Methods in Cellular Imaging

Advances in technology have revolutionized the development of light microscopy techniques in biomedical research, thus improving visualization of the microstructure of cells and tissues under physiological conditions. Fluorescence microscopy methods are non-contact and non-invasive and provide high spatial and temporal resolution that other laboratory techniques cannot. This well-illustrated book targets graduate students and scientists who are new to the state-of-the-art fluorescence microscopy techniques used in biological and clinical imaging. It explains basic concepts and imaging procedures for wide-field, confocal, multiphoton excitation, fluorescence resonance energy transfer (FRET), lifetime imaging (FLIM), spectral imaging, fluorescence recovery after photobleaching (FRAP), optical tweezers, total internal reflection, high spatial resolution atomic force microscopy (AFM), and bioluminescence imaging for gene expression. The usage of these techniques in various biological applications, including calcium, pH, membrane potential, mitochondrial signaling, protein-protein interactions under various physiological conditions, and deep tissue imaging, is clearly presented. The authors describe the approaches to selecting epifluorescence microscopy, the detectors, and the image acquisition and processing software for different biological applications. Step-by-step directions on preparing different digital formats for light microscopy images on websites are also provided.

Inhalt

Basics of Fluorescence.- Fluorophores and Their Labeling Procedures for Monitoring Various Biological Signals.- Detectors for Fluorescence Microscopy.- Basics of a Light Microscopy Imaging System and Its Application in Biology.- Laser Scanning Confocal Microscopy Applied to Living Cells and Tissues.- Functional Imaging of Mitochondria Within Cells.- Diffusion Measurements by Fluorescence Recovery After Photobleaching.- Processing Microscope-Acquired Images for Use in Multimedia, Print, and the World Wide Web.- Basic Principles of Multiphoton Excitation Microscopy.- Building a Two-Photon Microscope Using a Laser Scanning Confocal Architecture.- Two-Photon Microscopy in Highly Scattering Tissue.- Multiphoton Laser Scanning Microscopy and Dynamic Imaging in Embryos.- In vivo Diffusion Measurements Using Multiphoton Excitation Fluorescence Photobleaching Recovery and Fluorescence Correlation Spectroscopy.- Cellular Response to Laser Radiation in Fluorescence Microscopes.- Measurement of Fluorescence Resonance Energy Transfer in the Optical Microscope.- Frequency-Domain Fluorescence Lifetime Imaging Microscopy: A Window on the Biochemical Landscape of the Cell.- Wide-Field, Confocal, Two-Photon, and Lifetime Resonance Energy Transfer Imaging Microscopy.- One- and Two-Photon Confocal Fluorescence Lifetime Imaging and Its Applications.- Biological Applications of Time-Resolved, Pump-Probe Fluorescence Microscopy and Spectroscopy in the Frequency Domain.- Spectral Microscopy for Quantitative Cell and Tissue Imaging.- Total Internal Reflection Fluorescence Microscopy.- Laser Traps in Cell Biology and Biophysics.- Bioluminescence Imaging of Gene Expression in Living Cells and Tissues.- Imaging Living Cells and Mapping Their Surface Molecules with the Atomic Force Microscope.