Optical Imaging Techniques in Cell Biology

Optical Imaging Techniques in Cell Biology, Second Edition covers the field of biological microscopy, from the optics of the microscope to the latest advances in imaging below the traditional resolution limit. It includes the techniques—such as labeling by immunofluorescence and fluorescent proteins—which have revolutionized cell biology. Quantitative techniques such as lifetime imaging, ratiometric measurement, and photoconversion are all covered in detail.

Expanded with a new chapter and 40 new figures, the second edition has been updated to cover the latest developments in optical imaging techniques. Explanations throughout are accurate, detailed, but as far as possible non-mathematical. This edition includes appendices with useful practical protocols, references, and suggestions for further reading. Color figures are integrated throughout.

Guy Cox is a professor within the Electron Microscopy Unit at the University of Sydney, Australia.

The Light MicroscopeLenses and MicroscopesThe Back Focal Plane of a LensGood ResolutionResolution: Rayleigh’s ApproachAbbeAdd a Drop of OilKöhler IlluminationOptical Contrasting TechniquesDarkfieldPhase ContrastPolarizationDifferential Interference ContrastHoffman Modulation ContrastWhich Technique Is Best?Fluorescence and Fluorescence MicroscopyWhat Is Fluorescence?What Makes a Molecule Fluorescent?The Fluorescence MicroscopeOptical ArrangementLight SourceFilter Sets: Excitation Filter, Dichroic Mirror, and BarrierFilterImage CaptureOptical Layout for Image CaptureColor RecordingAdditive Color ModelSubtractive Color ModelCCD CamerasFrame-Transfer ArrayInterline-Transfer ArrayBack IlluminationBinningRecording ColorFilter WheelsFilter MosaicsThree CCD Elements with Dichroic BeamsplittersBoosting the SignalThe Confocal MicroscopeThe Scanning Optical MicroscopeThe Confocal PrincipleResolution and Point Spread FunctionLateral Resolution in the Confocal MicroscopePractical Confocal MicroscopesThe Light Source: LasersGas LasersSolid-StateLasersSemiconductor LasersSupercontinuum LasersLaser DeliveryThe Primary BeamsplitterBeam ScanningPinhole and Signal Channel ConfigurationsDetectorsThe Digital ImagePixels and VoxelsContrastSpatial Sampling: The Nyquist CriterionTemporal Sampling: Signal-to-Noise RatioMultichannel ImagesAberrations and Their ConsequencesGeometrical AberrationsSpherical AberrationComaAstigmatismField CurvatureChromatic AberrationChromatic Difference of MagnificationPractical ConsequencesApparent DepthNonlinear MicroscopyMultiphoton MicroscopyPrinciples of Two-Photon FluorescenceTheory and PracticeLasers for Nonlinear MicroscopyAdvantages of Two-Photon ExcitationConstruction of a Multiphoton MicroscopeFluorochromes for Multiphoton MicroscopySecond Harmonic MicroscopySummaryHigh-Speed Confocal MicroscopyTandem Scanning (Spinning Disk) MicroscopesPetràn SystemOne-Sided Tandem Scanning Microscopes (OTSMS)Microlens Array: The Yokogawa SystemSlit-Scanning MicroscopesMultipoint-Array ScannersStructured IlluminationDeconvolution and Image ProcessingDeconvolutionDeconvolving Confocal ImagesImage ProcessingGrayscale OperationsImage ArithmeticConvolution: Smoothing And SharpeningThree-Dimensional Imaging: Stereoscopy and ReconstructionSurfaces: Two-And-A-Half DimensionsPerception of the 3D WorldMotion ParallaxConvergence and Focus of Our EyesPerspectiveConcealment of One Object by AnotherOur Knowledge of the Size and Shape of Everyday ThingsLight and ShadeLimitations of Confocal MicroscopyStereoscopyThree-Dimensional ReconstructionTechniques That Require Identification of "Objects"Techniques That Create Views Directly from Intensity DataSimple ProjectionsWeighted Projection (Alpha Blending)Green Fluorescent ProteinStructure and Properties of GFPGFP VariantsApplications of GFPHeat ShockCationic Lipid ReagentsDEAE–Dextran And PolybreneCalcium Phosphate CoprecipitationElectroporationMicroinjectionGene GunPlants: AgrobacteriumFluorescent Staining, Teresa Dibbayawan, Eleanor Kable, and Guy CoxImmunolabelingTypes of AntibodyRaising AntibodiesLabelingFluorescent Stains for Cell Components and CompartmentsQuantitative FluorescenceFluorescence Intensity MeasurementsLinearity CalibrationMeasurementColocalizationRatio ImagingCell LoadingMembrane PotentialFast-Response DyesSlow-Response DyesFluorescence Recovery after PhotobleachingAdvanced Fluorescence Techniques: FLIM, FRET, and FCSFluorescence LifetimePractical Lifetime MicroscopyFrequency DomainTime DomainFluorescence Resonant Energy Transfer (FRET)Why Use FRET?Identifying And Quantifying FretIncrease in Brightness of Acceptor EmissionQuenching of Emission from the DonorLifetime of Donor EmissionProtection from Bleaching of DonorFluorescence Correlation Spectroscopy (FCS)Raster Image Correlation SpectroscopyEvanescent Wave MicroscopyThe Near-Field and Evanescent WavesTotal Internal Reflection MicroscopyNear-Field MicroscopyBeyond the Diffraction Limit4Pi and Multiple-Objective MicroscopyStimulated Emission Depletion (STED)Structured IlluminationStochastic TechniquesSuper-Resolution SummaryAppendix A: Microscope Care and MaintenanceCleaningThe Fluorescent IlluminatorAppendix B: Keeping Cells Alive under the Microscope, Eleanor Kable and Guy CoxChambersLightMovementFinallyAppendix C: Antibody Labeling of Plant and Animal Cells: Tips and Sample Schedules, Eleanor Kable and Teresa DibbayawanAntibodies: Tips on Handling and StoragePipettes: Tips on HandlingAntibodies and Antibody TitrationsExampleImmunofluorescence ProtocolMethodMultiple Labeling and Different SamplesPlant MaterialProtocolDiagram Showing Position of Antibodies on Multiwell SlideAppendix D: Image Processing with ImageJ, Nuno MorenoIntroductionDifferent Windows in ImageJImage LevelsColors and Look-UpTablesSize CalibrationImage MathQuantificationStacks and 3D RepresentationFFT and Image ProcessingMacro Language in ImageJIndex