UID:
almafu_9958127522302883
Umfang:
1 online resource (609 p.)
Ausgabe:
First edition.
ISBN:
0-12-420201-2
Serie:
Methods in cell biology, volume 123
Inhalt:
This new volume, number 123, of Methods in Cell Biology looks at methods for quantitative imaging in cell biology. It covers both theoretical and practical aspects of using optical fluorescence microscopy and image analysis techniques for quantitative applications. The introductory chapters cover fundamental concepts and techniques important for obtaining accurate and precise quantitative data from imaging systems. These chapters address how choice of microscope, fluorophores, and digital detector impact the quality of quantitative data, and include step-by-step protocols fo
Anmerkung:
Description based upon print version of record.
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Front Cover; Quantitative Imaging in Cell Biology; Copyright; Contents; Contributors; Preface; Chapter 1: Concepts in quantitative fluorescence microscopy; 1.1. Accurate and Precise Quantitation; 1.2. Signal, Background, and Noise; 1.3. Optical Resolution: The Point Spread Function; 1.4. Choice of Imaging Modality; 1.5. Sampling: Spatial and Temporal; 1.5.1. 2D sampling; 1.5.2. 3D sampling; 1.5.3. Temporal sampling; 1.6. Postacquisition Corrections; 1.6.1. Background subtraction; 1.6.2. Flat-field correction; 1.6.3. Photobleaching; 1.6.4. Storing and processing images for quantitation
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1.7. Making Compromises1.8. Communicating Your Results; Acknowledgment; References; Chapter 2: Practical considerations of objective lenses for application in cell biology; Introduction; 2.1. Optical Aberrations; 2.1.1. On-axis aberrations; 2.1.2. Off-axis aberrations; 2.2. Types of Objective Lenses; 2.2.1. Optical corrections; 2.2.2. Numerical aperture; 2.3. Objective Lens Nomenclature; 2.4. Optical Transmission and Image Intensity; 2.5. Coverslips, Immersion Media, and Induced Aberration; 2.5.1. Optical path length; 2.5.2. Correction collars; 2.5.3. Cover glass; 2.5.4. Immersion media
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2.6. Considerations for Specialized Techniques2.7. Care and Cleaning of Optics; Conclusions; References; Chapter 3: Assessing camera performance for quantitative microscopy; 3.1. Introduction to Digital Cameras for Quantitative Fluorescence Microscopy; 3.2. Camera Parameters; 3.2.1. Quantum efficiency; 3.2.2. Noise; 3.2.3. Poisson noise; 3.2.4. Camera noise; 3.2.5. Fixed-Pattern noise; 3.2.6. Digitization, bit depth, and dynamic range; 3.2.7. Amplification; 3.2.8. sCMOS considerations; 3.3. Testing Camera Performance: The Photon Transfer Curve; 3.3.1. Photon transfer theory
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3.3.2. PTC collection protocolReferences; Chapter 4: A Practical guide to microscope care and maintenance; Introduction; 4.1. Cleaning; 4.1.1. Before cleaning; 4.1.2. Objectives; 4.1.2.1. Proper use of objective lenses; 4.1.2.2. Objective lens inspection and cleaning; 4.1.2.3. Temperature; 4.1.3. Fluorescence filters; 4.1.3.1. Excitation and emission filters; 4.1.3.2. Mirrors; 4.1.4. Camera; 4.1.5. The dust is still there!; 4.2. Maintenance and Testing; 4.2.1. Computer maintenance; 4.2.2. Check the transmitted light pathway; 4.2.3. Measure intensity of fluorescence light sources
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4.2.4. Flatness of fluorescence illumination4.2.5. Color registration; 4.2.6. Vibration; 4.2.7. Measure the point spread function; 4.2.8. Test performance of motorized components and software; 4.3. Considerations for New System Installation; Acknowledgments; References; Chapter 5: Fluorescence live cell imaging; 5.1. Fluorescence Microscopy Basics; 5.2. The Live Cell Imaging Microscope; 5.3. Microscope Environmental Control; 5.3.1. Temperature; 5.3.2. Media composition and pH; 5.3.3. Imaging chambers; 5.4. Fluorescent Proteins; 5.4.1. Protocol for analyzing FP photobleaching
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5.5. Other Fluorescent Probes
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English
Weitere Ausg.:
ISBN 0-12-420138-5
Sprache:
Englisch
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