See Fig. 1B)
3.2 Chemiluminescence Spectra (See Fig. 1C)
3.3 Preparation of Mammalian Cells Expressing Marine Luciferases
3.4 Bioluminescence Spectra (See Fig. 1C)
3.5 Passive Diffusion Kinetics of Cy5-CTZ and Cy5 into Mammalian MDA-MB-231 Cells (See Fig. 2a)
3.6 Determination of Luciferase-Specific Bioluminescence of Cy5-CTZ (See Fig. 2b)
3.7 Kinetic Profiles of Bioluminescence Intensities (Fig. 3a)
3.8 Attenuation of the BL Intensities of Cy5-CTZ in Whole Blood (Fig. 3b)
4 Notes
References
Chapter 11: Azide- and Dye-Conjugated Coelenterazine Analogues for Imaging Mammalian Cells
1 Introduction
2 Materials
2.1 Components for Synthesis of CTZ
2.2 Components for Bioluminescence Assay
2.3 Instrumentation
2.4 Software
3 Methods
3.1 General Procedure for Synthesis
3.1.1 Synthesis of 5-(4-(2-Azidoethoxy)phenyl)-3-benzylpyrazin-2-amine (See Fig. 1b Compound (3))
3.1.2 Synthesis of 3-(4-(2-Bromoethoxy)Phenyl)-1,1-Diethoxypropan-2-One (See Fig. 1b Compound (5))
3.1.3 Synthesis of 3-(4-(2-Azidoethoxy)Phenyl)-1,1-Diethoxypropan-2-One (See Fig. 1b Compound (6))
3.1.4 General Procedure (A) for Preparation of Azide-Modified CTZ Analogues (See Fig. 1 Compound (9) and Compound (10))
3.1.5 Synthesis of 6-(4-(2-Azidoethoxy)Phenyl)-8-Benzyl-2-(4-Hydroxybenzyl)Imidazo[1,2-a]Pyrazin-3(7H)-One (See Fig. 1b Compou...
3.1.6 Synthesis of 2-(4-(2-Azidoethoxy)Benzyl)-8-Benzyl-6-(4-Hydroxyphenyl)Imidazo[1,2-a]Pyrazin-3(7H)-One (See Fig. 1b Compou...
3.1.7 General Procedure (B) for Preparation of Amino-Modified CTZ Analogues (See Fig. 1b Compound (11) and Compound (12))
3.1.8 General Procedure (C) for Preparation of FITC-Modified CTZ Analogues (See Fig. 1c Compound (13), Compound (14), and Comp...
3.1.9 Synthesis of 5-(3-(2-(4-(8-Benzyl-2-(4-Hydroxybenzyl)-3-Oxo-3,7-Dihydroimidazo[1,2-a]Pyrazin-6-yl)Phenoxy)Ethyl)Thiourei...
3.1.10 Synthesis of 5-(3-(2-(4-(8-Benzyl-6-(4-Hydroxyphenyl)-3-Oxo-3,7-Dihydroimidazo[1,2-a]Pyrazin-6-yl)Methyl)Phenoxy)Ethyl)...
3.1.11 Synthesis of 5-((6-((2-(4-((8-Benzyl-6-(4-Hydroxyphenyl)-3-Oxo-3,7-Dihydroimidazo[1,2-a]Pyrazin-2-yl)Methyl)Phenoxy)Eth...
3.1.12 General Procedure (D) for Preparation of Nile Red-Modified CTZ Analogues (See Fig. 1c Compound (16) and Compound (17))
3.1.13 Synthesis of N-(2-(4-(8-Benzyl-2-(4-Hydroxybenzyl)-3-Oxo-3,7-Dihydroimidazo[1,2-a]Pyrazin-6-yl)Phenoxy)Ethyl)-2-((9-(Di...
3.1.14 Synthesis of N-(2-(4-((8-Benzyl-6-(4-Hydroxyphenyl)-3-Oxo-3,7-Dihydroimidazo[1,2-a]Pyrazin-2-yl)Methyl)Phenoxy)Ethyl)-2...
3.2 Determination of Relative Bioluminescence Intensities of CTZ Analogues with Conventional Marine Luciferases (See Fig. 2a)
3.3 Determination of the Optical Spectra of Azide-Modified CTZ Analogues (See Fig. 2b)
3.4 Measurement of the BRET Spectra of Dye-Conjugated CTZ Analogues with ALuc16 (See Fig. 2c)
3.5 Measurement of the BRET Spectra of Dye-Conjugated CTZ Analogues with RLuc8.6-535 (See Fig. 2d, e)
3.6 Relative Bioluminescence Intensity Matrix of Selected CTZ Analogues with New ALuc Variants (See Fig. 3a)
3.7 Selective Imaging of Living COS-7 Cells Expressing Each Luciferase with a Specific Luciferin (See Fig. 3b)
4 Notes
References
Chapter 12: Luciferase-Specific Coelenterazine Analogues for Optical Cross Talk-Free Bioassays
1 Introduction
2 Materials
2.1 Components for Synthesis of the Selected CTZ (6-et-OH-CTZ, 6-pi-OH-CTZ and 6-pi-OH-2H-CTZ) (See Note 2)
2.2 Components for Bioluminescence Assay
2.3 Instrumentation
2.4 Software
3 Methods
3.1 General Procedure for Synthesis
3.1.1 Synthesis of 3-Benzyl-5-((Trimethylsilyl)Ethynyl)Pyrazin-2-Amine (2) (See Fig. 1b Compound (2))
3.1.2 Synthesis of 3-Benzyl-5-Ethynylpyrazin-2-Amine (3) (See Fig. 1b Compound (3))
3.1.3 Synthesis of 3-Benzyl-5-(4-((tert-Butyldimethylsilyl)Oxy)Phenyl)Ethynyl)Pyrazin-2-Amine (5) (See Fig. 1b Compound (5))
3.1.4 General Procedure for Preparation of Compounds (9)-(11)
3.1.5 Synthesis of 8-Benzyl-2-(4-Hydroxybenzyl)-6-((4-Hydroxyphenyl)Ethynyl)Imidazo[1,2-a]Pyrazin-3(7H)-One (9) (See Fig. 1b C...
3.1.6 Synthesis of (E)-2,8-Dibenzyl-6-(4-Hydroxystyryl)Imidazo[1,2-a]Pyrazin-3(7H)-One (10) (See Fig. 1b Compound (10)) (6-pi-...
3.2 Luciferase Specificity of Newly Synthesized CTZ Analogues (See Figs. 1c and 2a)
3.3 Luciferase-Selective Property of CTZ Analogues in Lysates and Living Mammalian Cells (See Fig. 2b, c)
3.4 Multiplex Imaging of Ligand-Driven Events with Single-Chain Probes and the Specific CTZ Analogues (See Fig. 3)
4 Notes
References
Part IV: Imaging with Organic Fluorescent Probes
Chapter 13: Live Imaging of Virus-Infected Cells by Using a Sialidase Fluorogenic Probe
1 Introduction
2 Materials
2.1 Chemicals and Reagents
2.2 Mammalian Cells
2.3 Culture Media
2.4 Proteins and Viruses
2.5 Primers
2.6 Instruments
3 Methods
3.1 Live Imaging of Virus-Infected Cells or Drug-Resistant Virus-Infected Cells
3.2 Selective Imaging of Drug-Resistant Virus-Infected Cells
3.3 Highly Efficient Isolation of a Drug-Resistant Virus
3.4 Live Imaging and Immunostaining of NA-Expressed Cells by Using the New Sialidase Fluorogenic Probe and Anti-NA Monoclonal ...
4 Notes
References
Chapter 14: Live-Cell Imaging of Sirtuin Activity Using a One-Step Fluorescence Probe
1 Introduction
2 Materials
2.1 Cell Culture and Imaging
2.2 SIRT Inhibitors, Nicotinamide (NAM), EX-527, AGK2
2.3 Synthesis of KST-F-DA
3 Methods
3.1 Preparation of Cell-Membrane-Permeable SIRT FL Probe, KST-F-DA
3.1.1 Synthesis of 4-[(4-(Dimethylamino)Phenyl)Diazenyl]Benzenepropionic Acid (Dabcyl-PH (2))
3.1.2 Synthesis of 2,5-Dioxopyrrolidin-1-yl 4-[(4-(Dimethylamino)Phenyl)Diazenyl]Benzenepropionate (Dabcyl-PH-SE (3))
3.1.3 Synthesis of Fmoc-Lys(Dabcyl-PH)-OH (5)
3.1.4 Synthesis of (7)
3.1.5 Synthesis of FITC-DA-TEG-NBoc (9)
3.1.6 Synthesis of FITC-DA-TEG-NH2 (10)
3.1.7 Synthesis of Ac-KST-OH (12)
3.1.8 Synthesis of KST-F-DA (TFA Salt)
3.2 SIRT1 FL Imaging Using SIRT Inhibitors
3.3 SIRT1 FL Imaging Under Starvation Condition
4 Notes
References
Chapter 15: Live-Cell Fluorescence Imaging of Microtubules by Using a Tau-Derived Peptide
1 Introduction
2 Materials
2.1 Synthesis of TP and Conjugation with TMR
2.2 Live-Cell Imaging
2.3 Cell Viability Assay (WST Assay)
3 Methods
3.1 Synthesis of Tau-Derived Peptide (TP)
3.1.1 Fmoc-Solid Phase Synthesis of TP
3.1.2 Deprotection and Cleavage from Resin
3.1.3 Purification of the Peptide
3.2 Synthesis of TMR-Labeled TP (TP-TMR)
3.2.1 Conjugation of TMR-5-Maleimide to TP
3.2.2 Purification of TP-TMR
3.3 Live-Cell Imaging of MTs
3.3.1 Cell Culture and Passage
3.3.2 Imaging of MTs in Living Cells Using TP-TMR
3.4 Cell Viability Assay (WST Assay)
4 Notes
References
Chapter 16: Discovery of the Environment-Sensitive Near-Infrared (NIR) Fluorogenic Ligand for Ξ±1-Adrenergic Receptors Imaging ...
1 Introduction
2 Materials
2.1 Instrumentation
2.2 Reagents and Labware for FL Intensity Measurement and FL Imaging
3 Methods
3.1 Synthesis of Azide-Derived Quinolines 1a-c
3.1.1 Synthesis of Compound 9
3.1.2 Synthesis of Compounds 10a-c
3.1.3 Synthesis of Compounds 1a-c
3.2 Synthesis of Azide-Derived Quinolines 1d-f
3.2.1 Synthesis of 12a-c
3.2.2 Synthesis of Compounds 13a-c
3.2.3 Synthesis of Compounds 1d-f
3.3 Synthesis of Alkyne-Derived Cy5 (2)
3.3.1 Synthesis of Compound 5
3.3.2 Synthesis of Compound 6
3.3.3 Synthesis of Compound 7
3.3.4 Synthesis of Fluorescent Ligand 2
3.4 Synthesis of Fluorescent Ligands 3a-f
3.5 Specific FL Response to Ξ±1-AR Proteins or Cells Expressing Ξ±1-AR In Vitro
3.6 FL Imaging on Living Cells
3.7 FL Imaging on Tissue Sections
3.8 FL Imaging In Vivo
4 Notes
References
Chapter 17: Rapid and Sensitive Detection of Cancer Cells with Activatable Fluorescent Probes for Enzyme Activity
1 Introduction
2 Materials
2.1 Live-Cell Imaging
2.2 FL Imaging of Cancer in Mouse Models
2.3 Ex Vivo FL Imaging of Cancer in Clinically Resected Tissues
3 Methods
3.1 Live-Cell Confocal Imaging of Aminopeptidase Activity of GGT
3.2 Live-Cell Confocal Imaging of Carboxypeptidase Activity of PSMA
3.3 Ex Vivo FL Imaging of Metastatic Cancer in a Mouse Model
3.4 Ex Vivo FL Imaging of Cancer in Surgically Resected Human Tissues
4 Notes
References
Chapter 18: Detection of Intracellular Reactive Oxidative Species Using the Fluorescent Probe Hydroxyphenyl Fluorescein
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Retrovirus Infection
2.3 Detection of ROS
3 Methods
3.1 Cell Culture
3.2 Retrovirus Infection
3.3 Detection of ROS in Live Cells
3.4 Image Analysis
4 Notes
References
Chapter 19: Development of Near-Infrared Fluorescent Mg2+ Probe and Application to Multicolor Imaging of Intracellular Signals
1 Introduction
2 Materials
2.1 Synthesis of KMG-500 Series
2.2 Metal Salts and pH-Buffer Components for KMG-500 Series Response Characterization
2.3 Cell Culture and Staining
2.4 Fluorescent Probes for Simultaneous Use with KMG-501
2.5 Instrumentation
3 Methods
3.1 Synthesis of KMG-500 Series (See Fig. 1)
3.1.1 General Procedure for Synthesis
3.1.2 Synthesis of Compound 1
3.1.3 Synthesis of Compound 2 and 2b
3.1.4 Synthesis of Compound 1a and 2a
3.1.5 Synthesis of Compound 1b
3.1.6 Synthesis of Compound 4a and b
3.1.7 Synthesis of KMG-501 (See Note 5)
3.2 Evaluation of Molecular Properties of the KMG-500 Series
3.2.1 Molar Extinction Coefficients of KMG-500 Series
3.2.2 FL Quantum Yields (phi) of KMG-500 Series
3.2.3 Mg2+ Concentration-Dependent FL-Emission Spectra of KMG-500 Series
3.2.4 The Metal Ion Selectivity of KMG-500 Series
3.2.5 The pH Dependence of KMG-500 Series
3.2.6 Cell Membrane Permeability of KMG-500 Series
3.3 Measurements of Mg2+ Signals in Living Cells Using KMG-501
3.3.1 Evaluation of Intracellular Localization of KMG-501
3.3.2 Time-Lapse Measurement of Intracellular Mg2+ Concentration Change Using KMG-501
3.3.3 Multicolor Imaging of ATP, Mitochondrial Membrane Potential, and Mg2+
4 Notes
References
Chapter 20: Long-Term Mg2+ Imaging in Live Cells with a Targetable Fluorescent Probe
1 Introduction
2 Materials
2.1 Reagents
2.2 Cell Media
2.3 Fluorescent Probes
2.4 Instrumentation
3 Methods
3.1 Preparation of Cell Sample
3.2 Long-Term Live-Cell Imaging (See Note 5) (See Fig. 2)
3.3 Analysis of Imaging Data
4 Notes
References
Part V: Imaging with BRET Probes
Chapter 21: Highly Bright NIR-BRET System for Imaging Molecular Events in Live Cells
1 Introduction
2 Materials
2.1 Reagents and Lab Equipment
2.2 Instrumentations
2.3 Software
2.4 Animals
3 Methods
3.1 Construction of Three Different Mammalian Expression Plasmids Encoding RLuc8.6-535SG, iRFP-RLuc8.6-535SG, or iRFP-ER-RLuc8...
3.2 Bioluminescence Spectra Measurement of CTZ Derivatives with RLuc8.6-535SG (See Fig. 1d, e)
3.3 Bioluminescence Imaging of Living Single Mammalian Cells Using Microscope CCD-Camera (See Fig. 1f)
3.4 Characterization of the Optimal BRET Spectra of CTZ Derivatives (See Fig. 2a, b)
3.5 Characterization of the BRET Efficiency of iRFP-RLuc8.6-535SG (d0-d8) According to the Flexible Linker Lengths (See Fig. 2...
3.6 Establishment of a Mammalian Cell Line Stably Expressing the Probe
3.7 Ligand-Driven BL Intensities of MDA-MB-231 Cells Encoding iRFP-RLuc8.6-535SG or iRFP-ER-RLuc8.6-535SG (See Fig. 2d)
3.8 Dose-Dependent BL Intensities of the MDA-MB231 Cells Encoding iRFP-ER-RLuc8.6-535SG (See Fig. 2e)
3.9 Quantitative Determination of the SubstrateΒ΄s Sensitivity Based on Cell Numbers (See Fig. 3a)
3.10 Evaluation of Lung-Trapped Tumor Cells Expressing iRFP-RLuc8.6-535SG by NIR-BRET Imaging in Living Mice (See Fig. 3b)
3.11 Bioluminescence Imaging of Tumor Xenografts Expressing iRFP-RLuc8.6SG in Mouse (See Fig. 3c)
3.12 Ex Vivo Imaging of Tumor Xenografts Expressing iRFP-RLuc8.6SG (See Fig. 3d)
4 Notes
References
Chapter 22: Ligand-Activatable BRET9 Probes for Imaging Molecular Events in Living Mammalian Cells
1 Introduction
2 Materials
2.1 Reagents and Lab Supplies
2.2 Instrumentations
2.3 Software
3 Methods
3.1 Construction of a Series of Plasmid Vectors Encoding BRET9 Probes
3.2 Characterization of BRET9 Series Probes in the Presence or Absence of Rapamycin (See Fig. 2a)
3.3 Determination of the Bioluminescence Spectra of v2_mChe and v2_mPlum in the Presence of Rapamycin (See Fig. 2b)
3.4 Establishment of MDA-MB-231 Cells Stably Expressing the Control and Acting Probes
3.5 Determination of Rapamycin Dose-Response Curves Using MDA-MB-231 Cells Stably Expressing v2_mChe Clones #1 and #2 (See Fig...
3.6 In Vivo BL Imaging of Rapamycin Recognition of v2_mChe in Living Mice
4 Notes
References
Chapter 23: Manipulation of Actin Cytoskeleton by Intracellular-Targeted ROS Generation
1 Introduction
2 Materials
2.1 Cell Culture, Transfection, and Luciferin Treatment
2.2 Spectrum Measurement
2.3 Cytochemical Staining and Image Acquisition
3 Methods
3.1 Cell Culture, Transfection, and Luciferin Treatment
3.2 Spectrum Measurement
3.3 Cytochemical Staining and Image Acquisition
4 Notes
References
Chapter 24: Bioluminescence Imaging of Neuronal Network Dynamics Using Aequorin-Based Calcium Sensors
1 Introduction
2 Materials
2.1 Expression Vector and Animals
2.2 Materials for the Preparation of Acute Slices
2.3 Materials for Overnight Transduction of Acute Brain Slices with Sin-GA
2.4 Instrumentation
3 Methods
3.1 Preparation of Acute Slices from Neocortex and Transduction with Sin-GA
3.2 Bioluminescence Imaging
3.3 Data Acquisition and Handling
3.4 Data Analysis
4 Notes
References
Chapter 25: Method for Detecting Emission Spectral Change of Bioluminescent Ratiometric Indicators by a Smartphone
1 Introduction
2 Materials
2.1 DNA Plasmids
2.2 Purified Proteins from E. coli
2.3 Capturing BL Images with a Smartphone
3 Methods
3.1 Preparation of Reaction Buffers for Ca2+ Titration
3.2 Setting of Smartphone
3.3 Setting of Samples with Indicators and Taking BL Image
3.4 Color Ratio Analysis
4 Notes
References
Chapter 26: Bioluminescence Resonance Energy Transfer (BRET) Imaging in Living Cells: Image Acquisition and Quantification
1 Introduction
2 Materials
2.1 Microscope System
2.2 Cell Culture
2.3 Image Acquisition
3 Methods
3.1 Cell Culture
3.2 Camera Setup
3.3 BRET Measurement
3.4 Image Analysis
4 Notes
References
Part VI: Imaging with FRET Probes
Chapter 27: GPCR Signaling Regulation in Dictyostelium Chemotaxis
1 Introduction
2 Materials
2.1 Cell Culture, Media, Buffer, and Solutions
2.2 Small-Population Assay
2.3 Micropipette Assay
2.4 Confocal Microscope
2.5 Live Imaging and Translocation Assay of Heterotrimeric G Proteins
2.6 Fractionation Assay
2.7 Immunoprecipitation (Pull-Down) Assay
2.8 FRET Assay
2.9 Immunoblotting
3 Methods
3.1 Preparation of Chemotactically Competent Cells
3.1.1 Starvation of D. discoideum Cells Without External cAMP Pulsing
3.1.2 Starvation of D. discoideum Cells with External cAMP Pulsing
3.2 Detection of the Phosphorylation of cAR1 Receptor and GΞ±2 upon LigandΒ΄s Stimulation
3.3 Immunoblotting
3.4 Small-Population Assay
3.5 Micropipette Assay
3.5.1 Observation of Chemotactic Cells
3.5.2 Quantitative Analysis of the Chemotactic Responses
3.6 Live Imaging of Heterotrimeric G Proteins
3.7 Fractionation Assay
3.8 Coimmunoprecipitation Assay Between Heterotrimeric G Protein and Gip1
3.9 Gip1-Mediated G Protein Translocation
3.9.1 Observation of Gip1-Mediated G Protein Translocation
3.9.2 Quantification of the G Protein Translocation Responses
3.10 FRET Assay
3.10.1 FRET Changes Between GΞ±2 and GΞ² upon cAMP Stimulation
3.10.2 cAMP-Dose-Dependent FRET Changes Between GΞ±2 and GΞ²
3.11 Gradient Sensing Assay by PHAKT-GFP Measurements
4 Notes
References
Chapter 28: Live-Cell Imaging Technique to Visualize DAMPs Release During Regulated Cell Death
1 Introduction
2 Materials
2.1 Equipment
2.2 Optics
2.3 Reagents and Materials
2.4 Cells
2.5 Analysis Software
3 Methods
3.1 Assembly of the Microfabricated Well Array (MWA) Chip
3.2 Modification of the Glass Base Surface of the MWA Chip
3.3 Preparation of Detection Solution
3.4 Cell Preparation
3.5 LCI-S Calibration
3.6 Time-Lapse Scanning with LCI-S
3.7 Fluorescence Intensity Measurement Using the NIS-Elements Software
3.8 Estimation of the Disappearance or Appearance Timing of Each Signal
3.9 Quantitative Characterization of Secretion Dynamics in Two Modes
4 Notes
References
Chapter 29: Time-Lapse Imaging of Necroptosis and DAMP Release at Single-Cell Resolution
1 Introduction
2 Materials
2.1 FRET Analysis
2.2 Imaging
2.3 Analysis Software
3 Methods
3.1 Cells that Stably Express a FRET Biosensor
3.2 Data Collection for FRET Analysis of Necroptosis
3.3 Data Analysis of FRET for Necroptosis
3.4 Pseudocolor Montages of the FRET/CFP Ratio
3.5 Data Collection for Analyzing the DAMP Release Mode
3.6 Data Analysis for Determining the DAMP Release Mode
4 Notes
References
Part VII: Imaging with Advanced Instrumentation
Chapter 30: High-Throughput Whole-Plate Imaging of Cells for Multiple Biological Applications
1 Introduction
2 Materials
2.1 Cell Proliferation Analysis by Simple Bright-Field Imaging
2.1.1 Reagents and Lab Equipment Required
2.1.2 Instrumentations
2.1.3 Software
2.2 Live and Dead Cell Analysis by Multicolor FL Imaging
2.2.1 Materials
2.2.2 Instruments and Software
2.3 Multicolor FL Imaging of Intact Cells for Reporter Gene Expression Analysis
2.3.1 Materials
2.3.2 Instruments and Software
2.4 Imaging of Intact 3D Spheroids Using Multicolor Imaging (See Note 11)
2.4.1 Materials
2.4.2 Instruments and Software
2.5 Multicolor Imaging of Extracellular (EV)-Mediated MicroRNA Transfection in 3D Spheroids [6]
2.5.1 Materials and Reagents
2.5.2 Instruments and Software
3 Methods
3.1 Cell Proliferation Analysis by Simple Bright-Field Imaging (Fig. 1)
3.2 Live and Dead Cell Analysis by Multicolor FL Imaging (Fig. 2)
3.3 Multicolor FL Imaging of Intact Cells for Reporter Gene Expression Analysis
3.4 Imaging of Intact 3D Spheroids Using Multicolor Imaging
3.4.1 Preparation of Cell Culture Plate and Matrigel Media
3.4.2 Preparation of Single-Cell Suspension
3.4.3 Preparation of 3D Spheroid
3.5 Multicolor Imaging of Extracellular (EV)-Mediated MicroRNA Transfection in 3D Spheroids (Fig. 5)
4 Notes
References
Chapter 31: Pinhole Closure Improves Spatial Resolution in Confocal Scanning Microscopy
1 Introduction
2 Materials
2.1 Preparation of Cells Expressing Fluorescent-Labeled Proteins
2.2 Confocal Microscopy
2.3 Image Processing Software
3 Methods
3.1 Cell Culture and FP-Tagging Protein Expression
3.2 Image Acquisition Using a Confocal Scanning Microscope and an APD
3.3 Image Processing
4 Notes
References
Chapter 32: Workflows of the Single-Molecule Imaging Analysis in Living Cells: Tutorial Guidance to the Measurement of the Dru...
1 Introduction
2 Materials
2.1 Plasmid DNA (pDNA) Vector Encoding HaloTag/SNAP-Tag/GFP-Fusion
2.2 Cells
2.3 Medium, Buffer, and Reagents
2.4 FL Dyes (See Note 3)
2.5 Equipment for TIRFM (See Fig. 1)
3 Methods
3.1 Preparation of Coverslips for the SMI in Living Cells (See Note 7)
3.2 Transfection by Lipofectamine 3000 (See Note 8)
3.3 HaloTag/SNAP-tag Staining (See Note 11)
3.4 Single-Molecule Imaging (SMI)
3.5 Image Processing by ImageJ
3.6 Additional Image Processing by ImageJ for the Dual-Color Analysis
3.7 Single-Molecule Tracking (SMT) Analysis by AAS (See Note 19)
3.8 Analysis of the Single-Molecule Dynamics of GPCRs by Using smDynamicsAnalyzer
3.8.1 Installation of Igor and Building a Folder Structure to Use smDynamicsAnalyzer (See Note 20)
3.8.2 Respective Analysis of the AAS and G-Count Data Formats: Total Analysis of the Data in a Folder (See Fig. 4a)
3.8.3 Load: Load Input Data (See Fig. 4b)
3.8.4 Trace: Output XY Plots of Trajectories (See Fig. 4c)
3.8.5 MSD-dt: Output MSD-Deltat Plots (See Fig. 4d)
3.8.6 Hist D: Output Displacement Histograms (See Fig. 4e)
3.8.7 Intensity: Output Intensity Histograms (See Fig. 4f)
3.8.8 Density: Output Density Analysis Results (See Figs. 4g and 5)
3.8.9 Off-Rate: Output Decay Curves of the Duration of Trajectories (See Figs. 4h and 6)
3.8.10 On-Rate: Output Cumulative Event Number Plots of the Starting Time of Trajectories (See Figs. 4i and 6)
3.8.11 Stats: Calculate Statistics of all the Parameters Analyzed
3.8.12 Total Basic Analysis of the AAS and G-Count Data Formats (See Fig. 7)
3.8.13 Comparison Analysis of the AAS and G-Count Data Formats: Total Comparison Analysis and Multiple Comparison Tests (See F...
3.8.14 MSD-Deltat, D(MSD-Deltat), L(MSD-Deltat): Parameter Comparison of the MSD-Deltat plot analysis (See Fig. 8a-c)
3.8.15 Dstate and Dratio: Parameter Comparison of the Displacement Histogram Analysis (See Fig. 8d-k)
3.8.16 Intensity: Output the Intensity Analysis Results (See Fig. 8l-n)
3.8.17 Off-Rate: Output Mean-Decay Curves of the Duration of Trajectories and Violin/Box Plots of the Off-Rate Parameters (See...
3.8.18 On-Rate: Output Violin/Box Plot of the On-Rate Parameters (See Fig. 8t)
3.8.19 Density: Output Violin/Box Plot of the Density Analysis Parameters (See Fig. 8u)
3.8.20 2D Plots: Output 2D Plots of the Diffusion, Intensity, and Density Parameters of the Single Cell (See Fig. 8v)
3.8.21 Total Basic Analysis of the HMM Data Format (See Fig. 9)
3.8.22 Trace: Output XY Plots of the Trajectories (See Fig. 9b)
3.8.23 Intensity: Output Intensity Histograms (See Fig. 9c)
3.8.24 MSD-Deltat: Output MSD-Deltat Plots (See Fig. 9d)
3.8.25 Hist D: Output Displacement Histograms (See Fig. 9e)
3.8.26 Comparison Analysis of the HMM Data Formats: Total Comparison Analysis and Multiple Comparison Tests Are Carried Out (S...
3.8.27 MSD-Deltat: Parameter Comparison of the MSD-Deltat Plot Analysis (See Fig. 10a-d)
3.8.28 Dstate and Dratio: Parameter Comparison of the VB-HMM Analysis (See Fig. 10e-l)
3.8.29 Intensity: Output the Intensity Analysis Results (See Fig. 10m-p)
3.8.30 Colocalization Analysis of the HMM Data Formats (See Fig. 11)
3.8.31 Find Col.: Find Colocalization Trajectories from HMM Data (See Fig. 11a, b)
3.8.32 Hist D: Output Histograms of Displacement Between Frames (See Fig. 11c)
3.8.33 Param. D Ratio: Output the Category Plot of the Diffusion State Ratio (See Fig. 11d)
3.8.34 On-Rate: Output Cumulative Event Number Plots of the Starting Time of the Colocalization (See Fig. 11e)
3.8.35 Off-Rate: Output the Decay Curves of the Colocalization Duration (See Fig. 11f)
3.8.36 Param. Off-Rate: Output the Category Plots of the Parameters of the Off-Rate Analysis (See Note 56)
3.8.37 Intensity: Output the Intensity Histograms at the Colocalization Frames (See Note 56)
3.8.38 Compare Parameters of the Colocalization Analysis (See Fig. 12)
4 Notes
References
Index