Optical Imaging Techniques in Cell Biology

Since the word microscopy was coined in 1656, the evolution of the instrument has had a long and convoluted history. Plagued with problems of chromatic aberration, spherical aberration, and challenges with illumination and resolution, the microscope’s technical progression happened in a series of fits and starts until the late 19th century. After Ernst Abbe perfected the “how” of lens design, achieving the theoretical limit imposed by wavelength, there came a revolution in subject matter or “what” could be studied by microscope. Covering the entire field of microscopy, Optical Imaging Techniques in Cell Biology provides an overview of the technical evolution of the microscope and explains how the basics of optical microscopy led to the most advanced techniques employed today. The author addresses a vast array of topics including optical contrasting techniques, fluorescence, confocal versus widefield microscopes, lasers as a light source, and digital imaging, as well as the correction of aberrations that might arise. Building on this foundation, he then examines more advanced techniques such as quantitative fluorescence, fluorescence resonant energy, three-dimensional imaging, high-speed confocal microscopy, non-linear microscopy, and stimulated emission depletion. Delivering a truly comprehensive work encompassing the scope and breadth of the field, the author brings a new level of understanding to the student, technician, researcher, or investigator working in the fascinating realm of optical microscopy.

Table of Contents:
Introduction: – The Optical Microscope In Cell Biology Brief historical overview from Robert Hooke (cells, 1685) through Swammerdam, van Leeuwenhoek (sperm, bacteria, blood cells), Robert Browne (nucleus), Schwann & Schleiden (cell theory), Lister (corrected objectives), Abbe, etc. The Light Microscope Lenses and Microscopes The Back Focal Plane of a Lens Good Resolution Resolution: Rayleigh’s approach Diffraction: Abbe’s approach Add a Drop of Oil … Köhler Illumination Optical Contrasting Techniques Darkfield Phase Contrast Polarization Differential Interference Contrast Which Technique Is Best? Fluorescence and Fluorescence Microscopy What Is Fluorescence? What Makes a Molecule Fluorescent? The Fluorescence Microscope Optical Arrangement Light Source Filter Sets Image Capture Optical Layout for Image Capture Film Monochrome Color Films Additive Color Model Subtractive Color Model CCD Cameras Frame-Transfer Array Interline-Transfer Array Back Illumination Binning Recording Color Filter Wheels Filter Mosaics Three CCD Elements with Dichroic Beamsplitters The Confocal Microscope The Scanning Optical Microscope The Confocal Principle Resolution and Point Spread Function Lateral Resolution in the Confocal Microscope Practical Confocal Microscopes The Light Source: Lasers Gas Lasers Solid-State Lasers Semiconductor Lasers Laser Delivery The Primary Beamsplitter Beam Scanning Pinhole and Signal Channel Configurations Detectors The Digital Image Pixels and Voxels Contrast Spatial Sampling: The Nyquist Criterion Temporal Sampling: Signal-to-Noise Ratio Multichannel Images Aberrations and Their Consequences Geometrical Aberrations Spherical Aberration (SA) Coma Astigmatism Field Curvature Chromatic Aberration Chromatic Difference of Magnification Practical Consequences Apparent Depth Nonlinear Microscopy Multiphoton Microscopy Principles of Two-Photon Fluorescence Theory and Practice Lasers for Nonlinear Microscopy Advantages of Two-Photon Excitation Construction of a Multiphoton Microscope Fluorochromes for Multiphoton Microscopy Second Harmonic Microscopy High-Speed Confocal Microscopy Tandem-Scanning (Spinning Disk) Microscopes Petràn System One-Sided Nipkow Disk Microscopes (OTSMs) Microlens Array: The Yokogawa System Slit-Scanning Microscopes Slit–Slit Systems Spot–Slit Systems Multipoint-Array Scanners Structured Illumination Deconvolution and Image Processing Deconvolution Deconvolving Confocal Images Image Processing Gray-Scale Operations Image Arithmetic Convolution: Smoothing and Sharpening Three-Dimensional Imaging: Stereoscopy and Reconstruction Surfaces: Two-and-a-Half Dimensions Perception of the 3D World Limitations of Confocal Microscopy Stereoscopy Three-Dimensional Reconstruction Techniques that Require Identification of "Objects" Techniques that Create Views Directly from Intensity Data Simple projections Weighted projection (alpha blending) Green Fluorescent Protein Structure and Properties of GFP GFP Variants Applications of GFP Heat Shock Cationic Lipid Reagents DEAE–Dextran and Polybrene Calcium Phosphate Coprecipitation Electroporation Microinjection Gene Gun Plants: Agrobacterium Fluorescent Staining Immunolabeling Types of Antibody Raising Antibodies Labeling Fluorescent Stains for Cell Components and Compartments Quantitative Fluorescence Fluorescence Intensity Measurements Linearity Calibration Measurement Colocalization Ratio Imaging Cell Loading Membrane Potential Slow-Response Dyes Fluorescence Recovery after Photobleaching Advanced Fluorescence Techniques: FLIM and FRET Fluorescence Lifetime Practical Lifetime Microscopy Frequency Domain Time Domain Fluorescence Resonant Energy Transfer (FRET) Why Use FRET? Identifying and Quantifying FRET Increase in brightness of acceptor emission Quenching of emission from the donor Lifetime of donor emission Protection from bleaching of donor Fluorescence Correlation Spectroscopy (FCS) Breaking the Diffraction Limit Near-Field Microscopy Total Internal Reflection Microscopy (TIRF) 4-p and Multiple-Objective Microscopy Stimulated Emission Depletion (STED) Appendix 1 Microscope Care and Maintenance Cleaning The Fluorescent Illuminator Appendix 2 Keeping Cells Alive under the Microscope Chambers Light Movement Finally Appendix 3 Antibody Labeling of Plant and Animal Cells: Tips and Sample Schedules Antibodies: Tips on Handling and Storage Pipettes: Tips on Handling Antibodies and Antibody Titrations Example Primary antibody titration Immunofluorescence Protocol Method Multiple Labeling and Different Samples Plant Material Protocol Diagram showing position of antibodies on multiwell slide: Appendix 4 Image Processing with ImageJ Introduction Different Windows in ImageJ Image Levels Colors and Look-up Tables Size Calibration Image Math Quantification Stacks and 3D Representation FFT and Image Processing Macro Language in ImageJ