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Fluorescence Imaging principles and methods FLUORESCENCE IMAGING
Contents
Chapter 1: Introduction to fluorescence...... 1 Advantages of fluorescent detection ...... 1 Fluorescence process...... 2 Properties of fluorochromes...... 3 Excitation and emission spectra...... 3 Signal linearity ...... 5 Brightness ...... 5 Susceptibility to environmental effects ...... 6 Quantification of fluorescence ...... 7
Chapter 2: Fluorescence imaging systems ...... 9 Introduction...... 9 Excitation sources and light delivery optics ...... 10 Light collection optics...... 10 Filtration of the emitted light...... 10 Detection, amplification, and digitization ...... 11 Scanner systems ...... 12 Excitation sources...... 12 Excitation light delivery...... 13 Light collection ...... 15 Signal detection and amplification ...... 17 System performance...... 18 CCD camera-based systems ...... 19 Excitation sources and light delivery...... 20 Light collection ...... 20 Signal detection and amplification ...... 20 System performance...... 20 Amersham Pharmacia Biotech imaging systems...... 21
Chapter 3: Fluorochrome and filter selection ...... 25 Introduction...... 25 Types of emission filters...... 25 Using emission filters to improve sensitivity and linearity range...... 26 General guidelines for selecting fluorochromes and filters ...... 27 Single-colour imaging ...... 27 Multicolour imaging ...... 28
Chapter 4: Image analysis ...... 31 Introduction...... 31 Image display...... 31 Image documentation ...... 32