63-0035-28 Rev.AB, 2002-10 US$105hbFluorescence Imagingprinciples and methodshandbookS1´hνEXhνEMS0EnergyS1● iFLUORESCENCE IMAGINGContentsChapter 1: Introduction to fluorescence...........................................................11.1 Advantages of fluorescence detection.....................................................11.2 Fluorescence process ..............................................................................21.3 Properties of fluorochromes...................................................................31.3.1 Excitation and emission spectra..................................................31.3.2 Signal linearity............................................................................51.3.3 Brightness ...................................................................................51.3.4 Susceptibility to environmental effects........................................61.4 Quantitation of fluorescence..................................................................7Chapter 2: Fluorescence imaging systems .....................................................92.1 Introduction...........................................................................................92.1.1 Excitation sources and light delivery optics ..............................102.1.2 Light collection optics...............................................................102.1.3 Filtration of the emitted light....................................................102.1.4 Detection, amplification, and digitization.................................112.2 Scanner systems....................................................................................122.2.1 Excitation sources.....................................................................122.2.2 Excitation light delivery............................................................132.2.3 Light collection.........................................................................152.2.4 Signal detection and amplification............................................172.2.5 System performance..................................................................182.3 CCD camera-based systems..................................................................192.3.1 Excitation sources and light delivery.........................................202.3.2 Light collection.........................................................................202.3.3 Signal detection and amplification............................................202.3.4 System performance..................................................................202.4 Amersham Biosciences imaging systems...............................................21Chapter 3: Fluorochrome and filter selection ...............................................253.1 Introduction.........................................................................................253.2 Types of emission filters.......................................................................253.3 Using emission filters to improve sensitivity and linearity range ..........263.4 General guidelines for selecting fluorochromes and filters....................273.4.1 Single-color imaging..................................................................273.4.2 Mulicolor imaging ....................................................................28Chapter 4: Image analysis ..............................................................................314.1 Introduction.........................................................................................314.2 Image display .......................................................................................314.3 Image documentation...........................................................................324.4 Quantitation.........................................................................................334.4.1 One-dimensional gel/blot analysis.............................................334.4.2 Array and microplate analysis...................................................354.4.3 Two-dimensional protein gel analysis .......................................364.5 Background correction.........................................................................364.6 Image processing tools .........................................................................404.7 Amersham Biosciences image analysis software ...................................41FLUORESCENCE IMAGINGChapter 5: Fluorescence imaging applications ............................................475.1 Introduction to fluorescence imaging applications ...............................475.1.1 Fluorescent stains......................................................................475.1.2 Covalent fluorescence labelling of nucleic acids and proteins.....................................................................................485.1.3 Using naturally occurring fluorescent proteins..........................495.1.4 Chemifluorescence applications ................................................505.2 Detection of nucleic acids in gels..........................................................515.2.1 Nucleic acid gel stains...............................................................515.2.2 Instrument compatibility...........................................................525.2.3 Typical protocol........................................................................525.2.4 Expected results........................................................................545.3 Quantitation of nucleic acids in solution..............................................565.3.1 Stains for quantitation of nucleic acids in solution ...................565.3.2 Instrument compatibility...........................................................575.3.3 Typical protocol........................................................................575.3.4 Expected results........................................................................595.4 Southern and Northern blotting...........................................................605.4.1 Fluorogenic substrates for Southern and Northern detection....605.4.2 Instrument compatibility...........................................................615.4.3 Typical protocol........................................................................625.4.4 Expected results........................................................................655.5 Microarray