This third edition of a classic text in biological microscopy includes detailed descriptions and in-depth comparisons of parts of the microscope itself, digital aspects of data acquisition and properties of fluorescent dyes, the techniques of 3D specimen preparation and the fundamental limitations, and practical complexities of quantitative confocal fluorescence imaging. Coverage includes practical multiphoton, photodamage and phototoxicity, 3D FRET, 3D microscopy correlated with micro-MNR, CARS, second and third harmonic signals, ion imaging in 3D, scanning RAMAN, plant specimens, practical 3D microscopy and correlated optical tomography.

Foundations of Confocal Scanned Imaging in Light Microscopy.- Fundamental Limits in Confocal Microscopy.- Special Optical Elements.- Points, Pixels, and Gray Levels: Digitizing Image Data.- Laser Sources for Confocal Microscopy.- Non-Laser Light Sources for Three-Dimensional Microscopy.- Objective Lenses for Confocal Microscopy.- The Contrast Formation in Optical Microscopy.- The Intermediate Optical System of Laser-Scanning Confocal Microscopes.- Disk-Scanning Confocal Microscopy.- Measuring the Real Point Spread Function of High Numerical Aperture Microscope Objective Lenses.- Photon Detectors for Confocal Microscopy.- Structured Illumination Methods.- Visualization Systems for Multi-Dimensional Microscopy Images.- Automated Three-Dimensional Image Analysis Methods for Confocal Microscopy.- Fluorophores for Confocal Microscopy: Photophysics and Photochemistry.- Practical Considerations in the Selection and Application of Fluorescent Probes.- Guiding Principles of Specimen Preservation for Confocal Fluorescence Microscopy.- Confocal Microscopy of Living Cells.- Aberrations in Confocal and Multi-Photon Fluorescence Microscopy Induced by Refractive Index Mismatch.- Interaction of Light with Botanical Specimens.- Signal-to-Noise Ratio in Confocal Microscopes.- Comparison of Widefield/Deconvolution and Confocal Microscopy for Three-Dimensional Imaging.- Blind Deconvolution.- Image Enhancement by Deconvolution.- Fiber-Optics in Scanning Optical Microscopy.- Fluorescence Lifetime Imaging in Scanning Microscopy.- Multi-Photon Molecular Excitation in Laser-Scanning Microscopy.- Multifocal Multi-Photon Microscopy.- 4Pi Microscopy.- Nanoscale Resolution with Focused Light: Stimulated Emission Depletion and Other Reversible Saturable Optical Fluorescence Transitions Microscopy Concepts.- Mass Storage, Display, and Hard Copy.- Coherent Anti-Stokes Raman Scattering Microscopy.- Related Methods for Three-Dimensional Imaging.- Tutorial on Practical Confocal Microscopy and Use of the Confocal Test Specimen.- Practical Confocal Microscopy.- Selective Plane Illumination Microscopy.- Cell Damage During Multi-Photon Microscopy.- Photobleaching.- Nonlinear (Harmonic Generation) Optical Microscopy.- Imaging Brain Slices.- Fluorescent Ion Measurement.- Confocal and Multi-Photon Imaging of Living Embryos.- Imaging Plant Cells.- Practical Fluorescence Resonance Energy Transfer or Molecular Nanobioscopy of Living Cells.- Automated Confocal Imaging and High-Content Screening for Cytomics.- Automated Interpretation of Subcellular Location Patterns from Three-Dimensional Confocal Microscopy.- Display and Presentation Software.- When Light Microscope Resolution Is Not Enough:Correlational Light Microscopy and Electron Microscopy.- Databases for Two- and Three-Dimensional Microscopical Images in Biology.- Confocal Microscopy of Biofilms - Spatiotemporal Approaches.- Bibliography of Confocal Microscopy.