Fluorescence Imaging technical manual Fluorescence Imaging principles and methods principles and methods S1´ S1 ν h EX Energy ν h EM S0 tm 63-0035-28 Rev.A, 2000-12 US$105 #310-332 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 G i FLUORESCENCE IMAGING Quantification .................................................................................................33 One-dimensional gel/blot analysis.............................................................33 Array and microplate analysis ..................................................................35 Two-dimensional protein gel analysis .......................................................36 Background correction ....................................................................................36 Image processing tools.....................................................................................40 Amersham Pharmacia Biotech image analysis software ...................................41 Chapter 5: Fluorescence applications using Amersham Pharmacia Biotech imaging systems ...........................................45 Introduction.....................................................................................................45 Detection of nucleic acids in gel ......................................................................45 Nucleic acid gel stains...............................................................................45 Instrument compatibility...........................................................................47 Typical protocol........................................................................................47 Expected results ........................................................................................49 Detection of proteins in gel..............................................................................51 Protein gel stains.......................................................................................51 Instrument compatibility...........................................................................52 Typical protocols ......................................................................................53 Protein detection in one-dimensional gels ..........................................53 Protein detection in two-dimensional gels..........................................55 Expected results ........................................................................................58 Quantification of nucleic acids in solution ......................................................59 Dyes for quantification of nucleic acids in solution .................................59 Instrument compatibility...........................................................................60 Typical protocol........................................................................................60 Expected results ........................................................................................62 Quantification of proteins in solution..............................................................63 Dyes for quantification of proteins in solution .........................................63 Instrument compatibility...........................................................................64 Typical protocol........................................................................................64 Expected results ........................................................................................66 Southern and Northern blotting ......................................................................67 Fluorogenic substrates for Southern and Northern detection....................67 Instrument compatibility...........................................................................69 Typical protocol........................................................................................69 Expected results ........................................................................................72 Western blotting ..............................................................................................73 Western detection strategies ......................................................................73 Enzyme-amplified detection (chemifluorescence)................................73 Direct fluorescent detection................................................................75 Total protein stains for Western blots ................................................75 Instrument compatibility...........................................................................76 Typical protocols ......................................................................................77 Western blotting using a fluorogenic substrate...................................77 Western blotting using a fluorochrome-conjugated antibody .............80 Expected results ........................................................................................82 tm 63-0035-28 G ii FLUORESCENCE IMAGING Using covalent labels for nucleic acid and protein analysis..............................83 Nucleic acid labelling ...............................................................................84 Protein labelling .......................................................................................85 Instrument compatibility...........................................................................86 Applications and protocols .......................................................................86 Differential display analysis ...............................................................86 In-lane PCR product analysis.............................................................89