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Fluorescence Imaging

1. Additional capabilities with database module m Sample matching to user built libraries m A variety of clustering methods for dendrogram construction IMAGEMASTER 2D Premier tool for automated analysis of 2 D gels in proteomics Automated spot detection and measurement Batch processing of unlimited number of gels Grouping of multiple gel images into one experiment Gel averaging Multiple statistical tools Web site query Multiple reporting capabilities including Web page building Additional capability with database module m Data extraction queries m Similar spot queries and ratio queries to examine expression changes m Statistical tests to help identify significant results and patterns e 43 FLUORESCENCE IMAGING a5 8 1 Bl 0 _ je jele Beis uj Leet om tm 63 0035 28 44 Table 2 continued IMAGEMASTER ARRAY 2 Powerful facility for array anal
2. o 9 317 97 786 000 0 545 1 0 75 1496 pe a 318 36 343 500 3 237 4 0 83 4 o S e 319 9 945 500 1 79 4 0 74 8 e o 320 137 940 500 4 792 3 0 67 4 I amp 321 29 196 500 15 259 11 0 71 17 9 apo 322 7 966 000 6 102 3 0 85 2 e 323 1 495 500 50 58 25 0 82 4 PN EX ens 4 324 28 517 500 3 212 2 0 87 2 325 11 426 500 876 136 1 0 84 1 326 18 356 000 4 153 6 0 90 3 328 666 500 3276 41 5 0 82 3 329 5 664 000 19 92 8 0 82 4 330 5 642 500 3 119 1 0 87 1 Background correction Most image analysis software offers multiple choices for applying background correction to fluorescence measurements The nature of image background can vary significantly depending on a number of factors such as the fluorescent detection chemistry used the sample matrix i e gel membrane microplate and the integrity or quality of the sample itself Because fluorescent detection is extremely sensitive high background levels in the scanned image can be a common problem especially in the early stages of protocol development Fluorescence protocols require careful attention to cleanliness and sample handling to minimize background problems see Chapter 6 for tips The nature of the background signal should be assessed before proceeding with image analysis Fig 28 Background commonly appears as m unifo
3. e using Typhoon 8600 ng teet i 545 3 A watt Tes es b 4 tes a of Fy e e ki ET 4 Table 9 Fluorescent gel detection of protein Typhoon Fluorlmager Storm VDS CL Stain LOD LDR LOD LDR LOD LDR LOD LDR ng band fold ng band fold ng band fold ng band fold SYPRO Orange 2 1000 3 500 6 250 5 200 SYPRO Red 2 1000 2 500 3 250 ND ND SYPRO Ruby 3 500 5 ND 7 ND 3 200 A dilution series of BSA was loaded onto a one dimensional polyacrylamide gel 1 mm thick with 4 stacking gel and 10 resolving gel and electrophoresed using Hoefer miniVE System Results are expressed as limit of detection LOD and linear detection range LDR t ND Not determined tm 63 0035 28 58 CHAPTER 5 FLUORESCENCE APPLICATIONS Quantification of nucleic acids in solution Dyes for quantification of nucleic acids in solution The concentration of DNA or RNA in solution is conventionally determined by measuring the absorbance of the solution at 260 nm and 280 nm The accuracy of this method however is significantly affected by the presence of free nucleotides DNA or RNA and contaminants from the nucleic acid preparations Nucleic acids are more accurately quantified in solution using fluorescent dyes that bind with very high specificity and sensitivity Table 10 When bound to their target molecules DNA or RNA the fluorescence of these dyes is greatly enhanced Wherea
4. 103 Instrumetit OperatlOn a ioo nir 105 ERR 107 fep 109 Appendix 1 Frequently asked 110 115 Typhoon Storm and FluorImager Systems eere 115 ALGO JT 118 Appendix 2 Spectral characteristics of commonly used fluorophores and fluorescent 119 Appendix 3 Instrument compatibility and setup with common fluorophores and fluorescent proteins 127 Appendix 4 Instrument performance with common HELIO POTN OS 131 Ue ipae ees 133 References cited gn text iiiiouatacauiaa ia e D cte iir m EE ero RES 133 General fefereli cesso nno ep eol 135 nme EE 137 Fig 1 Fluorescently labelled DNA size ladders and PCR products loaded in the same lanes were electrophoretically separated in a polyacrylamide gel and imaged using Typhoon 8600 scanner Fluorescein green Cy 3 yellow ROX blue and Cy5 red labels were used in amounts varying from 0 25 to 5 fmol per band CHAPTER 1 INTRODUCTION TO FLUORESCENCE Chapter 1 INTRODUCTION TO FLUORESCENCE Advantages of fluorescent detection Fluorescent labelling and staining when combined with an appropriate imaging instrumen
5. 670BP30 526SP Fluorlmager Excitation nm 514 488 488 488 488 488 514 488 488 488 488 488 488 488 488 NA 488 Emission filter 610RG 530DF30 530DF30 530DF30 530DF30 570DF30 610RG 610RG 610RG 610RG 530DF30 530DF30 530DF30 570DF30 570DF30 NA 570DF30 Storm VDS CL Fluorescence mode Excitation Emission NA Blue Blue Blue Blue Blue Red Blue Blue Blue Blue Blue Blue Blue Blue Red Blue Transmission Transmission Transmission Transmission Transmission Transmission Transmission Transmission Transmission Transmission NA NA NA NA NA NA Reflection UV high UV low UV low UV low UV low UV high UV high UV high UV high UV high NA NA NA NA NA NA UV high e 127 FLUORESCENCE IMAGING Typhoon Fluorlmager Storm VDS CL Fluorophore max max Excitation Emission Excitation Emission Fluorescence nm nm nm filter nm filter mode Excitation Emission Substrates and stains for Western blotting DDAO phosphate 646 660 633 670BP30 NA NA Red NA NA ECF 440 560 532 526SP 488 570DF30 Blue Reflection UV high ECL Plus 430 503 CL CL 488 530DF 30 Blue CL CL SYPRO Rose Plus 350 610 NA NA NA NA NA Reflection UV high SYPRO Ruby blot 280 450 618 532 610BP30 488 610RG Blue Reflection UV high Multipurpose labels Alexa Fluor 350 346 442 NA NA NA NA NA R T UV low Alexa Fluor 4
6. Fluorescence excitation Fluorescence emission BODIPY TR X 400 e 300 y2 450 350 Cy3 5 400 450 Cy5 5 450 500 500 1 400 1 500 1 550 APPENDIX 2 588 617 1 1 I 1 1 550 600 650 700 750 800 Wavelength nm 489 506 1 1 1 1 450 500 550 600 650 700 Wavelength nm 581 596 1 1 1 1 550 600 650 700 750 800 Wavelength nm 675 694 1 1 1 1 600 650 700 750 800 850 Wavelength nm e 121 FLUORESCENCE IMAGING 743 767 Fluorescence excitation 500 550 600 650 700 750 800 850 Wavelength nm 558 583 900 Fluorescence excitation 300 350 400 450 500 550 600 650 Wavelength nm m e 440 560 Fluorescence excitation 1 1 1 1 300 350 400 450 500 550 600 650 Wavelength nm ECL Plus 430 503 700 700 Fluorescence excitation 1 1 1 1 1 300 350 400 450 500 550 600 650 Wavelength nm tm 23 4567 01 122 700 Fluorescence emission Fluorescence emission Fluorescence emission Fluorescence emission Fluorescence excitation Fluorescence emission Fluorescence excitation Fluorescence emission Fluorescence excitation Fluorescence emission Fluorescence excitation gt Fluorescence emission DDAO phosphate 646 660 1 1 1 I 500 550 600 650 700 750 800 Wavelength nm EBFP 380 440 1 1 1 300 350 400 450 500 550 600 650 700 Wavelength 434 477 1 1
7. m Assisted lane finding and automated band finding FluorSep m Reduction of cross contamination from multiple fluorochromes typically found in multichannel fluorescence images m Support for two to four channel images ImageQuant tools m Image processing options for single and multichannel image files Signal inversion m Noise filtration Image rotation IMAGEMASTER TOTALLAB Easy to use software for analysis of 1 D gels dot and slot blots and microplates m Automatic lane identification and easy to use functions for background subtraction band detection and molecular weight determination Designed for quantitative needs in basic array analysis Automatic colony counting facility Spot detection algorithm Volume and area measurements tm 63 0035 28 42 CHAPTER 4 IMAGE ANALYSIS U 7 Table 2 continued IMAGEMASTER 1D Comprehensive software for 1 D gel image analysis 123 4 5 6 7 9 9 0 1 2 Heese Shi se emp sealer AG unm m Option of two modules Prime entry level and Elite power user level m Automated lane and band detection m Account for distortion within and among gels Band matching and lane relationship studies Elite module only
8. 63 0035 28 106 Add optical filters to eliminate excitation light in laser based scanners Stray laser light that is reflected or scattered by the sample can be rejected from the collection pathway by adding an optical filter that rejects the laser light while allowing fluorescent emission light to pass through Change the PMT photomultiplier tube voltage to improve signal collection in laser based scanners For accurate quantification the sample signal should fall within the linear range of the system For intensely fluorescent samples that saturate the system decrease the PMT voltage to bring high intensity signals into the linear range of the scanner For weak samples increase the PMT voltage to increase the signal Otherwise you may lose sensitivity and accuracy of quantification at the lower end of the signal range Refer to the instrument user s manual for additional information Change the lens aperture to improve signal collection for CCD cameras For intense signals that saturate the system reduce or close the lens aperture to reduce the amount of light entering the camera For weak signals open the lens aperture to collect more light Adjust the focal plane to optimize fluorescent detection when using Typhoon scanner Different matrices e g thick agarose gels sandwich gels and microplates can change the spatial location and thus the focal plane of the fluorescently labelled target To achieve optimal results
9. GO XOU RO 58 Quantification of nucleic acids in solution 59 Dyes for quantification of nucleic acids in solution 59 Instrument compatibility co ententeosenen ense eese eene o e en 60 Typical protocolo OO tied OR URS OE UA IRR URS OR ART DOR 60 Expected results 4 5 rer oe Heo en eene e ten oa unu euer osa 62 Quantification of proteins in 8 63 Dyes for quantification of proteins in solution eene 63 Instrument compatibility 64 Typical protocol 64 Expected tesults ue peer is et e tex en e e ei eese ei 66 Southern and Northern blotting 67 Fluorogenic substrates for Southern and Northern detection 67 Instrument compatibility i ERROR PRECOR UR COR ORE CY UR ees teed 69 Typical protocol aieo etenim hiemem c em e m i yn PER en tee 69 Expected npn ee RO ERR ERREUR OR OR OLOR RU 72 Western E TEE TT CDD 73 Western detection strategies ssion aa ia a iaa etea eria 73 Enzyme amplified detection chemifluorescence 73 Direct fluorescent detection erret UR asa 75 Total protein stains for Western 75 Instrument c ripatibllity 76 Typical protocols 77 Western blotting usi
10. I However non radioactive alternatives including chemiluminescent and fluorescent detection chemistries are now widely accepted and much preferred as the result of their safety sensitivity and convenience Fluorescent Western detection employs either a direct or enzyme amplified format The greatest sensitivity is achieved using fluorogenic substrates in an enzyme amplified format with horseradish peroxidase or alkaline phosphatase 16 17 However direct fluorescent detection using labels such as fluorescein Cy3 and Cy5 conjugated to antibodies is simpler and provides more accurate quantification 16 Fluorescent stains optimized for protein blot detection can be used for the rapid and sensitive assessment of Western transfer efficiency Western detection strategies Enzyme amplified detection chemifluorescence The most common Western detection chemistries employ enzyme amplified detection schemes using either horseradish peroxidase HRP or alkaline phosphatase AP Fig 38 Fluorogenic substrates that are available for use with these enzymes are listed in Table 19 While the sensitivity of chemifluorescence based Westerns is comparable to that of chemiluminescence quantification is improved when compared with film detection Fig 39 Table 19 Fluorogenic substrates for Western blots Excitation Emission Fluorescence Substrate max nm max nm emission colour Enzyme DDAO phosphate 646 660 Red Alkaline phosphata
11. When imaging sandwich gels the position of the sample should be within the focal depth of the imaging instrument Make sure that the thickness of the glass electrophoresis plates is optimal for the imaging system by consulting the instrument user s guide Clean the glass with distilled water and a clean lint free cloth or Kimwipe tissue If visible spots remain clean the glass first with 75 ethanol and then with distilled water Household glass cleaners should not be used because they contain ingredients that fluoresce Use flat bottom microplates For microplates the shape of the well is critical for proper excitation and collection of fluorescent light Flat bottom wells provide the largest imaging area with uniform surface characteristics Microplates with clear bottoms and clear black or opaque walls should be used Image quality and quantification are improved when using Nalge Nunc PolySorp 96 well plates with removable strips Sample placement The placement of the sample onto the imager is important to prevent the introduction of fluorescent artefacts such as air bubbles dust or interference patterns Clean the glass platen glass tray before and after imaging Dust dried buffer and or fluorescent stains and skin oils from fingerprints increase background fluorescence which in turn can interfere with image quality and quantification Clean the glass with distilled water and a clean lint free cloth or Kimwipe If visibl
12. band and divide by the total signal in the lane to calculate the percent of the signal in the shifted bands Alternative protocol Eliminate Step 1 labelling and proceed with Steps 2 4 After electrophoresis separate the gel sandwich and stain the gel for 20 min with a 1 10 000 dilution of Vistra Green in TAE buffer Rinse the gel and wipe the excess liquid off the bottom of the glass plate Image the gel using Typhoon or FluorImager system Expected results The results of a multicolour bandshift analysis using two different DNA targets labelled with HEX and TAMRA and bacterial Mnt protein are shown in Figure 45 The gel was imaged using Typhoon 8600 e 95 FLUORESCENCE IMAGING tm 63 0035 28 96 Using naturally occurring fluorescent proteins Green fluorescent protein and its variants Green fluorescent protein GFP is widely used as a reporter molecule for the study of protein localization protein binding events and gene expression 27 Using recombinant DNA technology the coding sequence for GFP can be spliced with that of other proteins to create fluorescent fusion proteins GFP fusion proteins can then be used in vivo to localize proteins of interest to specific cell types and subcellular sites and in vitro to study protein protein interactions In gene expression studies when GFP expression is placed under the control of a specific promoter or DNA regulatory sequence GFPs serve as reporters of transcriptional
13. fluorescence imaging Material Type Vendor Membranes Membrane protection Glass electrophoresis plates Microplates Detection bags are a component of the ECF kits Hybond N membrane nucleic acids Amersham Pharmacia Biotech Hybond P membrane proteins Amersham Pharmacia Biotech Detection bags Amersham Pharmacia Biotech Low fluorescence glass plates Amersham Pharmacia Biotech Clear flat bottom polystyrene microplates Polysorp 96 well plates with removable strips tm 63 0035 28 102 Corning Costar Nalge Nunc Avoid generating air bubbles when casting gels Air bubbles affect light scatter and can cause artefacts that interfere with quantification Background fluorescence contributed by the gel matrix increases with gel thickness Therefore use the thinnest gel practical for your experiment When preparing agarose gels make sure the agarose is completely dissolved and well mixed before casting the gel Uneven agarose concentration will cause non uniform backgrounds that will affect quantification If a plastic gel tray is used be sure to remove the gel from the tray prior to scanning CHAPTER 6 PRACTICAL RECOMMENDATIONS Place membranes between low fluorescence transparent plastic bags To prevent contamination of the sample and glass tray or imaging platform use low fluorescence hybridization bags to sandwich the membrane Use low fluorescence glass plates of optimal thickness
14. 1 300 350 400 450 500 550 600 650 700 Wavelength nm EGFP 489 508 1 1 1 300 350 400 450 500 550 600 650 700 Wavelength nm Fluorescence excitation Fluorescence excitation Fluorescence excitation Fluorescence excitation gt Ethidium bromidet 526 605 400 450 500 550 600 650 700 750 800 Wavelength nm FAM 495 535 350 400 450 500 550 600 650 700 750 Wavelength nm Fluorescein 495 520 UN 350 400 450 500 550 600 650 700 750 Wavelength nm OE 525 557 350 400 450 500 550 600 650 700 750 Wavelength nm Fluorescence emission Fluorescence emission Fluorescence emission Fluorescence emission Fluorescence excitation Fluorescence emission Fluorescence excitation Fluorescence emission Fluorescence excitation Fluorescence emission Fluorescence excitation Fluorescence emission APPENDIX 2 EYFP 514 527 1 1 1 1 300 350 400 450 500 550 600 650 700 Wavelength nm FITC 495 535 1 1 1 1 350 400 450 500 550 600 650 700 750 Wavelength nm FluorX 494 520 1 1 1 I 1 300 350 400 450 500 550 600 650 700 Wavelength nm NanoOrange 470 570 350 400 450 500 550 600 650 700 750 Wavelength nm e 123 FLUORESCENCE IMAGING OliGreent 500 523 Fluorescence excitation 1 1 D 1 1 1 400 450 500 550 600 650 700 750 800 Wavelength nm Oregon Green 514 511 530 Fluorescence excitation 350 400 450 500 550 600 650 700 750 Wavelen
15. 13 111 linearity 18 21 112 long pass LP filter 25 112 M materials with low fluorescence properties 102 membranes 102 membrane protection 102 microplates 102 103 monochromatic 10 112 moving head scanners 15 multichannel experiment 17 multicolour imaging 28 N NanoOrange 63 123 127 131 Neodymium Yttrium Aluminium Garnet Nd YAG laser 12 Northern blotting 67 nucleic acid gel stains 45 127 131 nucleic acid labelling 84 numerical aperture NA 16 112 object quantification method 34 OliGreen 59 124 127 one dimensional gel blot analysis 33 optical filters 10 25 105 112 P parallax effect 14 112 PCR product analysis 89 photobleaching 6 112 photodestruction 6 photomultiplier tube PMT 7 11 18 112 photomultiplier tube voltage 106 phycobiliproteins 99 PicoGreen 59 124 127 131 protein gel stains 51 127 131 protein labelling 85 protein stains for Western blots 75 128 132 Q quantum efficiency 6 113 R R phycoerythrin 99 relative fluorescence units rfu 7 113 resolution 18 20 113 RiboGreen 59 124 127 131 S sensitivity 19 21 116 118 short pass SP filter 26 113 signal saturation 107 113 signal to signal ratio S N 27 113 Southern blotting 67 FLUORESCENCE IMAGING Stokes shift 4 113 Storm 22 SYBR Gold 46 125 127 131 SYBR Green I 46 125 127 131 SYBR Green II 46 59 125 12
16. 132 ECL Plus 73 122 128 132 emission 3 110 emission filters 25 110 emission spectrum 4 110 energy of the emitted photon 3 110 energy transfer 84 enzyme amplified detection chemifluorescence 73 109 epi illumination 20 110 ethidium bromide 46 123 127 131 excitation 2 110 excitation spectrum 3 110 excited state lifeline 3 extinction coefficient 5 111 F f theta lens 14 filters 10 25 105 filtration 10 fluorescein 75 82 115 123 129 132 fluorescein isothiocyanate FITC 85 123 129 e 137 FLUORESCENCE IMAGING tm 63 0035 28 138 fluorescence 2 112 fluorescent dyes 2 fluorescent indicator dyes 101 Fluorlmager 595 23 fluorochromes 2 111 fluorochrome separation 40 fluorophores 2 111 focal plane 106 full width at half maximum transmission 26 111 G galvanometer based system 14 ghost image 116 glass electrophoresis plates 102 glass plates 103 green fluorescent protein 96 129 132 H helium neon HeNe laser 12 HeNe laser helium neon 12 image analysis software 41 image documentation 32 image filtering 41 ImageMaster software 42 44 ImageMaster VDS CL 23 ImageQuant software 42 intensity 5 111 interference patterns 117 K Kapton tape 104 111 L label covalent 83 label multipurpose 128 lane quantification method 34 laser 12 111 light collection 15 light emitting diodes LEDs 12
17. 488 nm and 514 nm that are useful for excitation of many common fluorochromes The 488 nm line is especially well suited for fluorescein and other related blue excited dyes Argon ion lasers are relatively large gas lasers and require external cooling Helium neon or HeNe lasers which generate a single wavelength of light e g 633 nm are popular in many laser scanners including densitometers storage phosphor devices and fluorescence systems In fluorescence detection the helium neon laser can be used to excite the CyS fluorochrome These lasers are smaller than argon ion lasers and do not require independent cooling Neodymium Yttrium Aluminium Garnet Nd YAG solid state lasers when frequency doubled generate a strong line at 532 nm that is not readily available from other laser sources This excitation source is useful for imaging a wide range of different fluorochromes that excite efficiently at wavelengths between 490 nm and 600 nm Cooling is required to stabilize the output Diode lasers or semiconductor diode lasers are compact lasers Because of their small size and light weight these light sources can be integrated directly into the scanning mechanism of a fluorescence imager Diode lasers are inexpensive and are generally limited to wavelengths above 635 nm CHAPTER 2 FLUORESCENCE IMAGING SYSTEMS Light Emitting Diodes LEDs As a laser alternative the LED produces an output with a much wider bandwidth 2
18. 5 ROX 5 TAMRA 5 TET 5 Tetramethylrhodamine 5 Texas Red X 5 Fluorescent proteins Allophycocyanin 650 B phycoerythrin 546 R phycoerythrin 565 GFP wt 395 470 GFP S65T 488 EGFP 489 EYFP 514 DsRed 558 NA Not applicable 615 532 532 532 532 532 532 532 532 532 532 532 532 532 532 532 633 532 532 532 532 532 532 532 526SP 526SP 526SP 526SP 555BP20 555BP20 526SP 555BP20 526SP 580BP30 610BP30 580BP30 555BP20 580BP30 610BP30 670BP30 580BP30 580BP30 526SP 526SP 526SP 555BP20 580BP30 488 488 488 488 514 514 488 488 488 514 514 514 514 514 NA NA 514 514 488 488 488 488 514 CL Chemiluminescence only Not applicable for fluorescence UV high 580BP30 filter UV low 520BP30 filter R T Reflection membranes Transmission gels 530DF30 530DF30 530DF30 530DF30 570DF30 570DF30 530DF30 530DF30 530DF30 570DF30 610RG 570DF30 530DF30 570DF30 NA NA 570BP30 570BP30 530DF30 530DF30 530DF30 530DF30 570BP30 Blue Blue Blue Blue NA NA Blue NA Blue NA NA NA NA NA NA Red Blue Blue Blue Blue Blue NA NA NA NA NA Reflection Reflection Reflection Reflection NA UV low UV low UV low UV low UV high UV high UV low UV low UV low NA NA NA UV high NA NA NA NA NA UV low UV low UV low UV low NA e 129 FLUORESCENCE IMAGING tm 63 0035 28 130 Fluorophore Nuclei
19. 500 ND 200 100 500 1000 100 ND 300 ND bromide SYBR Gold 25 10 500 1000 40 10 500 1000 500 40 100 500 ND 20 ND 100 SYBR Green 25 10 500 1000 40 10 500 1000 500 40 100 500 ND 20 ND 100 Vistra Green 25 10 500 1000 40 10 500 1000 500 40 100 500 ND 20 ND 100 A dilution series of a DNA ladder was loaded onto a 1 agarose gel 3 mm or a 10 polyacrylamide gel 1 mm Results are expressed as limit of detection LOD and linear detection range LDR for agarose polyacrylamide t ND Not determined NA Not applicable Table 6 Fluorescent gel detection of single stranded DNA and RNA Typhoon Fluorlmager Storm VDS CL Stain LOD LDR LOD LDR LOD LDR LOD LDR pg band fold pg band fold pg band fold pg band fold Ethidium 5000 NDt 50 ND 10 000 ND 30 ND NAT NA 5000 ND 50 ND bromide SYBR Gold ND 250 ND 200 ND 300 ND 150 ND 1000 ND ND ND SYBR Green ND 250 ND 200 ND 300 ND 150 ND 1000 ND ND ND SYBR Green II 10000 ND 100 ND 10000 ND 100 ND 100 000 ND 20 ND ND ND Vistra Green ND 250 ND 200 ND 300 ND 100 ND 1000 ND 50 ND ND dilution series of a DNA oligonucleotide or RNA ladder was separated on a formaldehyde agarose gel or a denaturing polyacrylamide gel Results are expressed as limit of detection LOD and linear detection range LDR for agarose polyacrylamide t ND Not determined NA Not applicable tm 63 0035 28 50 CHAPTER 5 FLUORESCENCE APPLICATIONS Detection of protei
20. 63 0035 28 94 e Preparation of binding reaction Note Repeat the steps below for all protein DNA combinations to be tested including a negative control containing no protein Prepare the following mix Fluorescent oligonucleotide duplex 1 4 pmol Binding protein 1 0 pmol or as required H O to a total volume of 10 pl Incubate the reactions on ice for 30 min o Gel electrophoresis To 3 pl of the protein DNA mixture from each binding reaction add 1 pl of a 50 w v sucrose solution and mix gently Note Do not mix tracking dye with the sample Place tracking dye in a separate lane if needed see next step Load 2 pl of the protein DNA sucrose mixture onto a 6 non denaturing polyacrylamide gel Load tracking dye in a separate lane Fill the reservoirs with TAE buffer containing 1 mM MgCl and run the gel at 10 V cm at 4 C until the tracking dye has migrated approximately halfway down the gel Imaging Typboon 8600 Affix two Kapton tape strips over each spacer on the outside of the long glass plate Place water between the glass plate and the Typhoon glass platen to minimize the appearance of interference patterns Avoid trapping air bubbles between the glass plate and the Typhoon platen Select the appropriate settings for laser excitation and emission filter see Appendix 3 for appropriate settings Select a focal plane of 3 mm Fig 45 Multi label gel shift experiment First two l
21. Fig 24c tools for lane and band identification are available Quantification in this manner is inherently more flexible than a lane profile method since the user has more control in defining the area to be analysed and in choosing a method for background correction prior to quantification see section 4 4 All the image pixels bounded by each object are used for quantification While the absolute data differs between the methods the trends or relative differences between the measurements from each method are similar Fig 24a b c Counts x 10 m EE nn Background HER m Pixel r 4 4 4 1 35611 55 16 159 Rectangle 1 914863 7 16 81 1 750 235 49 16 03 2 48933 02 22 204 Rectangle 2 1212416 22 27 2 1 018 636 94 21 76 3 64490 90 29 264 Rectangle 3 1485657 27 29 3 1 326 294 64 28 33 4 39506 01 17 927 Rectangle 4 1000343 18 38 4 852 357 26 18 21 5 31835 27 14 446 Rectangle 5 830632 5 15 26 5 733 996 99 15 68 a b c Fig 24 Comparison of results from area versus volume analysis methods In panel a area refers to integration of signal from each peak identified in a trace through the gel lane with background taken as the lowest value in the wide line profile Volume analysis panel b produces a value of integrated signal from within a box surrounding each separate band in the gel lane A background value selected from a different region of the gel has been applied to
22. Fluorlmager A constant amount of S15 GFP S65T His 1 mM was incubated with varying amounts 0 0 95 mM left to right of S protein for 20 min at 20 C Samples were resolved by electrophoresis on a native 6 polyacrylamide gel Image kindly provided by Sang Hyun Park and Ronald Raines University of Wisconsin Madison WI tm 63 0035 28 98 Examples of applications using GFP Monitoring gene expression in yeast In this application FluorImager was used to analyse transient gene expression in transformed yeast cells expressing GFP as a reporter GFP transformed colonies were spotted and grown on agar plates Expression of GFP was observed by scanning the agar plate using 488 nm excitation Fig 46 Study of protein protein interactions When used as a probe in a fusion protein GFP functions as an independent domain without altering the properties of the protein of interest As such GFP and its variants are effective tools for in vivo and in vitro functional analyses of protein protein interactions For example GFP has been used to demonstrate the interaction between the S peptide and S protein fragments of ribonuclease A 30 In this study varying amounts of S protein were incubated with purified 15 peptide GFP S65 T His and the complexes were then separated from free components in a native polyacrylamide gel Fig 47 The image of the gel retardation assay was acquired using the 488 nm excitation source of FluorImag
23. MgCl 5 ul 2 5 mM 2 mM dGTP dATP dTTP 1 25 ul 50 uM each 1 mM dCTP CyDye dCTP dCTP 1 10 2 5 ul 50 uM dCTP Forward primer 0 5 uM Reverse primer 0 5 uM DNA template 70 ng Taq DNA polymerase 5 units ul 0 2 ul 1 unit Sterile ddH O to a final reaction volume of 50 ul PCR buffer 500 mM KCI 100 mM Tris Cl pH 9 0 eo PCR Place the samples into the thermal cycler and heat for 1 min at 95 C to denature them Run the following program for 30 cycles 95 C for 15 s 57 C for 15 s and 72 C for 30 s Complete the program by incubating the samples for 2 min at 72 C CHAPTER 5 FLUORESCENCE APPLICATIONS e Gel electrophoresis Prepare a 10 polyacrylamide gel in Tris borate EDTA TBE buffer 14 Mix 1 5 pl of the amplified product with TE buffer and 6x sample buffer for a final volume of 6 pl Load the samples onto the gel and run for 1 5 h at 100 V o Imaging Typboon 8600 Affix two Kapton tape strips over each spacer on the outside of the long glass plate Place water between the glass plate and the Typhoon glass platen to minimize the appearance of interference patterns Avoid trapping air bubbles between the glass plate and the Typhoon platen Select the appropriate settings for laser excitation and emission filter see Table 24 or Appendix 3 Select a focal plane of 3 mm Storm 830 860 Be sure to use Cy5 labelled primers Remove the glass plate that was treated with silane Cover
24. The name of a long pass filter may also include other designations such as OG orange glass RG red glass E emission LP long pass or EFLP edge filter long pass OG and RG are coloured glass absorption filters whereas E LP and EFLP filters are coated interference filters Coloured glass filters are less expensive and have more gradual transition slopes than coated interference filters e 25 FLUORESCENCE IMAGING a Transmission 96 bh o e Wavelength nm Fig 15 Transmission profile for a band pass 670BP30 filter The full width at half maximum FWHM transmission of 30 nm is indicated by the arrows tm 63 0035 28 26 Short pass SP filters reject wavelengths that are longer than a specified value and pass shorter wavelengths Like long pass filters short pass filters are named according to their cutoff point For example a 526SP filter rejects 50 of the maximum transmittance at 526 nm Fig 14b Band pass BP filters allow a band of selected wavelengths to pass through while rejecting all shorter and longer wavelengths A band pass filter provides very sharp cutoffs with very little transmission of the rejected wavelengths High performance band pass filters are also referred to as Discriminating Filters DF The name of a band pass filter is typically made up of two parts e the wavelength of the band centre For example the 670BP30 filter passes a band of light centred at
25. Wavelength nm Wavelength nm Alexa Fluor 660 Alexa Fluor 680 663 690 679 702 Fluorescence excitation Fluorescence emission Fluorescence excitation Fluorescence emission 400 450 500 550 600 650 700 750 80 400 450 500 550 600 650 700 750 800 Wavelength nm Wavelength nm Allophycocyanin BODIPY 630 650 650 660 632 640 Fluorescence excitation Fluorescence emission Fluorescence excitation Fluorescence emission 1 1 1 1 I 400 450 500 550 600 650 700 750 80 400 450 500 550 600 650 700 750 800 Wavelength nm Wavelength nm BODIPY 650 665 BODIPY FL 651 660 505 513 Fluorescence excitation Fluorescence emission Fluorescence excitation Fluorescence emission 400 450 500 550 600 650 700 750 80 300 350 400 450 500 550 600 650 700 Wavelength nm Wavelength nm tm 23 4567 01 120 Fluorescence excitation Fluorescence excitation Fluorescence excitation Fluorescence excitation BODIPY TMR X 535 574 350 400 450 500 550 600 650 700 Wavelength nm CBQCA 350 400 450 500 550 600 650 700 Wavelength nm Cy3 550 570 350 400 450 500 550 600 650 700 Wavelength nm Cy5 649 670 400 450 500 550 600 650 700 750 Wavelength nm Fluorescence emission Fluorescence emission Fluorescence emission Fluorescence emission Fluorescence excitation Fluorescence emission Fluorescence excitation Fluorescence emission Fluorescence excitation Fluorescence emission
26. acid solution for 5 15 min Longer destaining may result in a loss of sensitivity For SYPRO Ruby destain the gel for 30 min in deionized water o Imaging Place the wet gel directly onto the platen Typhoon and Storm glass tray FluorImager or platform VDS CL of the imager in a small amount of water Avoid trapping air bubbles between the gel and the glass CHAPTER 5 FLUORESCENCE APPLICATIONS In the Scanner Control Setup window choose the appropriate laser and emission filter combinations Table 8 For Typhoon imaging choose platen for the focal depth setting Acquire the image according to the recommended instrument set up Analysis See Chapter 4 for information concerning image analysis Protein detection in two dimensional gels Two dimensional gel electrophoresis is used to analyse complex mixtures of proteins through the combined resolving power of two electrophoretic methods 13 In the first dimension proteins are resolved according to their isoelectric points by isoelectric focusing IEF The isoelectric point of each protein relates to the pH at which the net charge of the molecule is zero Following IEF of the proteins SDS PAGE is used as the second dimension to further resolve the proteins according to their molecular weights The result is a complex pattern of spots corresponding to the many different protein molecules present in the original sample Fluorescent gel stains have been dev
27. acrylamide or urea Use powder free gloves to eliminate fluorescent talcum particles Purify stock buffer solutions if necessary Dust in buffer solutions and gels can cause minor spikes in the background thus affecting image quality and quantification Filter solutions to remove dust and store the solutions in clean rinsed containers Spectroscopic grade solvents should be used in the preparation of buffers because of their low autofluorescence When appropriate autoclave or filter sterilize solutions and buffer stocks to eliminate the possibility of microbial contamination Use filter filled pipette tips e 101 FLUORESCENCE IMAGING Table 30 Recommended materials Use appropriate staining containers for post staining For post staining procedures utilize containers that will not interfere with your stain It is known that SYPRO and SYBR stains are adsorbed by glass surfaces while propylene containers are better suited for these stains Refer to the dye manufacturer s product information for details on handling specific dyes Use sample support materials with low fluorescence properties Gels membranes glass plates and microplates all autofluoresce to some extent Using materials with low fluorescence properties improves the limit of detection and linear range New materials should always be tested to determine their fluorescence properties before they are used in experiments See Table 30 for materials recommended for use in
28. activity GFP is uniquely suited as a reporter molecule in these applications because it can be expressed in many different cell types and organisms with no need for additional substrates or cofactors Fluorescence from GFP is direct stable and readily observed using common modes of fluorescence detection 28 Wild type GFPs are not optimal for some reporter gene applications For example when excited by the 488 nm argon ion laser blue light commonly used in fluorescence microscopy and fluorescence activated cell sorter FACS the fluorescence intensity from wild type GFPs is relatively low In addition a significant lag in the development of fluorescence after protein synthesis can occur and complex photoisomerization of the GFP chromophore may result in the loss of fluorescence Furthermore wild type GFPs are expressed at low levels in many higher eukaryotes Numerous GFP variants have therefore been engineered to overcome these limitations 29 For example several GFP variants are available with a significantly larger extinction coefficient for excitation at 488 nm and a modified gene sequence with codon usage that is preferentially found in highly expressed eukaryotic proteins The spectral properties of green fluorescent protein and its variants are given in Table 25 CHAPTER 5 FLUORESCENCE APPLICATIONS Table 25 Spectral properties of GFP and its variants Fluorescence Extinction Approximate Excitation Emission emission co
29. all calculations Volume analysis from automated lane and band finding panel c with a specific background is calculated around each individual band using the lowest value tm 63 0035 28 34 Fig 25 Approaches to software analysis of arrays In panel a simple arrays low density dot blots microplates are analysed using a grid or series of rectangles to surround each array element In panel b dedicated array software packages employ spot finding and or flexible array templates that find best fits to enclosing spot elements a CHAPTER 4 IMAGE ANALYSIS Array and microplate analysis Arrays range from simple dot blots with a few spots to high density gene expression arrays with thousands of closely spaced elements Arrays are typically configured in regular and predictable patterns of rows and columns Simple arrays and microplates can be analysed manually using grid tools or a series of ellipse objects to identify each element of the array Fig 25a Automated high throughput analysis of high density arrays requires sophisticated software packages complete with algorithms for automated spot finding Fig 25b data normalization comparisons between different arrays and database input of analysis results Array software frequently employs a quality metrics system to assist in the identification of poorly arrayed contaminated or improperly detected spots Tools for elemental display and graphical analysis provide easy vi
30. and 526SP emission filter The plot shows signal linearity over a range of 100 amol to 44 fmol CHAPTER 1 INTRODUCTION TO FLUORESCENCE excited state before the fluorescent light is emitted The Stokes shift is fundamental to the sensitivity of fluorescent techniques because it allows emission photons to be detected against a low background spectrally removed from excitation photons See Appendix 2 for excitation and emission spectra of many commonly used fluorophores Signal linearity The intensity of the emitted fluorescent light is a linear function of the amount of fluorochrome present when the wavelength and intensity of the illuminating light are constant e g when using a controlled laser light source Although the signal becomes non linear at very high fluorochrome concentrations linearity is maintained over a very wide range of concentrations In fact measurement down to 100 amol is not unusual with linearity extending over several orders of magnitude Fig 5 Brightness Fluorochromes differ in the level of intensity brightness they are capable of producing This is important because a dull fluorochrome is a less sensitive probe than a bright fluorochrome Brightness depends on two properties of the fluorochrome m its ability to absorb light extinction coefficient m the efficiency with which it converts absorbed light into emitted fluorescent light quantum efficiency The brightness of a fluorochrome is propor
31. electrophoresis the gel was stained for 30 min Amount of DNA ladder loaded per lane ranged from 120 000 pg 4 pg in two fold serial dilutions CHAPTER 5 FLUORESCENCE APPLICATIONS Gels attached to one of the electrophoresis plates Place the glass plate directly onto the platen Typhoon in the extended universal holder tray FluorImager or platform VDS CL of the imager For optimal image quality on Typhoon place Kapton tape supplied with the Typhoon accessory kit over each spacer on the outside of the long plate Place water between the glass plate and Typhoon platen to minimize the appearance of interference patterns Choose 3 mm for the focal height setting For Storm place the gel directly in contact with the platen In the Scanner Control Setup window choose the appropriate laser and emission filter combinations Table 4 Analysis See Chapter 4 for information concerning image analysis Expected results Typical results for the fluorescent detection of nucleic acids in agarose gels are given in Tables 5 and 6 Figure 31 shows the detection of DNA in an agarose gel stained with Vistra Green and imaged using Typhoon 8600 e 49 FLUORESCENCE IMAGING Table 5 Fluorescent gel detection of double stranded DNA Typhoon Fluorlmager Storm VDS CL Stain LOD LDR LOD LDR LOD LDR LOD LDR pg band fold pg band fold pg band fold pg band fold Ethidium 100 ND
32. filter rejects light with wavelengths that are shorter than the cutoff light of a single frequency single wavelength or single colour a set of images that can be viewed as a composite when overlaid or viewed as individual images Each separate image of the set represents a single channel the statistical uncertainty inherent in a measurement such as the standard deviation associated with measured background counts NA a number that expresses the ability of a lens to resolve fine detail in an object being observed The NA is related to the angular aperture of the lens and the index of refraction of the medium found between the lens and the specimen a glass designed to specifically attenuate reflect and transmit only selected wavelengths of light a shift in the apparent position of an object that occurs when it is viewed from different vantage points or photodestruction the irreversible destruction of an excited fluorophore upon exposure to an intense light source resulting in loss of the emission light intensity brightness photomultiplier tube a photoelectric device that converts light into electric current and amplifies the current a quantum of light This concept is based on Planck s quantum theory of light which states that the energy of an oscillating system can have only discrete quantized values the basic unit of programmable gray or colour in a digital image The physical size of a pixel depends
33. in a single well or dilute the tracking dye with sample buffer The sample may have stained unevenly Make sure you mix staining solutions thoroughly use a large excess of staining solution and rock or shake the gels during staining if possible How do reduce the appearance of diffraction patterns in the image from a glass plate placed on a platen Diffraction patterns are caused by the interface between two different pieces of glass To reduce their appearance use two Kapton strips supplied in the Typhoon accessory kit positioned over the spacers on the outside edges of the 3 mm thick plate to raise the sandwich gel slightly above the glass platen Fill the gap between the platen and the bottom of the 3 mm electrophoresis glass plate with distilled water If water is used be sure to avoid trapping air bubbles between the sandwich gel and the glass platen Rest one side of the sandwich gel on the glass platen and slowly lower it When you can no longer lower the sandwich gel using your fingers insert the Wonder Wedge tool supplied in the Typhoon accessory kit between the glass platen and the 3 mm electrophoresis glass plate then slowly remove the wedge After scanning use the Wonder Wedge to help remove the sandwich gel e 117 FLUORESCENCE IMAGING tm 63 0035 28 118 Is my image suitable for quantification Display the scanned image in ImageQuant and use the Gray Color Adjust Pixel Locator or Create Graph featur
34. in which the excited state is created by a chemical reaction a 488 495 513 526 Excitation 400 450 500 550 600 650 700 Wavelength nm b 520 532 605 Emission 400 450 500 550 600 650 700 Wavelength nm Fig 3 Excitation a and emission b spectra of fluorescein green DNA bound TOTO orange and DNA bound ethidium bromide red Curves are normalized to the same peak height The wavelength at which maximum excitation a or maximum emission b occurs is shown above each curve The position at which 488 nm laser light intersects with each of the three excitation spectra is indicated The curves are approximations based on data collected at Amersham Pharmacia Biotech or presented in references land 2 CHAPTER 1 INTRODUCTION TO FLUORESCENCE Excited state lifetime The excited state of a fluorophore is characterized by a very short half life usually on the order of a few nanoseconds During this brief period the excited molecules generally relax toward the lowest vibrational energy level within the electronic excited state Fig 2 The energy lost in this relaxation is dissipated as heat It is from the resulting relaxed singlet excited state S that fluorescence emission originates Emission When a fluorochrome molecule falls from the excited state to the ground state light is often emitted at a characteristic wavelength The energy of the emitted photon hv is the difference between the e
35. interest the background signal is typically reduced with only slight attenuation of the signal from the fluorochrome Therefore 532 Excitation 300 350 400 450 500 550 600 Wavelength nm Fig 16 Excitation of fluorescein green and Cy3 orange using 532 nm laser light The absorption spectra of Cy3 and fluorescein are overlaid with the 532 nm wavelength line of the Nd YAG laser 560LP Emission Y 500 550 600 650 700 Wavelength nm Fig 17 Emission filtering of Cy3 fluorescence using either a 580BP30 dark gray area or a 560LP filter light and dark gray areas CHAPTER FLUOROCHROME AND FILTER SELECTION the use of an appropriate band pass filter should improve the overall signal to noise ratio S N To determine if a filter is needed scans should be performed with and without the filter while other conditions remain constant The resulting S N values should then be compared to determine the more efficient configuration Interchangeable filters can also be used in fluorescence scanners to attenuate the sample signal itself so that it falls within the linear range of the system Although scanning the sample at a reduced PMT voltage can attenuate the signal the response of the PMT may not be linear if the voltage is set below the instrument manufacturer s recommendation If further attenuation is necessary to prevent saturation of the PMT the addition of an appropriate emission filter can decrease the signal
36. least 1 h at room temperature Incubate the blot with primary antibody against the target protein for 1 h then wash the membrane thoroughly Incubate the blot with enzyme conjugated secondary antibody for 1 h then wash the membrane thoroughly After the final washing step position the blot in an open low fluorescence bag or page protector CHAPTER 5 FLUORESCENCE APPLICATIONS e Application of substrate Add 50 100 pl of substrate per cm of membrane Incubate for 5 min Note The blot can be air dried to slow or stop signal development After developing seal the blot in a low fluorescence bag or page protector o Imaging Place the sealed developed wet blot or dry blot sample side down on the glass platen Storm Typhoon glass tray FluorImager or platform VDS CL of the imager Note Water can be used between the plastic bag and the platform to minimize the occurrence of interference patterns in the image Use a glass plate to hold the blot flat during imaging Acquire the image according to the recommended instrument setup The choice of pixel size and PMT voltage settings will depend on the individual experiment Adjust the PMT voltage setting to prevent signal saturation Analysis See Chapter 4 for information concerning image analysis e 79 FLUORESCENCE IMAGING m 63 0035 28 80 Western blotting using a fluorochrome conjugated antibody Amersham Pharmacia Biotech pro
37. of chlorophyll molecules located in the photosynthetic reaction centre Because of their role in light collection phycobiliproteins possess exceptional spectral properties quantum yields up to 0 98 and molar extinction coefficients of up to 2 4 x 10 cm M Phycobiliproteins have been covalently conjugated to antibodies and other proteins to generate probes that are readily detectable and which may be useful for Western blotting applications Table 28 Properties of phycobiliproteins Fluorescence Extinction Excitation Emission emission coefficient Quantum Protein max nm max nm colour M cm yield Allophycocyanin 650 660 Red 700 000 0 68 B phycoerythrin 546 575 Orange red 2 410 000 0 98 R phycoerythrin 565 578 Orange red 1 960 000 0 82 FLUORESCENCE IMAGING J Instrument compatibility The broad excitation spectra particularly of the R phycoerythrin conjugates allow phycobiliproteins to be efficiently excited using different types of imaging instrumentation with different excitation sources Table 29 Allophycocyanin conjugates are ideal for use with helium neon HeNe laser excitation 633 nm Table 29 Instrument settings for use with phycobiliproteins Typhoon Fluorlmager Storm Protein Excitation Emission Excitation Emission Fluorescence nm filter nm filter mode Allophycocyanin 633 670BP30 NA NA Red B phycoerythrin 532 580BP30 514 570BP30 Blue R phycoerythrin 532 580BP30 514 570BP30 B
38. on the resolution of the image a process in which the signal intensity of a sample is calculated quantum yield 9 the efficiency with which a fluorochrome converts absorbed light to emitted light the ratio of the number of photons emitted to the number of photons absorbed rfu resolution saturation sensitivity threshold short pass filter spectral cross contamination signal to noise ratio spatial resolution Stokes shift trans illumination transmission uniformity wavelength of light GLOSSARY relative fluorescence units the arbitrary units in which fluorescence intensity is reported by the fluorescence imaging systems see amplitude resolution or spatial resolution the reception of excess light by a photosensitive detector resulting in loss of signal discrimination or detection threshold a measure of the lowest signal that can be accurately detected by an instrument an optical filter that transmits light of wavelengths that are shorter than a specified cutoff value while rejecting light of wavelengths that are longer than the cutoff the presence of fluorescent signal from more than one fluorochrome in a single optical channel spectral contamination in a single optical channel that cannot be separated by optical filtering S N a measure of the brightness of a desired fluorescent signal relative to the brightness of the background the number of data points sampled per unit lengt
39. pixel values by using a pixel measurement tool Alternatively data from a line profile across the image will display signal intensity versus pixel co ordinate or distance Use these tools to determine if any signal has saturated the detector at the high end of the intensity scale Use background correction and analysis tools that are appropriate for the image For discussion and suggestions see Chapter 4 e 107 FLUORESCENCE IMAGING tm 63 0035 28 108 absorption absorption spectrum algorithm amplitude resolution aperture autofluorescence background band pass filter beamsplitter brightness CCD chemifluorescence chemiluminescence coherent GLOSSARY Glossary TERMS DEFINED the transfer of energy from a photon of light to a fluorochrome molecule a plot of the absorption light wavelength versus the amount of light absorbed by a fluorochrome a mathematical or computational procedure for solving a recurrent problem or gray level quantification describes the minimum difference that is distinguishable between levels of light intensity fluorescence detected from a sample an optical opening that admits light an inherent or intrinsic property of a material to fluoresce undesired signal often resulting from autofluorescence or light scatter from a matrix or sample support an optical filter that transmits a band of light between two specified wavelength cutoffs The fi
40. radiation Although in theory the more intense source will yield the greater fluorescence in actual practice photodestruction of the sample can occur when high intensity light is delivered over a prolonged period of time Susceptibility to environmental effects The quantum efficiency and excitation and emission spectra of a fluorochrome can be affected by a number of environmental factors including temperature ionic strength pH excitation light intensity and duration covalent coupling to another molecule and noncovalent interactions e g insertion into double stranded DNA Many suppliers provide information on the characteristics of their fluorescent reagents under various conditions A significant effect known as photodestruction or photobleaching results from the enhanced chemical reactivity of the fluorochrome when excited Since the excited state is generally much more chemically reactive than the ground state a small fraction of the excited fluoro chrome molecules can participate in chemical reactions that alter the molecular structure of the fluorochrome and create a molecule with reduced fluorescence The rate of these reactions depends on the sensitivity of the particular fluorochrome to bleaching the chemical environment the excitation light intensity the dwell time of the excitation beam and the number of repeat scans CHAPTER 1 INTRODUCTION TO FLUORESCENCE Quantification of fluorescence As discussed previous
41. reprobing With a dual label approach however the DNA probes from the two tissue types are labelled with different fluorochromes and used simultaneously with the same gene array In this way experimental error is reduced because only one array is used and hybridization conditions for the two probes are identical Additionally by using a 2 channel scan expression data is rapidly collected from both tissues thus streamlining analysis Other applications are equally amenable to dual label analysis For example Figure 18 shows a two colour Western blot experiment where two protein targets are differentially probed using antibodies conjugated with two different fluorescent tags The use of multicolour imaging can greatly improve the accuracy for applications such as DNA fragment sizing This technique is usually performed by loading a DNA size ladder and an unknown DNA sample in adjacent lanes of a gel Because variations in lane to lane migration rate can occur during electrophoresis errors in size estimation may result By labelling the standard and the unknown fragments with two fluorochromes whose spectra can be differentiated co resolution of the unknown and the size ladder can be achieved in the same lane Fig 19 The process for multicolour image acquisition varies depending on the imaging system An imager with a single detector takes consecutive images using different emission filters and in some cases different excitation light Wh
42. the fluorochrome at a constant intensity with a fixed wavelength of light The apex of the emission peak occurs at the wavelength whose energy equals the difference between the energy of the base level of the excited state and that of a favored vibrational level in the ground state Fig 4a The shape of the emission band is approximately a mirror image of the longest wavelength absorption band Fig 4b providing that the vibronic structures of the excited and ground states are similar In theory the transition 1 in excitation and transition 1 in emission Fig 4a should occur at the same wavelength However this is usually not the case in solution mainly due to solvent relaxation 3 The emission spectrum is always shifted toward a longer wavelength lower energy relative to the excitation spectrum as shown for the spectra of the three fluorochromes in Figure 3 The difference in wavelength between the apex of the emission peak and the apex of the excitation peak is known as the Stokes shift This shift in wavelength energy represents the energy dissipated as heat during the lifetime of the 600 000 500 000 400 000 300 000 Intensity 200 000 100 000 0 0 4 8 12 16 20 24 28 32 36 40 44 DNA fmol Fig 5 Fluorescence linearity A 24 mer DNA oligonucleotide 5 end labelled with fluorescein two fold serial dilutions was detected in denaturing polyacrylamide gel sandwich using Typhoon scanner with 532 nm excitation
43. ways as outlined below Lane profile Area m Wide line across sample track gel lane m Peaks identified Signal integrated across line Area under the curve calculated Benefits objectivity speed a Object quantification Volume Bands identified manually by user Bands bounded by separate objects Total signal inside each band object used Volume total integrated signal Benefits flexibility accuracy user created objects Lane quantification c Volume Wide line across sample track gel lane Bands identified as separate objects Total signal inside each band object used Volume total integrated signal Benefits objectivity speed accuracy CE e 33 FLUORESCENCE IMAGING The lane profile quantification method uses a wide line spanning the width of a gel lane to generate a profile from the average signal at each row of pixels perpendicular to the line Fig 24a The accuracy of this approach is greatest when the wide line includes most of the target signal across the width of the lane Each peak is identified the area under each peak or curve is integrated and the resulting peak area is then reported In the object and lane quantification methods analysis targets i e bands spots slots are enclosed using objects such as bands boxes rectangles polygons or ellipses Both manual Fig 24b and automated
44. will be skewed on the high side In this case aberrant noise from the background calculation can be disregarded by using a median value The region of the image selected to represent the background signal is important for accurate quantification In the same way the boundaries used to define analysis targets bands spots or slots will also impact the results of quantification If boundaries are too close to a particular band the signal from that target will be under represented In contrast a boundary that is set too far away from the target can overlap with other analyses bringing unexpected and undesired signal into the analysis e 39 FLUORESCENCE IMAGING Fig 30 Multicolour image processing using a fluorochrome separation routine Spectral contamination in this four colour image particularly evident in the blue and yellow channels a is reduced to give a better representation of the signal from each of the four fluorochrome labels b tm 63 0035 28 40 Image processing tools Software utilities for image processing functions improve the accuracy of quantification for both single colour and multicolour images Resolution of fluorescent signal overlap in multicolour images Overlap between the emission spectra of fluorochromes is a common and almost unavoidable aspect of multicolour imaging Even the best band pass filters cannot completely reject the emission from one fluorochrome when its emission spectrum ove
45. 0 volumes 250 500 ml per Ettan DALT II gel of SYPRO Ruby staining solution Wash the gel with 2 4 changes of deionized water for approximately 2 h and then with 1096 methanol and 796 acetic acid for at least 15 min The gel may be stored in the latter solution o Imaging Place the wet gel directly onto the platen Typhoon and Storm glass tray FluorImager or platform VDS CL of the imager in a small amount of water Avoid trapping air bubbles between the gel and the glass In the Scanner Control Setup window choose the appropriate laser and emission filter combinations Table 8 For Typhoon imaging choose platen for the focal depth setting Acquire the image according to the recommended instrument set up Analysis Analyse the image using ImageMaster 2 D software e 57 FLUORESCENCE IMAGING The expected limits of detection LOD and linear detection ranges LDR for protein quantification in gels are given in Table 9 Images from a one dimensional SDS PAGE and a two dimensional protein separation are shown in Figure 32 and Figure 33 respectively Fig 32 Proteins in an SDS PAGE gel were stained with SYPRO Orange and imaged using Typhoon 8600 Amount of BSA per lane ranged es from 1630 ng to 0 8 ng prepared in two fold serial dilutions Fig 33 coli proteins in 2 D SDS PAGE gel were stained with SYPRO Ruby and imaged
46. 01 Norway Tel 23 18 58 00 Fax 23 18 68 00 Portugal Tel 021 417 70 35 Fax 021 417 31 84 Russian Federation Tel 7 095 232 0250 956 1137 Fax 7 095 230 6377 oO Southeast Asia Tel 60 3 724 2080 Fax 60 3 724 2090 Spain Tel 935 944 950 Fax 935 944 955 Sweden Tel 018 612 1900 Fax 018 612 1910 Switzerland Tel 01 802 81 50 Fax 01 802 81 51 UK Tel 0800 616928 Fax 0800 616927 USA Tel 1 800 526 3593 Fax 1 877 295 8102 Amersham Pharmacia Biotech UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Pharmacia Biotech AB SE 751 84 Uppsala Sweden Amersham Pharmacia Biotech Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Pharmacia Biotech Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Molecular Dynamics Inc 928 East Arques Avenue Sunnyvale CA 94086 USA Amersham Pharmacia Biotech Inc 2000 AII rights reserved All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group which supplies them A copy of these terms and conditions is available on request www apbiotech com
47. 1 12504 1994 Park S H and Raines R T Prot Sci 6 2344 2349 1997 Garamszegi N et al BioTechniques 23 864 872 1997 REFERENCES General References Fluorescence principles and methods Guilbault G G ed Practical Fluorescence Second Edition Marcel Dekker New York 1990 Hemmil I A Applications of Fluorescence in Immunoassays John Wiley and Sons Inc New York 1991 Lakowicz J R ed Topics in Fluorescence Spectroscopy Vols 1 5 Plenum Publishing New York 1991 1997 Lakowicz J R Principles of Fluorescence Spectroscopy Second Edition Plenum Publishing New York 1999 Mathies R A et al Optimization of High Sensitivity Fluorescence Detection Anal Chem 62 1786 1791 1990 Rost R D W Chapter 2 Fluorescence Physics and Chemistry in Fluorescent Microscopy Vol 1 Cambridge University Press New York 1992 Royer C A Approaches to Teaching Fluorescence Spectroscopy Biophys J 68 1191 1195 1995 Sharma A and Schulman S G Introduction to Fluorescence Spectroscopy John Wiley and Sons Inc New York 1999 Taylor D L et al eds Applications of Fluorescence in the Biomedical Sciences A R Liss New York 1986 Fluorescence imaging instrumentation Bass M ed Handbook of Optics McGraw Hill 1994 Saleh B E A and Teich M C eds Fundamentals of Photonics John Wiley and Sons New York 1991 Skoog D A et al in P
48. 3 0035 28 16 With moving head systems emitted light is collected directly below the point of sample excitation Again it is important to collect as much of the emitted light as possible to maintain high sensitivity This can be achieved by using large collection lenses or lenses with large numerical apertures NA Since the NA is directly related to the full angle of the cone of light rays that a lens can collect the higher the NA the greater the signal resolution and brightness 5 Moving head designs can also include confocal optical elements that detect light from only a narrow vertical plane in the sample This improves sensitivity by focusing and collecting emission light from the point of interest while reducing the background signal and noise from out of focus regions in the sample Fig 10 Additionally the parallel motion of moving head designs removes other artefacts associated with galvanometer based systems such as spatial distortion and the flaring or blooming associated with high activity samples Sample Glass platen rm _____ Objective lens Ax x Aar Pinhole Detector Fig 11 Use of a beamsplitter or dichroic filter with two separate PMTs Light from a dual colour sample enters the emission optics as a combination of wavelengths A dichroic beamsplitter distinguishes light on the basis of wavelength Wavelengths above the beamsplitter range pass through those below are reflected I
49. 30 433 539 NA NA NA NA Blue R T UV low Alexa Fluor 488 495 520 532 526SP 488 530DF30 Blue R T UV low Alexa Fluor 532 532 554 532 555BP20 514 570DF30 NA R T UV high Alexa Fluor 546 556 573 532 580BP30 514 570DF30 NA NA NA Alexa Fluor 568 578 603 532 610BP30 514 610RG NA NA NA Alexa Fluor 594 590 617 532 610BP30 NA NA NA NA NA Alexa Fluor 633 632 647 633 670BP30 NA NA Red NA NA Alexa Fluor 660 663 690 633 670BP30 NA NA Red NA NA Alexa Fluor 680 679 702 633 670BP30 NA NA Red NA NA BODIPY 630 650 632 640 633 670BP30 NA NA Red NA NA BODIPY 650 665 651 660 633 670BP30 NA NA NA NA NA BODIPY FL 505 513 532 526SP 488 530DF30 Blue R T UV low BODIPY TMR X 535 574 532 580BP30 514 570DF30 NA R T UV high BODIPY TR X 588 617 532 610BP30 NA NA NA NA NA Cy2 489 506 532 526SP 488 530DF30 Blue R T UV low Cy3 550 570 532 580BP30 514 570DF30 NA NA NA Cy3 5 581 596 532 610BP30 514 610RG NA NA NA Cy5 649 670 633 670BP30 NA NA Red NA NA Cy5 5 675 694 633 670BP30 NA NA Red NA NA Cy7 743 767 NA NA NA NA NA NA NA tm 63 0035 28 128 Fluorophore Excitation max nm Emission max nm Typhoon Fluorlmager Storm VDS CL APPENDIX 3 Excitation nm Emission filter Excitation nm Emission filter Fluorescence mode Excitation Emission Multipurpose labels continued 95 95 95 FAM 4 FITC 4 Fluorescein 4 FluorX 4 HEX 5 JOE 5 Oregon Green 488 4 Oregon Green 514 5 Rhodamine Green 5 Rhodamine Red X
50. 32 FLUORESCENCE IMAGING Quantificatio a qe PE UTITUR UD TUTUP GU DER Oe dq edd aeu ned 33 One dimensional gel blot analysis eee 33 Array and microplate analysis erret nenne 35 Two dimensional protein gel analysis eene 36 Backeround Correcto aed rr n HERO ER ees Eee ANTE ees ebur erae eben uen eed 36 Image processing tools erre t ee Fe een ir ates 40 Amersham Pharmacia Biotech image analysis software essssse 41 Chapter 5 Fluorescence applications using Amersham Pharmacia Biotech imaging systems 45 Introductio Penererepereyerepenepertperty 45 Detection of nucleic acids in gel 45 Nucleic acid gel innar em ea em ee dr end 45 Instrument compatibility iier tee ere ni e Ree ates 47 Typical protocol Ans icon eo eter PD enemies cta Ged 47 Expected t sultsza abaco nnm anh eerie enn er ens ia 49 Detection of proteins in gel eee etre EEE AEREN EER Ea 51 aii ri redirent re EFE bro e Ree ere EXT 51 Instrument compatibility eer entr mr eir rer ener erede 52 Typical protocols ze irr Er reete ret pex een ei peniteat a een 53 Protein detection in one dimensional gels esses 53 Protein detection in two dimensional gels eese 55 Expected xesults
51. 532 526SP 488 530DF30 Blue PicoGreen 532 526SP 488 530DF30 Blue RiboGreen 532 526SP 488 530DF30 Blue 3 mm focal depth setting should be used on Typhoon when imaging microplates Typical protocol Amersham Pharmacia Biotech products available for this application Product Product number m Imaging systems Typhoon 8600 see catalogue Storm 840 860 see catalogue FluorImager 595 see catalogue Other materials required Product Vendor m PicoGreen nucleic acid stain Molecular Probes Inc m Clear polystyrene 96 well microplate Corning Costar Corp Suitable clear flat bottomed low fluorescence microplates should be used Image quality and quantification for Storm and Typhoon are improved when using Nalge Nunc PolySorp 96 well plates with removable strips so that the wells sit flat directly on the platen tm 63 0035 28 60 CHAPTER 5 FLUORESCENCE APPLICATIONS Sample preparation Using TE buffer dilute the DNA sample solution to a final volume of at least 50 pl for FluorImager and Storm or 80 pl for Typhoon Note Using a higher dilution of the experimental sample ensures that any contaminants are maximally diluted Each microplate well requires a minimum total volume of 100 pl for FluorImager and Storm or 160 pl for Typhoon Note The performance of PicoGreen is minimally affected by the presence of contaminants such as salts urea ethanol chloroform detergents proteins and agarose For addit
52. 60 nm and a wide range of power from low to moderate output Because LED light emissions are doughnut shaped and not collimated the source must be mounted very close to the sample using lenses to tightly focus the light LEDs are considerably smaller lighter and less expensive than lasers They are available in the visible wavelength range above 430 nm Excitation light delivery Because light from a laser is well collimated and of sufficient power delivery of excitation light to the sample is relatively straightforward with only negligible losses incurred during the process For lasers that produce multiple wavelengths of light the desired line s can be selected by using filters that exclude unwanted wavelengths while allowing the selected line to pass at a very high transmission percentage Excitation filters are also necessary with single line lasers as their output is not 100 pure Optical lenses are used to align the laser beam and mirrors can be used to redirect the beam within the instrument One of the main considera tions in delivering light using a laser scanning system is that the light source is a point while the sample typically occupies a relatively large two dimensional space Effective sample coverage can be achieved by rapidly moving the excitation beam across the sample in two dimensions There are two ways to move and spread the point source across the sample which are discussed below FLUORESCENCE IMAGING
53. 63 0035 28 78 Preliminary preparations and general handling instructions m For superior results with low fluorescence background the optimal antibody dilution for detection of the target protein must be determined 18 Although blocking and washing are important they are only temporary measures until the optimal antibody dilutions are determined For a primary antibody or antiserum of unknown activity use a dot or slot blot to quickly determine optimal antibody dilutions m An excess of buffer should be used for washing steps following blocking and antibody incubations The blot should be agitated during washing and the recommended time interval per wash should be adhered to strictly m PVDF membranes should be kept wet at all times m Successful fluorescent detection protocols require careful control of background by thoroughly blocking and washing the blot A minimum of 2 5 ml of wash solution should be used for every cm of membrane The blot should be incubated in a dish that is sufficiently large for the blot to circulate freely with orbital shaking Alkaline phosphatase based chemistries require particular attention to cleanliness transfer pads and all dishes and containers that come into contact with the blot should be cleaned using a combination of boiling water and ethanol when appropriate eo Preparation of blot Transfer the separated proteins from the gel to the PVDF membrane Block the membrane for at
54. 670 nm Fig 15 e the full width at half maximum transmission FWHM For example a 670BP30 filter passes light over a wavelength range of 30 nm 655 nm 685 nm with an efficiency equal to or greater than half the maximum transmittance of the filter Band pass filters with an FWHM of 20 30 nm are optimal for most fluorescence applications including multi label experiments Filters with FWHMs greater than 30 nm allow collection of light at more wavelengths and give a higher total signal however they are less able to discriminate between closely spaced overlapping emission spectra in multichannel experiments Filters with FWHMs narrower than 20 nm transmit less signal and are most useful with fluorochromes with very narrow emission spectra Using emission filters to improve sensitivity and linearity range When selectable emission filters are available in an imaging system filter choice will influence the sensitivity and dynamic range of an assay In general if image background signal is high adding an interchangeable filter may improve the sensitivity and dynamic range of the assay The background signal from some matrices gels and membranes has a broad relatively flat spectrum In such cases a band pass filter can remove the portion of the background signal comprising wavelengths that are longer or shorter than the fluorochrome emissions By selecting a filter that transmits a band at or near the emission peak of the fluorochrome of
55. 7 131 SYPRO Orange 51 125 127 131 SYPRO Red 51 125 127 131 SYPRO Rose Plus 75 125 128 SYPRO Ruby 51 125 127 131 SYPRO Ruby blot 75 126 128 SYPRO Tangerine 51 126 127 T tetramethylrhodamine 85 126 129 trans illumination 20 113 two dimensional protein gel analysis 36 Typhoon 8600 22 U uniformity 19 113 V Vista Green 45 126 127 131 wavelength 3 113 Western blotting 73 X xenon arc lamp 10 e 139 Trademarks and legal information AlkPhos Direct Cy CyDye ECL Plus Ettan FluorImager FluoroLink FluorSep Hoefer Hybond Hyperfilm ImageMaster ImageQuant Immobiline IPGphor Molecular Dynamics Personal Densitometer Rainbow Ready To Run Storm Typhoon and Vistra Green are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries Amersham is a trademark of Nycomed Amersham plc Pharmacia and Drop Design are trademarks of Pharmacia Corporation Array Vision is a trademark of Imaging Research Inc Alexa Fluor BODIPY NanoOrange OliGreen Oregon Green PicoGreen Rhodamine Green Rhodamine Red RiboGreen SYBR SYPRO Texas Red and TOTO are trademarks of Molecular Probes Inc Coomassie is a trademark of Imperial Chemical Industries Ltd FAM HEX ROX TAMRA and TET are trademarks of Perkin Elmer Corp Kapton is a trademark of DuPont Corporation Kimwipe is a trademark of Kimberly Clark PolySorp is a trademark of Nalge Nun
56. 70BP30 NA NA Red NA NA phosphate ECF 532 526SP 488 570DF30 Blue Reflection UV high ECL Plus cL CL 488 530DF30 Blue CL CL Labels Typhoon Fluorlmager Storm VDS CL Substrate Excitation Emission Excitation Emission Flourescence nm filter nm filter mode Excitation Emission Fluorescein 532 526SP 488 530DF30 Blue Reflection UV low Cy3 532 580BP30 514 570BP30 NA NA NA Cy5 633 670BP30 NA NA Red NA NA Stains Typhoon Fluorlmager Storm VDS CL Substrate Excitation Emission Excitation Emission Flourescence nm filter nm filter mode Excitation Emission SYPRO Rose NA NA NA NA NA Reflection UV high Plus SYPRO Ruby 532 610BP30 488 610RG Blue Reflection UV high blot NA Not applicable t Chemiluminescence only Not applicable for fluorescence m 63 0035 28 76 CHAPTER 5 FLUORESCENCE APPLICATIONS Typical protocols Western blotting using a fluorogenic substrate Amersham Pharmacia Biotech products available for this application Product Product number m Hoefer miniVE Vertical Electrophoresis System 80 6418 77 m Hoefer EPS 301 Power Supply 18 1130 01 m Hybond P PVDF membranes RPN2020F m ECL Plus Western Blotting Detection System RPN2132 m ECF Western Blotting Kit RPN5780 m Imaging systems Typhoon 8600 see catalogue Storm 840 860 see catalogue FluorImager 595 see catalogue ImageMaster VDS CL see catalogue Other materials required Product Vendor DDAO phosphate Molecular Probes Inc FLUORESCENCE IMAGING m
57. Copyright 1980 W H Freeman and Company Reprinted with permission tm 63 0035 28 4 The photon energy at the apex of the excitation peak equals the energy difference between the ground state of the fluorochrome S and a favored vibrational level of the first excited state S of the molecule Fig 4a In some cases the excitation spectrum shows a second peak at a shorter wavelength higher energy that indicates transition of the molecule from the ground state to the second excited state S The width of the excitation spectrum reflects the fact that the fluoro chrome molecule can be in any of several vibrational and rotational energy levels within the ground state and can end up in any of several vibrational and rotational energy levels within the excited state In practice a fluoro chrome is most effectively excited by wavelengths near the apex of its excitation peak For example as shown in Figure 3a the efficiencies with which the three fluorochromes are excited by 488 nm laser light vary as indicated by the relative height of each excitation curve at 488 nm compared with the height at maximum absorption Emission spectrum The relative probability that the emitted photon will have a particular wavelength is described in the fluorochrome s emission spectrum Fig 3b a plot of the relative intensity of emitted light as a function of the emission wavelength In practice the emission spectrum is generated by exciting
58. Fig 8 Galvanometer controlled scanning mechanism Light is emitted from the laser in a single straight line The galvanometer mirror moves rapidly back and forth redirecting the laser beam and illuminating the sample across its entire width X axis The f theta lens reduces the angle of the excitation beam delivered to the sample The entire sample is illuminated either by the galvanometer mechanism moving along the length of the sample Y axis or the sample moving relative to the scanning mechanism m 63 0035 28 14 Galvanometer based systems Galvanometer based systems use a small rapidly oscillating mirror to deflect the laser beam effectively creating a line source Fig 8 By using relatively simple optics the beam can be deflected very quickly resulting in a short scan time Compared to confocal systems galvonometer based scanners are useful for imaging thick samples due to the ability to collect more fluorescent signal in the vertical dimension However since the excitation beam does not illuminate the sample from the same angle in every position a parallax effect can result The term parallax here refers to the shift in apparent position of targets predominately at the outer boundaries of the scan area Additionally the arc of excitation light created by the galvanometer mirror produces some variations in the effective excitation energy reaching the sample at different points across the arc These effects can be minim
59. Fluorescent and Luminescent Probes for Biological Activity Second Edition Academic Press San Diego 1999 Marriott G Meth Enzymol 291 1 529 1998 Tsien Y The Green Fluorescent Protein Ann Rev Biochem 67 509 544 1998 Wells S and Johnson I Fluorescent Labels for Confocal Microscopy in Three Dimensional Confocal Microscopy Volume Investigation of Biological Systems Stevens J K et al eds Academic Press San Diego pp 101 129 1994 Index A absorption 2 109 absorption spectrum 3 109 Alexa Fluor 75 allophycocyanin 99 argon ion laser 12 array and microplate analysis 35 Array Vision software 44 B B phycoerythrin 99 background 36 109 116 background correction 36 band pass BP filter 26 109 bandshift assay 92 beamsplitter 10 17 109 brightness 5 109 C CBQCA 63 CCD camera based system 19 charge coupled device CCD 11 109 chemifluoresence enzyme amplified detection 73 109 chemiluminescence 2 109 collimated 10 110 cone angle 10 110 confocal optics 16 110 cutoff point 25 110 Cy2 86 121 128 132 Cy3 86 121 128 132 Cy3 5 86 121 128 132 CyS 86 121 128 132 5 5 86 121 128 INDEX Cy7 122 128 CyDye 75 D DDAO phosphates 67 73 differential display analysis 86 diode laser 12 110 direct fluorescence detection 75 dwell time 6 7 105 110 dynamic range 21 110 E ECE 67 73 122 127 128
60. ND Cy2 7 5 7 5 30 ND Cy3 0 2 4 NA ND Cy3 5 0 2 ND NA ND Cy5 0 2 ND 1 ND FAM 0 4 0 4 50 ND Fluorescein 0 4 0 4 50 ND HEX 0 2 2 NA ND ROX 0 2 12 NA ND TAMRA 0 2 4 NA ND TET ND 1 NA ND Fluorescent proteins Limit of detection ng protein band in SDS polyacrylamide gel GFP wt 13 2 13 ND GFP S65T ND 0 3 8 ND EGFP ND 0 3 8 ND ND Not determined NA Not applicable t First number from assay performed using Costar flat bottomed plate Second number from assay performed using Nunc Separable Strips Detection limits or sensitivities for Western blots depend on multiple experimental factors including the types and concentrations of protein target and antibodies used Each new detection protocol should be optimised for concentrations of both primary and secondary antibodies CL Chemiluminescence only Not applicable for fluorescence tm 63 0035 28 132 REFERENCES References References cited in text 1 10 11 12 13 14 15 Haugland R P Introduction to Fluorescence Techniques in Handbook of Fluorescent Probes and Research Chemicals Molecular Probes Inc Eugene OR pp 1 4 1996 Rye H S et al Nucl Acids Res 20 2803 2812 1992 Cantor C R and Schimmel P R Biophysical Chemistry Part 2 W H Freeman pp 433 465 1980 O Shea D Callen R W and Rhodes W T in Introduction to Lasers and Their Applications Addison Wesley Reading MA pp 51 78 1978 Smi
61. P30 514 570DF30 NA NA NA Cy3 5 532 610BP30 514 610RG NA NA NA Cy5 633 670BP30 NA NA Red NA NA Cy5 5 633 670BP30 NA NA Red NA NA NA Not applicable tm 63 0035 28 86 Applications and protocols Differential display analysis Differential display is a PCR based technique for studying broad scale gene expression 22 It enables direct side by side comparisons of complex expression patterns from multiple samples in a one dimensional gel format Using reverse transcription the technique resolves the 3 termini of messenger RNA mRNA molecules This step is followed by PCR amplification using additional upstream arbitrary primers PCR products are then separated on high resolution denaturing poly acrylamide gels from which bands of interest can be isolated and further analysed By using multiple primer combinations the differential display method can potentially screen all the expressed genes up to 15 000 different mRNAs in a mammalian cell More importantly the desired PCR product bands can be recovered from the gel and used as probes to isolate and genomic DNA for further molecular characterizations Fluorescent differential display offers fast results and easy quantification due to the proportional relationship between signal and quantity of message 23 Additionally fluorescently labelled PCR primers are stable for relatively long periods Fluorescence digital imaging of differential display gels provides a w
62. Qu manual Fluorescence Imaging principles and methods 63 0035 28 Rev A 2000 12 US 105 amersham phan macia biotech 4310 332 FLUORESCENCE IMAGING Contents Chapter 1 Introduction to 1 Advantages of fluorescent detection eseria a EErEE EEEE 1 Fluorescence prOGess re er ie 2 Properties ol fl orochfomes c eee ente evite novi 3 Excitation and emission Spectra iere Ie Ee HE ETE EHE LER EHE EEG 3 Signal aeter era eee eee 5 Brightness E EN EE ERR TED ped Fen 5 Susceptibility to environmental effects sess 6 Quantification of fluorescence iit eret pe co ei oi Ea 7 Chapter 2 Fluorescence imaging systems 9 Motigo Testo 9 Excitation sources and light delivery optics ccssccssscessecesseceeseceeseeeesees 10 Light treten d eara ntu eee peor edere xcu en 10 Filtration ofthe emitted light aerei 10 Detection amplification and digitization esses 11 Scanner EES EAE EEEE Y FLY VE EXE Y Exe Ce PERDER YS 12 Excitation SOULCES 4 opino DR RO iste ERREUR ERE EE EERR RE PY TEE TERRE CP ERE E CEDE 12 Excitation light Geli very zi eerte retenta ien ertt repe dents 13 Light collectiomass iia
63. TEMS Detection amplification and digitization For detection and quantification of emitted light either a photomultiplier tube PMT or a charge coupled device CCD can be used In both cases photon energy from emitted fluorescent light is converted into electrical energy thereby producing a measurable signal that is proportional to the number of photons detected After the emitted light is detected and amplified the analogue signal from a PMT or CCD detector is converted to a digital signal The process of digitization turns a measured continuous analogue signal into discrete numbers by introducing intensity levels The number of intensity levels is based on the digital resolution of the instrument which is usually given as a number of bits or exponents of 2 8 bit 12 bit and 16 bit digital files correspond to the number of intensity levels allocated within that image file 256 4096 and 65 536 respectively Digital resolution defines the ability to resolve two signals with similar intensities Since only a limited number of intensity levels are available it is unavoidable that this conversion process introduces a certain amount of error To allow ample discrimination between similar signals and to keep the error as low as possible the distribution of the available intensity levels should correspond well to the linear dynamic range of a detector There are two methods of distributing intensity levels A linear even distribution
64. abel LOD LDR LOD LDR LOD LDR LOD LDR ng band fold ng band fold ng band fold ng band fold DDAO phosphate 4 10 4 10 8 10 4 10 4 10 4 10 ECL Plus CL CL 5 30 1 2 30 CL CL Fluorescein 15 30 20 15 30 20 NA NA ND ND Cy3 30 20 30 20 NA NA ND ND Cy5 15 30 20 NA NA 15 30 20 NA NA Results are expressed as limit of detection LOD and linear detection range LDR LOD values are given as ng of tubulin protein Detection limits or sensitivities for Western blots depend on multiple experimental factors including the type and concentrations of protein target and antibodies used Each new Western detection protocol should be optimized for concentrations of both primary and secondary antibodies t NA Not applicable ND Not determined CL Chemiluminescence only Not applicable for fluorescence Temm lt Fig 42 Detection of tubulin using ECL Plus fluorescent signal The blot was imaged using Storm 860 Beta tubulin was detected in a serial two fold dilution of rat brain homogenate that was purified by SDS PAGE and blotted to Hybond P PVDF membrane m 63 0035 28 82 CHAPTER 5 FLUORESCENCE APPLICATIONS Using covalent labels for nucleic acid and protein analysis Because of their characteristic spectral properties fluorochromes that are covalently attached to nucleic acids proteins and antibodies permit the identification and measurement of
65. acquisition using a fluorescence imaging device creates one or more data files for each sample analysed The size of these files will vary depending on sample size and the digital resolution used for acquisition Software is used to display the image adjust the contrast annotate and print the image Image analysis tools allow fragment sizing quantification matching pattern analysis and generation of analysis reports Some software packages also provide access to libraries or a database for sample matching and querying Image utility functions address correction of spectral overlap in multicolour images image filtering rotation pixel inversion and image cropping The purpose of this chapter is to provide an overview of features common to image analysis software packages and to illustrate how the software is applied to different image analysis needs Image display One of the basic functions of an image analysis software package is to enable viewing adjustment and assessment of the acquired image Currently image files usually have at least a 12 bit or 16 bit data structure which means as many as 65 356 gray levels are possible Computer displays printers and humans are only capable of distinguishing approximately 256 gray levels It is necessary for the software to adjust the gray scale so that the objects of interest in the image can be seen Software features allow the user to fine tune the display range without affecting the origi
66. adjust the focal point of the optics Maintain the instrument under proper environmental conditions Keep the instrument in a clean relatively dust free environment and away from direct sunlight heat and air conditioning ducts Maintain the instrument s proper temperature and humidity requirements To avoid electrical noise connect the instrument to a dedicated properly grounded AC circuit An uninterruptible power supply is recommended to prevent malfunction and loss of data caused by unexpected power failures power surges or AC line fluctuations Refer to the instrument user s manual for additional information CHAPTER 6 PRACTICAL RECOMMENDATIONS Data evaluation The digital image acquired from a fluorescent sample should be evaluated for pixel saturation before proceeding to analysis It is also important to apply an appropriate background correction method to the quantification process Check the image for signal saturation If the instrument s control software displays a preview image of the sample monitor the preview and check for saturated data In Scanner Control software saturated data appear as red areas in the image If key areas of the image are saturated and you want to perform quantification on the image you must rescan the fluorescent sample using a lower PMT voltage setting Once the image is captured or acquired it can be displayed using image analysis software Adjust the image contrast settings and assess
67. al for multicolour detection Two of these Cy3 and CyS are popular labels for two channel fluorescence experiments such as gene expression arrays Fluorescence imaging instrumentation with 532 nm and 633 nm laser excitation sources are ideally suited for CyDye imaging Protocol Amersham Pharmacia Biotech products available for this application Imaging systems Typhoon 8600 FluorImager 595 Storm 830 860 Product Product number m Hoefer SE 400 Sturdier Vertical Unit 80 6154 86 Low fluorescence glass plate set 80 6442 14 Hoefer EPS 301 Power Supply 18 1130 01 Cy3 mono Reactive Dye Pack PA23001 5 mono Reactive Dye Pack PA25001 Cy3 dCTP PA53021 Cy5 dCTP 55021 PCR Nucleotide US77212 dNTP Set 100 mM solutions 27 2035 01 Taq DNA Polymerase cloned T0303Y see catalogue see catalogue see catalogue FLUORESCENCE IMAGING m 63 0035 28 90 e Preparation of sample Prepare the reactions in one of the following ways A PCR with CyDye 5 end labelled oligonucleotide primer Stock Volume Final PCR Buffer 5 ul lx 25 mM 3 ul 1 5 mM 10 mM dATP dGTP dTTP dCTP 1 ul 200 uM each Forward primer CyDye labelled 0 5 uM Reverse primer 0 5 uM DNA template 70 ng Taq DNA polymerase 5 units ul 0 2 ul 1 unit Sterile ddH O to a final reaction volume of 50 ul PCR Buffer 500 mM KCI 100 mM Tris Cl pH 9 0 B PCR with CyDye labelled dCTP Stock Volume Final PCR Buffer 5 ul 25 mM
68. anes contain 0 4 180 bp DNA fragmen HEX green or TAMR pmol o ts label A red two different ed with either The third and fourth lanes contain the same two labelled DNA fragments after i protein The fifth lane ncubat ion with Mnt contains mixtures of the bound labelled fragments to demonstrate m Itiplexing in the sa me gel ane yellow colour indicates overlay between green and red signal A 532 nm excitation was used Wi th 555BP30 and 580BP30 emission filters Samples courtesy of Chris Man Washington University School of Medicine Department of Genetics St Louis MO USA CHAPTER 5 FLUORESCENCE APPLICATIONS Storm 830 860 Be sure to use Cy5 labelled primers Remove the glass plate that has been treated with silane Cover the gel with plastic wrap being careful not to trap air bubbles or create wrinkles Place the gel face down on the glass platen Other options transfer the gel to Whatman 3MM filter paper and dry it Use Bind Silane to fix the gel to one glass electrophoresis plate and dry the gel directly on the glass plate Select the appropriate instrument settings for the fluorochrome label used see Table 24 or Appendix 3 Fluorlmager 595 Position the gel sandwich in the universal tray Select the instrument settings appropriate for the fluorochrome label used o Analysis Using image analysis software determine the signal from each shifted
69. as fluorescent antibodies oligonucleotide hybridization probes and PCR primers can be stored for six months or longer antibodies labelled with I become unusable in about a month and P labelled nucleotides and oligonucleotides decay significantly in about a week Because of their long shelf life fluorescently labelled reagents can be prepared in large batches that can be standardized and used for extended periods See licensing information on inside back cover FLUORESCENCE IMAGING Energy S i Vex hvem So Fig 2 Jablonski diagram illustrating the processes involved in creating an excited electronic singlet state by optical absorption and subsequent emission of fluorescence Excitation Vibrational relaxation Emission tm 63 0035 28 2 This minimizes inter assay reagent variability when used in applications such as DNA and protein sizing and quantification enzyme assays immunoassays PCR based genetic typing assays and DNA sequencing Additionally the need for frequent reagent preparation or purchase is eliminated Low hazard Most fluorochromes are easy to handle and in the majority of cases the simple use of gloves affords adequate protection With radioactive materials however lead or acrylic shields may be required In addition since fluorochromes can be broken down by incineration storage or disposal problems are minimal Radioactive wastes on the other hand require shielded storage lon
70. ates are given in Table 18 Fig 37 Southern blot of EcoR digested human genomic DNA B actin cDNA was abelled and detected with ECF Random Prime Labelling and Detection System and imaged on Typhoon 8600 Amount of DNA per lane ranged from 10 4 ug to 0 32 ug prepared in two fold serial dilutions Table 18 Fluorescence based quantification of DNA in genomic Southern blots Typhoon Fluorlmager Storm VDS CL Substrate LOD LDR LOD LDR LOD LDR LOD LDR pg band fold pg band fold pg band fold pg band fold DDAO 0 25 50 NA NA 0 25 50 NA NA phosphate ECF 0 5 25 0 25 50 0 25 50 0 25 ND Results are expressed as limit of detection LOD and linear detection range LDR LOD values are given as amount of target detected t Not applicable tm 63 0035 28 72 Fluorescence 550 570 nm 4 Fluorescent product Excitation Substrate Alkaline v phosphatase Phosphate group 2 antibody 1 antibody Target protein DA Fig 38 Schematic of the ECF Western Blotting Kit Proteins are detected by chemifluorescence using alkaline phosphatase labelled anti species secondary antibody Signal is developed with the ECF substrate CHAPTER 5 FLUORESCENCE APPLICATIONS Western blotting Immunodetection of proteins that have been electrophoretically separated and then immobilized on a membrane is traditionally accomplished using isotope labelled antibodies e g
71. bes Inc e 53 FLUORESCENCE IMAGING tm 63 0035 28 54 Sample preparation For information on sample preparation refer to Amersham Pharmacia Biotech Technical Manual Protein Electropboresis 12 eo Gel electrophoresis Load the prepared samples onto the gel For denaturing conditions use a gel and or running buffer that contains 196 SDS Refer to Amersham Pharmacia Biotech Technical Manual Protein Electrophoresis for further details 12 e Staining the gel For SYPRO Red or Orange prepare a working stain solution by diluting the stain stock solution as supplied 1 5000 in a 7 596 acetic acid solution Prepare enough stain solution to cover the gel 5 10 times the gel volume For SYPRO Tangerine prepare a working stain solution by diluting the stain stock solution as supplied 1 5000 in 50 mM phosphate 150 mM NaCl pH 7 0 For SYPRO Ruby use the stain stock solution as supplied directly without dilution Note For larger gels prepare approximately 10 times the gel volume for staining in order to avoid a loss of sensitivity Stain the gel in a polypropylene container with gentle agitation for 30 min longer staining times may be needed for high percentage acrylamide gels SYPRO stains are not compatible with glass or metal staining trays Cover the staining container with aluminium foil to prevent photobleaching of the stains For SYPRO Red or Orange destain the gel in a 7 5 acetic
72. bilities of a fluorescence imaging instrument The broad selection of available fluorophores also facilitates the design of multi label or multicolour experiments Table 23 See Appendix 3 for an extended list of multipurpose labels e 53 FLUORESCENCE IMAGING Table 23 Properties of common fluorescent labels Extinction Quantum yield Formula weight g mol Colour of Excitation Emission coefficient for protein Bis Fluorophore fluorescence nm max nm M cm conjugates reactive reactive FluorX Green 494 520 68 000 0 30 586 60 Cy2 Green 489 506 150 000 0 12 713 78 896 95 Cy3 Orange 550 570 150 000 0 15 765 95 949 11 Cy3 5 Scarlet 581 596 150 000 0 15 1102 37 1285 54 Cy5 Far Red 649 670 250 000 0 28 791 99 975 15 Cy5 5 Near IR 675 694 250 000 0 28 1128 41 1311 58 Cy7 Near IR 743 767 250 000 0 28 818 02 1001 19 Nucleic acid labelling The fluorescent labelling of DNA and RNA molecules can be achieved in a number of ways The automated chemistry of oligonucleotide synthesis permits the covalent attachment of fluorophores at virtually any position in the single stranded DNA Oligonucleotides can also be designed to exhibit fluorescence resonance energy transfer ET properties 19 In this case the oligonucleotide is modified to contain a pair of fluorochromes donor and acceptor spaced at a defined distance from each other in the DNA molecule Alternatively oligonucleotides ca
73. c International Tween is a trademark of ICI Americas Inc Microsoft and Excel are trademarks of Microsoft Corporation Whatman is a trademark of Whatman International Ltd The Polymerase Chain Reaction PCR is covered by patents owned by Roche Molecular Systems and F Hoffmann La Roche Ltd A license to use the PCR process for certain research and development activities accompanies the purchase of certain reagents from licensed suppliers such as the purchase of certain reagents from licensed suppliers such as Amersham Pharmacia Biotech Limited and affiliates when used in conjunction with an authorized thermal cycler Asia Pacific Tel 852 2811 8693 Fax 852 2811 5251 Australasia Tel 61 2 9894 5152 Fax 61 2 9899 7511 Austria Tel 01 576 0616 25 Fax 01 576 0616 27 Belgium Tel 0800 73 888 Fax 03 272 1637 Canada Tel 1 800 463 5800 Fax 1 800 567 1008 Central East and Southeast Europe Tel 43 1 982 3826 Fax 43 1 985 8327 Denmark Tel 45 16 2400 Fax 45 16 2424 Finland Tel 358 0 9512 39 40 Fax 358 0 9512 17 10 France Tel 01 69 35 67 00 Fax 01 69 41 96 77 Germany Tel 0761 4903 106 Fax 0761 4903 405 Italy Tel 02 27322 1 Fax 02 27302 212 Japan Tel 81 3 5331 9336 Fax 81 3 5331 9370 Latin America Tel 55 11 3667 5700 Fax 55 11 3667 87 99 Middle East and Africa Tel 30 1 96 00 687 Fax 30 1 96 00 693 Netherlands Tel 0165 580 410 Fax 0165 580 4
74. c acid gel stains ds DNA Ethidium Bromide SYBR Gold SYBR Green Vistra Green Nucleic acid gel stains ss DNA Ethidium Bromide SYBR Gold SYBR Green SYBR Green II RNA Vistra Green Protein gel stains SYPRO Orange SYPRO Red SYPRO Ruby Nucelic acids solution stains PicoGreen RiboGreen Protein solution stains NanoOrange APPENDIX 4 Appendix 4 INSTRUMENT PERFORMANCE WITH COMMON FLUOROPHORES AND FLUORESCENT PROTEINS Typhoon Fluorlmager Storm VDS CL Limit of detection pg band in agarose polyacrylamide gel 100 ND 200 100 NA 100 ND 25 10 40 10 500 40 ND 20 25 10 40 10 500 40 ND 20 25 10 40 10 500 40 ND 20 Limit of detection pg band in agarose polyacrylamide gel 5000 ND 10 000 ND NA 5000 ND ND 250 ND 300 ND 1000 ND ND 250 ND 300 ND 1000 ND 10 000 ND 10 000 ND 100 000 ND ND ND 250 ND 300 ND 1000 ND Limit of detection ng band 3 6 5 2 3 ND 5 7 Limit of detection ng ml 10 2 5 5 50 ND ND 1 10 ND Limit of detection ug ml 1 0 3 0 5 1 ND e 131 FLUORESCENCE IMAGING Fluorophore Typhoon Fluorlmager Storm VDS CL Substrates for Northern and Southern detection Limit of detection pg target DDAO phosphate 0 25 NA 0 25 NA ECF 0 5 0 25 0 25 0 25 Substrates for Western blotting Limit of detection ng target DDAO phosphate 4 NA 4 NA ECF 8 4 4 4 ECL Plus CL 5 1 2 CL Multipurpose labels Limit of detection fmol DNA band in polyacrylamide gel Alexa Fluor 430 NA 200 100
75. ce emission Fluorescence excitation Fluorescence excitation Fluorescence excitation Fluorescence excitation APPENDIX 2 SYBR Goldt 495 537 250 300 350 400 450 500 550 600 Wavelength nm Fluorescence emission SYBR Green IIt N 520 250 300 350 400 450 500 550 600 Wavelength nm Fluorescence emission SYPRO Red 550 630 wii 250 300 350 400 450 500 550 600 650 700 750 Wavelength nm Fluorescence emission SYPRO Ruby 280 250 300 350 400 450 500 550 600 650 700 750 Wavelength nm Fluorescence emission e 125 FLUORESCENCE IMAGING SYPRO Ruby blot SYPRO Ruby IEF 280 618 280 610 Fluorescence excitation S Fluorescence emission Fluorescence excitation Fluorescence emission D 1 1 D 1 1 250 300 350 400 450 500 550 600 650 700 750 250 300 350 400 450 500 550 600 650 700 750 Wavelength nm Wavelength nm SYPRO Tangerine TAMRA 490 640 555 580 Fluorescence excitation Fluorescence emission Fluorescence excitation Fluorescence emission 1 1 1 1 1 250 300 350 400 450 500 550 600 650 700 750 400 450 500 550 600 650 700 750 800 Wavelength nm Wavelength nm Tetramethylrhodamine Texas Red X 555 580 595 615 Fluorescence excitation Fluorescence emission Fluorescence excitation Fluorescence emission 400 450 500 550 600 650 700 750 800 400 450 500 550 600 650 700 750 800 Wavelength nm Wavelength nm Vistra Green ae Spectra were obtained for the product of the enz
76. cia Biotech offers a variety of imaging instrumentation including laser scanning and CCD based systems A brief description of each instrument is given in Table 1 For more information please visit www apbiotech com e 21 FLUORESCENCE IMAGING tm 63 0035 28 22 Table 1 Amersham Pharmacia Biotech imaging systems TYPHOON 8600 High performance laser scanning system Excitation sources 532 nm Nd YAG and 633 nm HeNe lasers Filters 6 emission filters and 2 beamsplitters up to 13 emission filter positions Detection 2 high sensitivity PMTs Imaging modes 4 colour automated fluorescence detection direct chemiluminescence storage phosphor Scanning area 35 x 43 cm Sample types Gel sandwiches agarose and polyacrylamide gels blots microplates TLC plates and macroarrays STORM 830 840 oR 860 Variable mode laser scanning system Excitation sources 450 nm LED and or 633 nm laser diode Filters 2 built in emission filters Detection High sensitivity PMT Imaging modes Blue and or red excited fluorescence storage phosphor and chemifluorescence Scanning area 35 x 43 cm Sample types Gels blots microplates TLC plates and macroarrays CHAPTER 2 FLUORESCENCE IMAGING SYSTEMS Table 1 continued FLUORIMAGER 595 Dedicated fluorescence laser scanning system Excitation sources 488 nm and 514 nm laser lines of argon ion laser Filters 4 selectable emission filte
77. d fluorescent light as possible Laser light that is reflected or scattered by the sample is generally rejected from the collection pathway by a laser blocking filter designed to exclude the light produced by the laser source while passing all other emitted light Light collection schemes vary depending on the nature of the excitation system With galvanometer systems the emitted fluorescence must be gathered in a wide line across the sample This is usually achieved with a linear lens fibre bundle or light bar positioned beneath the sample that tracks with the excitation line collecting fluorescence independently at each pixel Although this system is effective it can produce image artefacts At the edges of the scan area where the angle of the excitation beam relative to the sample is farthest from perpendicular some spatial distortion may occur Where very high signal levels are present stimulation of fluorescence from sample areas that are adjacent to the pixel under investigation can result in an inaccurate signal measurement from that pixel an artefact known as flaring or blooming FLUORESCENCE IMAGING Fig 10 Illustration of confocal optics Fluorescence from the sample is collected by an objective lens and directed toward a pinhole aperture The pinhole allows the emitted light from a narrow focal plane red solid lines to pass to the detector while blocking most of the out of focus light black dashed lines m 6
78. d light becomes a function of how long the excitation beam continues to illuminate those fluorochrome molecules dwell time If the dwell time is long relative to the lifetime of the excited state each fluorochrome molecule can undergo many excitation and emission cycles Measuring fluorescent light intensity emitted photons can be accomplished with any photosensitive device For example for detection of low intensity light a photo multiplier tube or PMT can be used This is simply a photoelectric cell with a built in amplifier When light of sufficient energy hits the photocathode in the PMT electrons are emitted and the resulting current is amplified The strength of the current is proportional to the intensity of the incident light The light intensity is usually reported in arbitrary units such as relative fluorescence units rfu For additional information please see the General References section of this manual FLUORESCENCE IMAGING im 63 0035 28 8 Fig 6 Components of a general fluorescence imaging system CHAPTER 2 FLUORESCENCE IMAGING SYSTEMS Chapter 2 FLUORESCENCE IMAGING SYSTEMS Introduction All fluorescence imaging systems require the following key elements Excitation source Light delivery optics Light collection optics Filtration of the emitted light Detection amplification and digitization The design and components of a typical fluorescence detection system are illustrated in Fi
79. d this range to 200 900 nm System performance The performance of a laser scanner system is described in terms of system resolution linearity uniformity and sensitivity Resolution can be defined in terms of both spatial and amplitude resolution Spatial resolution refers to the number of data points sampled per unit length or area It is a function of the diameter of the light beam when it reaches the sample and the distance between adjacent measurements Spatial resolution is dependent on but not equivalent to the pixel size of the image Spatial resolution improves as pixel size reduces Systems with higher spatial resolution can not only detect smaller objects but can also discriminate more accurately between closely spaced targets However an image with a 100 pm pixel size will not have a spatial resolution of 100 pm The pixel size refers to the collection sampling interval of the image According to a fundamental sampling principle the Nyquist Criterion the smallest resolvable object in an image is no better than twice the sampling interva 6 Thus to resolve a 100 pm sample the sampling interval must be at most 50 pm Amplitude resolution or gray level quantification describes the minimum difference that is distinguishable between levels of light intensity or fluorescence detected from the sample 7 For example an imaging system with 16 bit digitization can resolve and accurately quantify 65 536 different values of lig
80. ducts available for this application Product Product number m Hoefer miniVE Vertical Electrophoresis System 80 6418 77 m Hoefer EPS 301 Power Supply 18 1130 01 Hybond P PVDF membranes RPN2020F CyDye FluoroLink Antibody Labelling Kits see catalogue Fluor Linked secondary antibodies see catalogue m Imaging systems Typhoon 8600 see catalogue Storm 840 860 see catalogue FluorImager 595 see catalogue ImageMaster VDS CL see catalogue Preliminary preparations and general handling instructions m An excess of buffer should be used for washing steps following blocking and antibody incubations The blot should be agitated during washing and the recommended time interval per wash should be adhered to strictly m PVDF membranes should be kept wet at all times m Successful fluorescent detection protocols require careful control of background by thoroughly blocking and washing the blot A minimum of 2 5 ml of wash solution should be used for every cm of membrane The blot should be incubated in a dish that is sufficiently large for the blot to circulate freely with orbital shaking m Depending on the fluorochrome air drying a direct fluorescent Western blot may be possible and may even improve the signal to noise ratio of the acquired image The suitability of drying the blot should be determined with each fluorochrome CHAPTER 5 FLUORESCENCE APPLICATIONS eo Preparation Transfer the separated proteins from th
81. e LDR First number from assay performed using Costar flat bottomed plate Second number from assay performed using Nunc Separable Strips ND Not determined CHAPTER 5 FLUORESCENCE APPLICATIONS Quantification of proteins in solution Dyes for quantification of proteins in solution Protein concentration in solution can be determined directly by measuring the absorbance of the solution at 280 nm or indirectly by using colourimetric assays Both methods however have some limitations For example the sensitivity of the absorbance method is limited because detection depends on the number of aromatic amino acid residues present Colourimetric methods such as the Bradford and Lowry assays do not work well in the presence of contaminants and must be read within a very limited period of time 14 High protein to protein signal variability is also common with colourimetric detection Proteins can be more accurately detected in solution using fluorescent dyes Table 13 As free molecules the dyes are not very fluorescent but when they bind to proteins they exhibit enhanced fluorescence Because they are typically quite specific for their target molecules these fluoro chromes work well even in the presence of various contaminants For example the dye NanoOrange binds specifically to the detergent coating on proteins and to hydrophobic regions of proteins and is not affected by the presence of contaminating nucleic acids or red
82. e gel to the PVDF membrane Block the membrane for at least 1 h at room temperature Incubate the blot for 1 h with primary antibody against the target protein Wash the membrane Note If the primary antibody is fluorochrome labelled skip the next step Incubate the blot with fluorochrome linked secondary antibody for 1 h and wash After the final washing step either seal the blot in a low fluorescence bag or page protector or air dry if appropriate e Imaging Place the sealed wet blot or dry blot sample side down on the glass platen Storm Typhoon glass tray FluorImager or platform VDS CL of the imager Note Water can be used between the plastic bag and the platform to minimize the occurrence of interference patterns in the image Use a glass plate to hold the blot flat during imaging Acquire the image according to the recommended instrument setup The choice of pixel size and PMT voltage settings will depend on the individual experiment Adjust the PMT voltage setting to prevent signal saturation o Analysis See Chapter 4 for information concerning image analysis 81 FLUORESCENCE IMAGING Typical results for fluorescent Western detection are given in Table 22 A Western blot developed with ECL Plus substrate and imaged using Storm is shown in Figure 42 Table 22 Expected results for fluorescent Western detection of tubulin Typhoon Fluorlmager Storm VDS CL Substrate L
83. e of 80 v v acetone 1 v v 2 mercapto ethanol and leave in the freezer for a few hours Collect the precipitate as before and discard the supernatant Air dry the pellet and resuspend in 10 ml of 8 M urea 2 w v CHAPS with sonication to aid solubilization Clarify the extract by centrifugation at 105 000 x g for 30 min eo Gel electrophoresis Prepare Immobiline Drystrip gels 18 cm pH 4 7 and IPGphor system according to manufacturer s instructions Dilute protein extract 100 pg of total protein in 350 pl with rehydration solution 8 M urea 2 w v CHAPS 20 mM DTT 2 v v pH 4 7 IPG buffer trace of bromophenol blue Load proteins using rehydration loading for 12 h at 20 C Separate samples using the following running conditions 500 V for 500 Vhr 1000 V for 1000 Vhr 8000 V for 60 000 Vhr After the first dimension separation is complete equilibrate each strip first with SDS equilibration solution containing 1 w v DTT for 15 min then with 2 5 w v iodoacetamide for 15 min Load the equilibrated strips onto a 1 mm 12 5 Laemmli gel cast for the Ettan DALT II system 13 Run the two dimensional gel at 5 W gel for 45 min and then at 26 67 W gel until the bromophenol blue dye front runs off the gel CHAPTER 5 FLUORESCENCE APPLICATIONS e Gel staining Incubate the gel in 40 ethanol 10 acetic acid for 30 60 min then incubate overnight at room temperature with gentle agitation in 5 1
84. e spots remain clean the glass first with 75 ethanol and then with distilled water Household glass cleaners should not be used for cleaning because they contain ingredients that fluoresce Volatile organic solvents such as acetone and the excessive use of ethanol should be avoided since they can damage the glass surface e 103 FLUORESCENCE IMAGING tm 63 0035 28 104 Protect glass from scratches Scratches in the glass platen or tray will scatter laser light and collect dirt solutions that will interfere with data collection and quantification Gently place samples on the glass to prevent scratches When handling glass electrophoresis plates on a glass tray or platen take care not to scratch the platen Handle the sample with powder free gloves Dust and powder fluoresce and scatter light which cause image artefacts To avoid this wear powder free gloves and rinse gloves with distilled water before handling samples and preparing reagents Change gloves often to prevent contamination of samples and reagents Use a low fluorescence bag sheet protector for placing membranes on the glass platen A bag sheet protector is used to cover membranes to avoid contaminating the glass platen or tray and to prevent contamination of the membrane Lay one edge of the membrane down inside an open bag sheet protector then slowly lower the entire membrane while working any bubbles out to the edges of the membrane Close the bag sheet pr
85. een Ultrasensitive gel stain for ss or dsDNA or RNA SYBR Green 497 520 Green Ultrasensitive gel stain for dsDNA and oligonucleotides SYBR Green IlI 497 520 Green Ultrasensitive gel stain for RNA and ssDNA Vistra Green 495 520 Green Ultrasensitive gel stain for dsDNA and oligonucleotides Table 4 Instrument settings for use with nucleic acid gel stains Typhoon Fluorlmager Storm VDS CL Stain Excitation Emission Excitation Emission Fluorescence nm filter nm filter mode Excitation Emission Ethidium bromide 532 610BP30 514 610RG NA Transmission UV high SYBR Gold 532 526SP 488 530DF30 Blue Transmission UV low SYBR Green 532 526SP 488 530DF30 Blue Transmission UV low SYBR Green 11 532 526SP 488 530DF30 Blue Transmission UV low Vistra Green 532 526SP 488 530DF30 Blue Transmission UV low NA Not applicable t UV high 580BP30 filter UV low 520BP30 filter tm 63 0035 28 46 CHAPTER 5 FLUORESCENCE APPLICATIONS stained with these dyes require no destaining before imaging Post stain processing of nucleic acids such as restriction digests and blot transfers is possible as these stains do not interfere with these techniques These stains also pose less of a health risk than ethidium bromide because they are less mutagenic Table 3 lists different nucleic acid gel stains and the nucleic acids with which each is compatible Instrument compatibility Fluorescence imaging systems from Amersham Phar
86. efficient Quantum relative Protein max nm max nm colour M cm yield brightness EBFP 380 440 Blue 31 000 0 18 1x ECFP 434 477 Blue 26 000 0 40 GFP wt 395 470 508 Green 1x GFP S65T 488 511 Green 4 6x EGFP 489 508 Green 55 000 0 60 35x EYFP 514 527 Yellow green 84 000 0 61 35x DsRed 558 583 Red 22 500 0 23 6x Instrument compatibility Table 26 Instrument settings for use with GFP and its variants Typhoon Fluorlmager Storm VDS CL Protein Excitation Emission Excitation Emission Fluorescence Excitation Emission nm filter nm filter mode EBFP NA NA NA NA NA NA ECFP NA NA NA NA Blue NA NA GFP wt 532 526SP 488 530DF30 Blue R T UV low GFP S65T 532 526SP 488 530DF30 Blue R T UV low EGFP 532 526SP 488 530DF30 Blue NA NA EYFP 532 555BP20 488 530DF30 NA NA NA DsRed 532 580BP30 514 570BP30 NA NA NA NA Not applicable t R T Reflection opaque samples Transmission clear samples FLUORESCENCE IMAGING Fig 46 Varying expression levels from GFP reporter constructs in yeast colonies Colonies were spotted on agar plates and incubated at 37 C The agar plate was placed in a microplate tray and scanned using Fluorlmager at a resolution of 100 um Image kindly provided by Drs John Phillips and Matt Ashby Acacia Biosciences Richmond CA Fig 47 GFP gel shift assay showing quantification of the interaction between S protein and S15 GFP S65T His using
87. eloped and optimized for sensitive detection of proteins resolved in this format Amersham Pharmacia Biotech products available for this application Product Product number Immobiline DryStrip pH 4 7 18 cm 17 1233 01 m IPG Buffer pH 4 7 17 6000 86 m IPGphor IEF System 80 6414 02 m Ettan DALT II Large Vertical System see catalogue m Imaging systems Typhoon 8600 see catalogue Storm 840 860 see catalogue FluorImager 595 see catalogue ImageMaster VDS CL see catalogue Other materials required Product Vendor m Protein samples prepared in appropriate loading buffer m SYPRO Ruby protein gel stain Molecular Probes Inc e 55 FLUORESCENCE IMAGING tm 63 0035 28 56 e Sample preparation Note The following is a general procedure for analysis of E coli proteins For additional information refer to Amersham Pharmacia Biotech Technical Manual 2 D Electrophoresis using Immobilized pH Gradients 13 Suspend 400 mg of lyophilized E coli in 10 ml of 8 M urea 4 w v CHAPS 20 mM triethanolamine Cl pH 8 0 20 mM dithiothreitol DTT 1 mM PMSE Sonicate the suspension for a few seconds per burst chill on ice between bursts Repeat until maximum clarification is observed Precipitate the sonicate overnight at 40 C with 80 ml acetone 10 ml 10096 w v trichloroacetic acid 1 ml 2 mercaptoethanol Collect the precipitate by centrifugation at 105 000 x g for 20 min Wash the pellet with the same volum
88. en two detectors are available the combined or mixed fluorescence from two different labels is collected at the same time and then resolved by filtering before the signal reaches the detectors Implementation of dual detection requires a beamsplitter filter to spectrally split the mixed fluorescent signal directing the resulting two 580BP30 610BP30 Emission 500 550 600 650 700 Wavelength nm Fig 20 Emission spectra of TAMRA orange and ROX red A 580BP30 filter dark gray was used for TAMRA and a 610BP30 filter light gray was used for ROX CHAPTER FLUOROCHROME AND FILTER SELECTION emission beams to separate emission filters optimal for each fluorochrome and finally to the detectors A beamsplitter or dichroic reflector is specified to function as either a short pass or long pass filter relative to the desired transition wavelength For example a beamsplitter that reflects light shorter than the transition wavelength and passes longer wavelengths is effectively acting as a long pass filter Fig 11 Fluorochrome selection in multicolour experiments When designing multicolour experiments two key elements must be considered the fluorochromes used and the emission filters available As with any fluorescence experiment the excitation wavelength of the scanner must fall within the absorption spectrum of the fluorochromes used Additionally the emission spectra of different fluorochromes selected for an experi
89. er SI system In another study using Storm system for imaging fusion proteins created between calmodulin CaM or calmodulin like protein CLM and the GFP S65T variant were used in a gel overlay assay to rapidly screen for interacting proteins 31 Expected results The detection limits for purified wild type GFP EGFP and GFP S65T Table 27 were determined by gel electrophoresis using Typhoon Storm and FluorImager systems The greatest sensitivity for detection of GFP S65T and EGFP was achieved using FluorImager system 488 nm excitation The same two GFPs were also detected at the lowest concentrations when imaged using Storm in the blue fluorescence mode followed by the wild type protein Wild type GFP was less compatible than the other variants when imaged using FluorImager system The linear range of detection for each GFP was between 1 5 and 3 orders of magnitude CHAPTER 5 FLUORESCENCE APPLICATIONS Table 27 Detection limits of GFP and variants Limit of detection ng Protein Typhoon Fluorlmager Storm GFP wt 13 2 13 GFP S65T ND 0 3 EGFP ND 0 3 ND Not determined Phycobiliproteins Phycobiliproteins are stable highly soluble fluorescent proteins derived from cyanobacteria and eukaryotic algae Table 28 These proteins contain covalently linked tetrapyrrole groups that play a biological role in collecting light and through fluorescence resonance energy transfer conveying it to a special pair
90. er on the outside of the long glass plate FLUORESCENCE IMAGING Fig 43 Differential display analysis cDNA from rat liver and lung tissue was labelled with Cy5 and electrophoresed on a 6 denaturing polyacrylamide gel The red box surrounds one species of cDNA that is differentially expressed in both tissue types The image was acquired using Typhoon 8600 tm 63 0035 28 88 Place water between the glass plate and the glass platen to minimize the appearance of interference patterns Avoid trapping air bubbles between the glass plate and the platen Select the appropriate settings for laser excitation and emission filter see Table 24 or Appendix 3 Select a focal plane of 3 mm Storm 830 860 Be sure to use Cy5 labelled primers Remove the glass plate that has been treated with silane Cover the gel with plastic wrap being careful not to trap air bubbles or create wrinkles Place the gel face down on the glass platen Other options transfer the gel to Whatman 3MM filter paper and dry it Use Bind Silane to fix the gel to one glass electrophoresis plate and dry the gel directly on the glass plate Select the appropriate instrument settings for the fluorochrome label used see Table 24 or Appendix 3 Fluorlmager 595 Position the gel sandwich in the universal tray Select the instrument settings appropriate for the fluorochrome label used see Table 24 or Appendix 3 o Recovering the gene fragments Us
91. es and their excitation and emission maxima and spectra as well as the appropriate instrument set up with Amersham Pharmacia Biotech fluorescence scanning systems e 27 FLUORESCENCE IMAGING Fig 18 Two colour fluorescent Western blot B galactosidase was detected using a Cy5 labelled secondary antibody red and tubulin was detected using an enzyme amplified chemistry with the fluorogenic ECF substrate green Storm 860 was used for image acquisition Fig 19 Three colour gel image of a DNA in lane sizing experiment The fluorochromes used were TAMRA yellow ROX red and fluorescein green The ROX and TAMRA bands are labelled DNA size ladders The fluorescein fragments are PCR products of unknown size Typhoon 8600 was used for image acquisition tm 63 0035 28 28 Multicolour imaging Multicolour imaging allows detection and resolution of multiple targets using fluorescent labels with different spectral properties The ability to multiplex or detect multiple labels in the same experiment is both time and cost effective and improves accuracy for some assays Analyses using a single label can require a set of experiments or many repetitions of the same experiment to generate one set of data For example single label analysis of gene expression from two different tissues requires two separate hybridizations to different gene arrays or consecutive hybridizations to the same array with stripping and
92. es eei be e e e tees ee esee eee ERE tue EAE 15 Signal detection and amplification esee 17 Systemi performance oes E epe E D acie E ERE CERE 18 camera based SY Steins 19 Excitation sources and light delivery eese 20 L3ght collection s Leiter mieten rere 20 Signal detection and amplification 20 System 20 Amersham Pharmacia Biotech imaging systems essere 21 Chapter 3 Fluorochrome and filter selection 25 Motigo Testo 25 Py PES Ok ener eee suc rco n EEEE PO CPUS 25 Using emission filters to improve sensitivity and linearity range 26 General guidelines for selecting fluorochromes and filters 27 Singlecolour imaging Pea eee 27 Multicolout imaging sssr eese curo ene evene to ets ere be b ere eee eara 28 Chapter 4 Image analysis esee 31 eee recepere oe ree east ee ree oen ves 31 Image ie e EISE Oaa NEESER EREEREER ENERALE FECE ERES 31 Image documentatiofi epe tree tote ei
93. es to assess the signal values across the image If saturated values are present in the image consider rescanning the sample using a lower PMT voltage setting VDS CL System Why can t focus on my image The sample may not be centred on the tray Centre the sample on the tray in the middle of the imaging area The autofocus algorithm requires a sharp edge as a reference for focusing If the lens is zoomed in and the edge of the sample is not visible autofocus will not function properly To avoid this situation place a piece of white paper e g a business card adjacent to the area of interest on the sample The object or sample may be too thick Make sure the object or sample is no thicker than 3 mm for an iris below 1 8 For thicker samples use a higher iris value and increase acquisition time accordingly Why does my image appear dirty fuzzy or uneven The sample tray or the optical surfaces may need cleaning The signal acquisition time may have been too short and should be increased The sample is too thick or is not flat on the surface of the tray Place a glass plate over dry samples to flatten them Remove any bubbles from below a wet gel Can sensitivity be improved by extending the exposure time The signal from a sample is integrated over time The sensitivity improves with exposure time but only up to a point The instrument noise dramatically affects the linearity of the CCD at low light intensity and long e
94. g Place the microplate in the microplate tray FluorImager or directly onto the platen Storm and Typhoon Acquire the image according to the recommended instrument set up for the fluorochrome used The choice of pixel size will depend on the individual experiment The PMT voltage setting should be adjusted to prevent signal saturation For Typhoon the 3 mm focal plane setting should always be selected for imaging microplates o Analysis Display the image using ImageQuant If saturated pixels are present the microplate should be rescanned at a lower PMT voltage setting Use the Gray Color Adjust function to adjust image contrast Ellipse objects can be used to quantify integrated signal from the microplate wells Draw an ellipse object within the inner walls of one well and copy it to the other wells Report the median values with background correction set to None In Microsoft Excel subtract the median value of the negative control well from each of the other wells This is important for good low end linearity Generate a standard curve from the protein standards used Note For the greatest accuracy the protein standards should be similar to the unknown protein i e similar size and source Determine the unknown protein concentration by extrapolating from the standard curve e 65 FLUORESCENCE IMAGING a 1 Fig 35 Detection of protein in solution using anoOrange and Typhoon 8600 with Nu
95. g term decay or regulated landfill disposal Commercial availability A variety of biologically important molecules are available cross linked to fluorochromes including monoclonal and polyclonal antibodies Some suppliers even offer a choice of a specific fluorochrome as label on a given molecule Other commercially available molecules include nucleotides and enzyme substrates such as fluorescent chloramphenicol for chloramphenicol acetyl transferase CAT assays and fluorescein digalactoside for B galactosidase assays lacZ gene Lower cost Long shelf life and lower costs for transportation and disposal of fluorochromes make fluorescent labelling in many cases less expensive than radiolabelling Fluorescence process Fluorescence results from a process that occurs when certain molecules generally polyaromatic hydrocarbons or heterocycles called fluorophores fluorochromes or fluorescent dyes absorb light The absorption of light by a population of these molecules raises their energy level to a brief excited state As they decay from this excited state they emit fluorescent light The process responsible for fluorescence is illustrated by a simple electronic state diagram Fig 2 Excitation When a photon of energy hv x supplied by an external source such as a lamp or a laser is absorbed by a fluorophore it creates an excited unstable electronic state S This process distinguishes fluorescence from chemiluminescence
96. gth nm Rhodamine Green 505 527 Fluorescence excitation 300 350 400 450 500 550 600 650 700 Wavelength nm RiboGreent 500 525 Fluorescence excitation 1 1 1 1 350 400 450 500 550 600 650 700 750 Wavelength nm tm 23 4567 01 124 Fluorescence emission Fluorescence emission Fluorescence emission Fluorescence emission Fluorescence excitation Fluorescence excitation Fluorescence excitation gt Fluorescence excitation Oregon Green 488 496 524 D D 1 1 1 350 400 450 500 550 600 650 700 750 Wavelength nm PicoGreent 502 523 D 1 1 1 350 400 450 500 550 600 650 700 750 Wavelength nm Rhodamine Red X 570 590 350 400 450 500 550 600 650 700 750 Wavelength nm ROX 578 604 AM 400 450 500 550 600 650 700 750 800 Wavelength nm Fluorescence emission Fluorescence emission Fluorescence emission Fluorescence emission Fluorescence excitation Fluorescence excitation Fluorescence excitation Fluorescence excitation R Phycoerythrin 565 578 1 1 1 1 I 350 400 450 500 550 600 650 700 750 Wavelength nm SYBR Green It 497 520 1 1 1 1 250 300 350 400 450 500 550 600 650 Wavelength nm SYPRO Orange 470 570 1 1 1 I 1 250 300 350 400 450 500 550 600 650 700 750 Wavelength nm SYPRO Rose Plus 350 610 ul 300 400 500 600 700 Wavelength nm Fluorescence emission Fluorescence emission Fluorescence emission Fluorescen
97. gure 6 The following paragraphs provide additional details concerning the elements that comprise the system Sample Light delivery optics Light collection optics Emission filter Excitation source Detection and amplification FLUORESCENCE IMAGING 532 Relative output a u 300 500 700 900 1100 Wavelength nm Fig 7 Spectral output of light from a xenon lamp and Nd YAG laser The relative output axis is scaled arbitrarily for the two light sources The 532 nm line of the Nd YAG laser is shown in green m 63 0035 28 10 Excitation sources and light delivery optics Light energy is essential to fluorescence Light sources fall into two broad categories wide area broad wavelength sources such as UV and xenon arc lamps and line sources with discrete wavelengths such as lasers Fig 7 Broad wavelength excitation sources are used in fluorescence spectrometers and camera imaging systems Although the spectral output of a lamp is broad it can be tuned to a narrow band of excitation light with the use of gratings or filters In contrast lasers deliver a narrow beam of collimated light that is predominantly monochromatic In most camera systems excitation light is delivered to the sample by direct illumination of the imaging field with the excitation source positioned either above below or to the side of the sample Laser based imaging systems on the other hand use more sophisticated optical paths comp
98. h high sensitivities for different fluoresecent labels are very cost effective because they enable analyses that require less label and consume less sample CCD camera based systems CCD charge coupled device based cameras are composed of an illumination system and a lens assembly that focuses the image onto the light sensitive CCD array Fig 13 CCD camera based systems are area imagers that integrate fluorescent signal from a continuously illuminated sample field Most of these systems are designed to capture a single view of the imaging area using lens assemblies with either a fixed or selectable focal distance Forced air cooler Multi stage peltier Fixed or zoom lens Emission filter wheel Upper excitation Upper excitation Sample Sample tray E Lower excitation FLUORESCENCE IMAGING m 63 0035 28 20 Excitation sources and light delivery Illumination or excitation in CCD camera systems is provided by ultraviolet UV or white light gas discharge tubes broad spectrum xenon arc lamps or high power narrow bandwidth diodes Light is delivered to the sample either from below trans illumination or from above epi illumination Even with the broadband light sources used in CCD camera systems wavelength selection is possible through the use of appropriate filters Light collection Lenses are used to collect fluorescent emission from the illuminated imaging field A lens s
99. h or area Spatial resolution is a function of the distance between adjacent measurements the difference in wavelength between the apex of the excitation spectrum shorter wavelength higher energy and the apex of the emission spectrum longer wavelength lower energy delivery of light through a sample with detection of the resulting signal from the opposite side the passage of light through a filter element describes the evenness of illumination or collection of light from an imaging area A the distance in nanometers between nodes in a wave of light Wavelength is inversely proportional to the energy of the light ec 1 E e 113 FLUORESCENCE IMAGING tm 63 0035 28 114 APPENDIX 1 Appendix 1 FREQUENTLY ASKED QUESTIONS Typhoon Storm and Fluorlmager Systems How do clean the glass sample tray glass platen or sample lid To clean the platen and sample lid Storm Typhoon or glass tray FluorImager dampen a lint free cloth with distilled water and wipe the surfaces Alternatively you can use a lint free cloth dampened with 75 ethanol to wipe the surfaces and then wipe the surface again using distilled water Because laboratory alcohol formulations may contain residue that is highly fluorescent make sure that surfaces cleaned with alcohol are always wiped with distilled water afterwards How is it possible to use 532 nm laser light to excite fluorescein and similar dyes Fluorochro
100. has the same spacing for all the intensity levels allowing measurement across the dynamic range with the same absolute accuracy However relative digitization error increases as signals become smaller A non linear distribution e g logarithmic or square root functions divides the lower end of the signal range into more levels while combining the high end signals into fewer intensity levels Thus the absolute accuracy decreases with higher signals but the relative digitization error remains more constant across the dynamic range FLUORESCENCE IMAGING m 63 0035 28 12 Scanner systems Excitation sources Most fluorescence scanner devices used in life science research employ laser light for excitation A laser source produces a narrow beam of highly monochromatic coherent and collimated light The combination of focused energy and narrow beam width contributes to the excellent sensitivity and resolution possible with a laser scanner The active medium of a laser the material that is made to emit light is commonly a solid state glass crystal liquid or gas 4 Gas lasers and solid state lasers both provide a wide range of specific wavelength choices for different imaging needs Other light sources used in imaging scanners include light emitting diodes LEDs which are more compact and less expensive than lasers but produce a wide band low power output Lasers Argon ion lasers produce a variety of wavelengths including
101. ht Fixed or interchangeable optical filters that are suitable for the emission profile of the fluorochromes are then used to refine the emitted fluorescence such that only the desired wavelengths are passed to the detector Matching a fluorochrome label with a suitable excitation source and emission filter is the key to optimal detection efficiency In this chapter details about the classes and use of emission filters are presented along with general guidelines for selecting fluorochromes and emission filters for both single colour and multicolour imaging Types of emission filters The composition of emission filters used in fluorescence scanners and cameras ranges from simple coloured glass to glass laminates coated with thin interference films Coated interference filters generally deliver excellent performance through their selective reflection and transmission effects Three types of optical emission filters are in common use Long pass LP filters pass light that is longer than a specified wavelength and reject all shorter wavelengths A good quality long pass filter is characterized by a steep transition between rejected and transmitted wavelengths Fig 14a Long pass filters are named for the wavelength at the midpoint of the transition between the rejected and transmitted light cutoff point For example the cutoff point in the transmission spectrum of a 560LP filter is 560 nm where 5096 of the maximum transmittance is rejected
102. ht intensity from a fluorescent sample Linearity of a laser scanner is the signal range over which the instrument yields a linear response to fluorochrome concentration and is therefore useful for accurate quantification A scanner with a wide dynamic range can detect and accurately quantify signal from both very low and very high intensity targets in the same scan The linear dynamic range of most laser scanner instruments is between 10 and 10 Fig 13 Components of a typical CCD camera based imaging device The sample can be illuminated in a variety of ways depending on the nature of the labels to be analysed The sample is then viewed by the camera The camera includes focusing optics to accommodate samples at different heights Emission filters can be inserted in the light path to select specific wavelengths and eliminate background CHAPTER 2 FLUORESCENCE IMAGING SYSTEMS Uniformity across the entire scan area is critical for reliable quantification A given fluorescent signal should yield the same measurement at any position within the imaging field Moving head scanners in particular deliver flat field illumination and uniform collection of fluorescent emissions across the entire scan area Sensitivity or detection threshold is a measure of the lowest fluorescent signal that can be detected by the instrument Increased sensitivity aids the detection of low abundance targets From an economical standpoint instruments wit
103. ide linear dynamic range and high sensitivity With its high resolution and magnification capabilities tightly spaced bands can be resolved and accurately excised and gel data can immediately be archived in a digitized format for future analysis CHAPTER 5 FLUORESCENCE APPLICATIONS Protocol Amersham Pharmacia Biotech products available for this application Product Product number m Hoefer SQ3 Sequencer 80 6301 16 Low fluorescence glass plate set see catalogue m Hoefer EPS 3501 Power Supply 18 1130 04 m Imaging systems Typhoon 8600 see catalogue Storm 840 860 see catalogue FluorImager 595 see catalogue Other materials required Product Fluorochrome labelled oligonucleotide primers for differential display amplification m Total RNA Preparation of sample Follow the recommended protocol for PCR amplification from total RNA 14 Prepare the amplified products for electrophoresis using denaturing formamide sample buffer with 5 mg ml of Dextran Blue 2000 Heat the samples at 85 C for 5 min and then place the tubes directly on ice eo Gel electrophoresis Before casting the gel treat one glass plate with silane 14 Prerun a 6 denaturing polyacrylamide gel at 35 W for 45 min using 0 6x TBE as the electrophoresis running buffer Load the samples onto the gel and run at 35 W for 1 5 2 h e Scanning the differential display gel Typboon 8600 Affix two Kapton tape strips over each spac
104. ing image analysis software print a 1 1 representation of the gel image on a transparency sheet Use the transparency sheet to locate the region of the gel containing the fragments and excise the fragments Expected results A differential display analysis using a Cy5 label and imaged using Typhoon 8600 is shown in Figure 43 Lung l Mi Liver pipes Lung np Liver Te SED ji CHAPTER 5 FLUORESCENCE APPLICATIONS In lane PCR product analysis Fluorescently labelled DNA fragments can be generated by PCR using modified oligonucleotide primers or deoxynucleotide triphosphates dNTPs 20 21 A wide selection of fluorochrome tags is available for oligonucleotide end labelling For PCR products that are generated using an end labelled PCR primer an equimolar relationship exists between the label and the DNA molecule In contrast PCR products that are produced using fluorescently modified dNTPs are internally labelled at multiple sites per molecule and consequently deliver the greatest sensitivity Fluorescent detection offers the advantages of sensitivity a wide linear dynamic range for quantification and the option for using multiple tags in analysis The CyDye series of fluorochromes are bright photostable molecules that are highly water soluble and insensitive to pH changes The labels are available in a range of intense colours with narrow emission bands making them ide
105. ion Note 60 Storm Image Analysis of Horseradish Peroxidase HRP Based Western Blots Using Amersham Pharmacia Biotech ECL Plus Substrate Amersham Pharmacia Biotech code number 63 0028 71 1999 Technical Note 59 Optimization of Amersham Pharmacia Biotech ECL Plus Detection of Western Blots for Storm Image Analysis Amersham Pharmacia Biotech code number 63 0028 81 1999 Ota N et al Nucl Acids Res 26 735 743 1998 Application Note 62 Fluorescent DNA Labelling by PCR Amersham Pharmacia Biotech code number 63 0028 73 1999 Application Note 67 Fluorescent Multiplex PCR and In lane Fragment Analysis Amersham Pharmacia Biotech code number 63 0031 84 2000 Liang P and Pardee A B Science 257 967 971 1992 Application Note 65 Fluorescent Differential Display Analysis Amersham Pharmacia Biotech code number 63 0031 03 2000 Fried M and Crothers D M Nucl Acids Res 9 6505 6525 1981 Application Note 103 Fluorescent Gel Mobility Shift Assay Amersham Pharmacia Biotech code number 63 0028 75 1995 Application Note 59 Red Fluorescence Electromobility Shift Assay with Extracts from Cell Lines and Lymph Nodes Amersham Pharmacia Biotech code number 63 0028 70 1999 Chalfie M et al Science 263 802 805 1994 Application Note 61 Green Fluorescent Protein Applications Amersham Pharmacia Biotech code number 63 0028 72 1999 Heim R et al Proc Natl Acad Sci USA 91 1250
106. ional information see the manufacturer s literature eo Staining the samples Prepare sufficient working solution of the PicoGreen reagent by diluting the stock solution as supplied 1 200 using TE buffer Note The PicoGreen working solution should be prepared in a plastic container on the day of the experiment Glass should not be used because PicoGreen may adsorb to glass surfaces Pipette the PicoGreen working solution into each well of the microplate using at least 50 pl for Fluorlmager and Storm or 80 pl for Typhoon Add an equal volume of the experimental DNA solution from step 1 1 to each well and mix thoroughly by pipetting Incubate 2 5 min at room temperature e Imaging Place the microplate into the microplate tray Fluorlmager or directly onto the platen Storm and Typhoon Acquire the image according to the recommended instrument setup for the fluorochrome used Select 200 micron pixel size setting The PMT voltage setting should be adjusted to prevent signal saturation For Typhoon the 3 mm focal plane setting should always be selected for imaging microplates e 61 FLUORESCENCE IMAGING Fig 34 Detection of DNA in solution using PicoGreen and Typhoon 8600 Lambda DNA was used at concentrations of 3 500 ng ml 429 ng ml 150 ng ml 52 5 ng ml 18 4 ng ml 6 4 ng ml 2 25 ng ml and 0 79 ng ml tm 63 0035 28 62 o Analysis Display the image using ImageQuant If satura
107. is with no film exposure step needed Fluorescence imaging thus provides a rapid convenient safe sensitive and quantitative alternative to radioactivity for performing this important procedure CHAPTER 5 FLUORESCENCE APPLICATIONS Protocol Amersham Pharmacia Biotech products available for this application Product Product number m Hoefer SE 400 Sturdier Vertical Unit 80 6154 86 Low fluorescence glass plate set 80 6442 14 m Hoefer EPS 301 Power Supply 18 1130 01 5 Oligolabelling Kit for Fluorescence RPN5755 m Cy3 mono Reactive Dye Pack PA23001 m Cy5 mono Reactive Dye Pack PA25001 m Imaging systems Typhoon 8600 see catalogue Storm 830 860 see catalogue FluorImager 595 see catalogue e Preparation of labelled DNA Label the oligonucleotides at their 5 ends with fluorescein according to the instructions provided with the 5 Oligolabelling Kit or using another label such as a CyDye and a comparable approach eo Preparation of DNA for annealing Note Repeat the steps below for all duplexes to be tested Prepare the following mix Labelled oligonucleotide 1 14 pmol Labelled oligonucleotide 2 14 pmol 10x annealing buffer 5 pl to a total volume of 50 nl 10x annealing buffer 200 mM Tris HCl pH 7 6 50 mM MgCl 1 mM DTT 0 1 mM EDTA Heat the reactions for 10 min at 70 C Incubate for 30 min at room temperature Place the reactions on ice until ready to use FLUORESCENCE IMAGING tm
108. ized with an f theta lens as illustrated in Fig 8 but when the angle of incident excitation light varies over the imaging field some spatial distortion can still occur in the resulting image Sample Sample tray f theta lens Galvo mirror G Laser Fig 9 Moving head scanning mechanism The light beam from the laser is folded by a series of mirrors and ultimately reflected onto the sample The sample is illuminated across its width as the scan head moves along the scan head rail X axis The entire sample is illuminated by the scan head laser and mirrors tracking along the length of the sample Y axis CHAPTER 2 FLUORESCENCE IMAGING SYSTEMS Moving head scanners Moving head scanners use an optical mechanism that is equidistant from the sample This means that the angle and path length of the excitation beam is identical at any point on the sample Fig 9 This eliminates variations in power density and spatial distortion common with galvanometer based systems Although scan times are longer with a moving head design the benefits of uniformity in both light delivery and collection of fluorescence are indispensible to accurate signal Sample Glass platen m Scan head rail quantification Mirror Scan head Mirror Laser Light collection The light collection optics in a scanner system must be designed to efficiently collect as much of the emitte
109. laten again and do not readjust the placement How do keep the sample from moving during the scan Remove any excess liquid from below the gel so that it does not move on the glass tray or glass platen Place a clean electrophoresis glass plate on top of a membrane or a blot that has been sealed between plastic sheets or page protectors Why is the sensitivity of my image unexpectedly poor The choices of excitation and emission filters may not have been optimal Make sure that the correct excitation light and the proper emission filters have been selected for the dyes used in the sample Check that the combination of excitation light and emission filter are compatible Refer to Appendix 3 and Chapter 3 for more details Why do have high image background or inaccurate readings The instrument may not have been warmed up before the sample was scanned Allow at least a 30 minute warm up time The instrument may be damaged and is no longer light tight If this has occurred do not continue to use the instrument Contact Technical Support to arrange for repair Dust fingerprints or other dirt may have contaminated the screen sample or glass platen Clean the glass platen If necessary for fluorescence scans filter the liquid samples reagents and components used to prepare the gels The sample support may be highly autofluorescent Use a low fluorescence material If using FluorImager the filter door may have been ope
110. lengths that are shorter and longer than the selected band while allowing wavelengths in the selected range centred around the fluorescent emissions of the sample to pass through to the collection pathway Increase the dwell time or accumulate multiple scans for mathematical processing Detection of weak fluorescent signals can be improved by increasing the dwell time because the instrument can excite and collect more emitted fluorescent light from the sample Multiple scans of the sample can also be accumulated and subjected to mathematical processing e g averaging summing or other accumulation methods This increases fluorescence sensitivity by reducing the amount of background fluorescence Averaged results for example represent the average of the constant signal and a reduction of random background effects averaged noise Methods for removing background signals whether due to residual laser light or sample matrix fluorescence enhance the dynamic range of an assay For example if the collection instrument has a dynamic range of 10 arbitrary fluorescence units such as rfu but the support material has a background of 100 rfu the effective dynamic range of the assay is only 10 rfu By selecting low fluorescence sample support material and using the various methods described above to lower the background to 10 rfu or less the effective dynamic range can be increased to 10 or greater e 105 FLUORESCENCE IMAGING tm
111. lification Module boosts sensitivity by coupling alkaline phosphatase to the fluorescein labelled DNA probe Alkaline phosphatase catalyses the formation of stable fluorophores that remain near the probe and emit light when detected using fluorescence imaging systems m 63 0035 28 68 The direct detection of a fluorescently labelled nucleic acid probe on a membrane usually does not provide adequate sensitivity for Southern or Northern analysis Consequently most non radioactive Southern and Northern detection schemes use a hapten to label the nucleic acid probe The hapten i e fluorescein digoxigenin or biotin provides a target recognized by an antibody or other binding molecule that is conjugated to an enzyme Signal amplification results from the conversion of multiple substrate molecules to fluorescent products by each enzyme An indirect detection scheme in which fluorescein is used as a hapten is illustrated in Figure 36 With some kits the probe can be directly labelled with a thermostable enzyme e g AlkPhos Direct systems Because this system bypasses the hapten detection step the signal development process is much faster Fluorescence 550 570 nm Excitation ET 3 Fluorescent product Substrate Alkaline phosphatase v Phosphate group Antifluorescein Fluorescein Solid support CHAPTER 5 FLUORESCENCE APPLICATIONS D Instrument compatibility Table 17 Compatibility of selected flu
112. lter rejects light with wavelengths shorter than the first cutoff and longer than the second cutoff a dichroic optical filter used to separate the fluorescent signal of two distinct fluorochromes from a mixed emission beam the level of fluorescence intensity of a fluorochrome Brightness depends on the extinction coefficient and the quantum efficiency charge coupled device a two dimensional photosensitive array that produces a pattern of charge that is proportional to the total integrated energy flux incident on each array element pixel the chemical and or enzymatic production of fluorescence the emission of light from a molecule as a result of a chemical reaction a property of light where all the waves are at the same frequency and phase Only light that is monochromatic can be completely coherent e 109 FLUORESCENCE IMAGING collimated light cone angle confocal imaging cutoff point dataset dichroic filter diode laser dwell time dynamic range emission tm 63 0035 28 110 light that is radiated in only one direction the full angle between the extreme off axis rays in a converging or diverging beam of light the detection of fluorescent light only from those points on a sample that are within the desired focal plane Confocality is controlled by an aperture pinhole placed in front of the detector that greatly reduces the passage of out of focus information both above and below the de
113. lue NA Not applicable tm 63 0035 28 100 CHAPTER 6 PRACTICAL RECOMMENDATIONS Chapter 6 PRACTICAL RECOMMENDATIONS Introduction There are a number of ways to improve the results of fluorescence imaging This chapter will describe useful recommendations for the various stages of a fluorescence imaging experiment from sample preparation to instrument operation and data analysis Sample preparation Careful sample preparation can minimize sample background fluorescence and non uniformity resulting in improved image quality and detection sensitivity Avoid using fluorescent indicator dyes Bromophenol blue xylene cyanol and other electrophoresis tracking dyes can fluoresce potentially masking the fluorescence of the bands of interest in the gel To avoid this problem use a non migrating sample loading buffer such as dextran blue If it is necessary to monitor migration during electrophoresis reduce the concentration of tracking dye to a minimum or load the tracking dye into a separate lane of the gel Avoid excessive exposure of fluorochromes to direct light To prevent photobleaching fluorochromes and fluorescently labelled samples should be protected from light Wrap aluminium foil around individual storage tubes plates or racks to reduce sample exposure to light during handling and storage Use chemicals of highest purity To minimize autofluorescence from contaminants use sequencing grade
114. lues imaging should be repeated at a reduced detector sensitivity setting Other image acquisition settings such as the scan area pixel size resolution and choice of laser or emission filter can also be adjusted to improve the resolution discrimination or strength of the desired signal Lane 1 profile O 9 t ream MEI a Lane 1 profile 100 MAM 100 Signal Signal Image documentation Investigators commonly annotate images with text numbers and other labels before archiving their files to disc or printing a copy for documentation Most imaging software packages offer solutions to simple documentation annotation and output of image files Enlargement zooming or magnification is often used to view in detail a subsection of a larger image Fig 22 A scaling function that fits the image to the size of the current program window is useful when the actual 100 size of an image is larger than the viewing area of the monitor For some applications image analysis software must be able to accommodate actual sample size or 1 1 printing For example excision and recovery of DNA fragments from fluorescent differential display analysis gels require a precise overlay of a printed copy of the fluorescent image with the original gel In other cases it may be desirable to subdivide large image files into separate smaller image file
115. ly the energy wavelength of the emitted fluorescent light is a statistical function of the available energy levels in the fluorochrome but it is independent of the intensity of the incident light In contrast the intensity of the emitted fluorescent light varies with the intensity and wavelength of incident light and the brightness and concentration of the fluorochrome When more intense light is used to illuminate a sample more of the fluorochrome molecules are excited and the number of photons emitted i e the number of electrons falling to the ground state increases If the illumination is very intense all the fluorochrome molecules are in the excited state most of the time saturation When the illumination wavelength and intensity are held constant as with the use of a controlled laser light source the number of photons emitted is a linear function of the number of fluorochrome molecules present Fig 5 At very high fluorochrome concentrations the signal becomes non linear because the fluorochrome molecules are so dense that excitation occurs only at or near the surface of the sample Additionally some of the emitted light is reabsorbed by other fluorochrome molecules self absorption The amount of light emitted by a given number of fluorochrome molecules can be increased by repeated cycles of excitation In practice however if the excitation light intensity and fluorochrome concentration are held constant the total emitte
116. m filtered images must be interpreted appropriately On the other hand intense fluorescent signal from dust and other contaminants can severely complicate analysis For these reasons the decision to filter and therefore alter an image prior to analysis must be carefully considered Amersham Pharmacia Biotech image analysis software Image analysis is an integral part of today s life science applications Amersham Pharmacia Biotech provides a comprehensive range of software products to address image analysis needs from basic documentation and routine purity screens to the querying of entire gene expression or 2 D gel datasets Table 2 Our image analysis software combined with our wide range of fluorescence imaging instrumentation deliver a complete system and a total solution to address a wide range of application needs e 41 FLUORESCENCE IMAGING Table 2 Amersham Pharmcia Biotech image analysis software IMAGEQUANT SOLUTIONS Powerful portfolio of software modules for 1 D gel and blot analysis ImageQuant m User defined signal integration of regions volume or lane profiles and peak analysis area m Support for up to four channel images m Text annotation of images and region of interest tool zi El Fragment analysis Molecular weight fragment size and isoelectric point determination Analysis of two channel images with in lane size standard
117. macia Biotech combine powerful excitation sources with efficient optics for sensitive fluorescence imaging of the common DNA gel stains including ethidium bromide Vistra Green SYBR Green and SYBR Gold 9 10 Setup for the various instruments is given in Table 4 Typical protocol Amersham Pharmacia Biotech products available for this application Product Product number Hoefer EPS 301 Power Supply 18 1130 01 Nucleic acid gel stains Ethidium bromide solution 10 mg ml 17 1328 01 Vistra Green nucleic acid gel stain RPN5786 m Electrophoresis units Ready To Run Separation Unit 80 6460 95 Hoefer HE 99X Max Submarine Unit 80 6061 57 Hoefer miniVE Vertical Electrophoresis System 80 6418 77 m Imaging systems Typhoon 8600 see catalogue Storm 840 860 see catalogue FluorImager 595 see catalogue ImageMaster VDS CL see catalogue FLUORESCENCE IMAGING tm 63 0035 28 48 e Sample preparation Prepare agarose gels that are no thicker than 3 mm if possible Mix the DNA samples with loading buffer Note When imaging small size nucleic acids or proteins avoid using bromophenol blue xylene cyanol and other electrophoresis tracking dyes because these dyes fluoresce and might mask the fluorescence of bands of interest on the gel To avoid this problem use a non migrating dye such as dextran blue in the sample loading buffer If it is necessary to monitor migration during electrophoresis reduce the concentration of tracki
118. mage acquisition However it is possible to collect multiple images by moving the lens assembly and CCD detector relative to the sample and then using software to stitch the images together to form a complete view of the sample In this way each segment of the image or tile can utilize the full resolution of the CCD CHAPTER 2 FLUORESCENCE IMAGING SYSTEMS Sensitivity and linearity CCD arrays are sensitive to light temperature and high energy radiation Dark current from thermal energy cosmic rays and the preamplifier causes system noise that can have a profound effect on instrument performance Cooling of the CCD significantly reduces noise levels and improves both sensitivity and linearity of the system For example active thermal cooling to 50 C improves the linear response of a CCD three to five fold Combining charges from adjacent pixels during acquisition can also enhance sensitivity although image resolution may suffer Dynamic range The dynamic range of a CCD is defined as the ratio of the full saturation charge to the noise level CCD cameras typically have a dynamic range of up to 10 An imaging system with a 15 x 15 pm pixel has a 225 area and a saturation level of about 180 000 If the system noise level is 10 then the dynamic range is the ratio of 180 000 10 or 18 000 1 thus demonstrating how system noise can limit the dynamic range Amersham Pharmacia Biotech imaging systems Amersham Pharma
119. may require considerable experimental manipulation Although conjugates can be prepared with very high degrees of substitution or labelling they frequently precipitate or bind non specifically Therefore to preserve function or binding specificity it is usually necessary to use labelling conditions that result in a submaximal fluorescence yield After the labelling reaction it is important to remove as much unconjugated dye as possible because the presence of free reactive dye can complicate subsequent experiments Several forms of reactive fluorochromes are commonly used Isothiocyanates such as fluorescein isothiocyanate FITC and tetramethylrhodamine isothiocyanate TRITC are amine reactive and widely used for preparing fluorescent antibody conjugates Succinimidyl esters are excellent reagents for amine modification and form extremely stable amide bonds The succinimidyl esters will also react with thiol groups Some fluorochrome derivatives of sulfonyl chlorides are also highly reactive with amines and react more mildly with thiol groups e 85 FLUORESCENCE IMAGING Instrument compatibility Table 24 Instrument settings for use with common fluorescent labels Typhoon Fluorlmager Storm VDS CL Fluorophore Excitation Emission Excitation Emission Fluorescence Excitation Emission nm filter nm filter mode FluorX 532 526SP 450 530DF30 Blue Transmission UV low Cy2 532 526SP 488 530DF30 Blue Transmission UV low Cy3 532 580B
120. me molecules have different rotational and vibrational energies associated with them these are represented in the excitation spectrum or the probability that the fluorochrome will be excited by a particular wavelength of light Refer to Chapter 1 Excitation of a fluorochrome at the peak of its excitation spectrum is most efficient since the majority of fluorochrome molecules are able to absorb this energy However a small population of fluorochrome molecules can also be excited at other regions of the excitation spectrum The emission profile for a fluorochrome is always independent of the wavelength used for excitation A small population of fluorescein molecules accepts 532 nm excitation energy even though the excitation maximum for the fluorochrome is 488 nm Fluorescein in turn is characterized by a fluorescence emission spectrum with a peak wavelength of 520 nm Efficient collection of fluorescein emissions on the short side of the spectrum below 532 nm is accomplished using a short pass filter 526SP high quality confocal optics and a highly sensitive PMT detector e 115 FLUORESCENCE IMAGING tm 63 0035 28 116 Why does my image contain a double or ghost image The sample may have moved after its initial placement on the glass plate or platen of the instrument If fluorescent material from the sample has contaminated the glass carefully remove the sample and clean the glass Place the sample on the glass plate or p
121. ment should be relatively well resolved from each other However some spectral overlap between emission profiles is almost unavoidable To minimize cross contamination fluorochromes with well separated emission peaks should be chosen along with emission filters that allow reasonable spectral discrimination between the fluorochrome emission profiles Figure 20 shows the emission overlap between two common fluorochromes and the use of band pass filters to discriminate the spectra For best results fluorochromes with emission peaks at least 30 nm apart should be chosen A fluorescence scanner is most useful for multicolour experiments when it provides selectable emission filters suitable for a variety of labels A range of narrow band pass filters that match the peak emission wavelengths of commonly used fluorochrome labels will address most multicolour imaging needs Software To reduce the wavelength cross contamination typically found in multichannel fluorescence images software processing can be used This involves applying a cross talk algorithm to the individual channels to yield a revised image set that more ideally represents the light emitted from the different labels in the sample Chapter 4 gives more details about fluorochrome separation software and image analysis software in general e 29 FLUORESCENCE IMAGING im 63 0035 28 30 CHAPTER 4 IMAGE ANALYSIS Chapter 4 IMAGE ANALYSIS Introduction Image
122. n be synthesized with an amino linker that can subsequently be labelled by reaction with an amine reactive form of the fluorochrome Kits are also available for modifying the 5 ends of pre existing oligonucleotides to generate reactive forms If DNA polymerization reactions are carried out in the presence of fluorochrome linked deoxynucleotide triphosphates dNTPs using enzymes such as the Klenow fragment or Tag DNA polymerase then DNA with multiple internal fluorescent labels can be generated End labelled DNA fragments can also be produced by PCR amplification using end labelled primers 20 21 m 63 0035 28 84 CHAPTER 5 FLUORESCENCE APPLICATIONS Protein labelling Most proteins peptides and antibodies can be directly labelled with fluorochromes via their available amine or thiol groups While virtually all proteins and antibodies have primary amine groups in their lysine side chains and at their N termini thiol groups are available only in cysteine side chains A wide variety of anti species antibodies are commercially available already conjugated to different fluorochromes Under ideal conditions a fluorescent conjugate retains the key function of the unlabelled biomolecule such as selective binding to a protein or nucleic acid target or modulation of a particular enzyme activity While conjugation of fluorochromes to biomolecules is usually a relatively straightforward reaction preparation of the optimal conjugate
123. n during the scan Close the filter door and repeat the scan Make sure there are no obstructions that prevent the door from closing completely The wrong light source or emission filters may have been used for the fluorescent sample APPENDIX 1 Why are there streaks or other artefacts in my image The instrument may not have been warmed up before the sample was scanned Allow at least a 30 minute warm up time Diagonal streaks may indicate a light leak during scanning Check for damaged panels on the instrument Contact Technical Support The glass sample tray or glass platen may be scratched If possible scan the sample on another area of the glass Contact Technical Support to order replacement glass and arrange for a service call Fingerprints appear in the scan Clean the glass sample tray or glass platen If the fingerprints are on the gel rinse the gel briefly in 0 1 Tween SDS Rinse the gel again in distilled water and then rescan it If the fingerprints persist you may need to prepare a new gel and handle it more carefully Dust specks appear in the scan Rinse wet gels in filtered distilled water to remove surface dust prior to scanning Filter liquid reagents that are used in gel and buffer preparation Make sure to dissolve agarose completely before pouring the gel Clean the glass sample tray or glass platen of the instrument with a damp lint free cloth The tracking dye is fluorescing Place the tracking dye
124. n orbital shaker Use at least the minimum suggested volume of buffer for washing steps m Always wear powder free gloves when handling membranes solutions and dishes used for washing m Adjust the hybridization or stringency wash temperature or add more washes if necessary For other factors that may affect the quality of detection refer to the troubleshooting guide included with the labelling and detection kit eo Preparation of blot Soutbern blots Separate the DNA samples in a neutral agarose gel then depurinate denature and neutralize the gel according to standard procedures 14 Transfer the samples to a Hybond N nylon transfer membrane Process the Southern blot through hybridization stringency washes and detection of the fluorescein hapten Nortbern blots Separate denatured RNA prepared in a glyoxal buffer in an agarose gel prepared in 1x MOPS buffer 14 Transfer the samples to a Hybond N nylon transfer membrane Process the Northern blot through hybridization stringency washes and detection of the fluorescein hapten CHAPTER 5 FLUORESCENCE APPLICATIONS e Application of substrate substrate Prepare ECF substrate as directed After the final washing step position the wet blot sample side up in an open low fluorescence bag or page protector Add the prepared substrate to the blot so that it is coated completely and evenly Cover the blot with the top sheet of the bag o
125. n this way two channels are created These two channels can then be filtered and detected independently CHAPTER 2 FLUORESCENCE IMAGING SYSTEMS Signal detection and amplification The first stage in fluorescent signal detection is selection of only the desired emission wavelengths from the label or dye In single channel or single label experiments emission filters are designed to allow only a well defined spectrum of emitted light to reach the detector Any remaining stray excitation or scattered light is rejected Because the intensity of the laser light is many orders of magnitude greater than the emitted light even a small fraction of laser light reaching the detector will significantly increase background Filtration is also used to reduce background fluorescence or inherent autofluorescence originating from either the sample itself or the sample matrix i e gel membrane or microplate In multichannel or multi label experiments using instrumentation with dual detectors additional filtering is required upstream of the previously described emission filter During the initial stage of collection in these experiments fluorescence from two different labels within the same sample is collected simultaneously as a mixed signal A dichroic beamsplitter must be included to spectrally resolve or split the contribution from each label and then direct the light to appropriate emission filters Fig 11 At a specified wavelength the beamsplit
126. nal image data or the results of quantification Contrast and brightness settings of the display can be adjusted to optimize the image view The ability to change both the high and low display value settings is important for viewing the range of gray or colour values of interest For example by increasing the low values image noise or background can be reduced Reducing the high value setting of the display increases image contrast such that weak signals can be visualized These adjustments are made separately to each channel in a multichannel image Multicolour software will also allow either side by side display of the individual channels or a multicolour overlay of all channels together 31 FLUORESCENCE IMAGING Fig 21 Effect of detector saturation on data quality A Cy5 labelled size standard was resolved in a 10 polyacrylamide gel and imaged on Typhoon 8600 Image acquired using a PMT setting of 1000 V panel a or 500 V panel b The line profiles through lane 1 of each image show the response of the PMT to the fluorescent signal collected Fig 22 Magnification or zooming to view details of an image tm 63 0035 28 32 Image analysis software can be used to ascertain if the image contains areas that are non quantifiable due to light saturation of the detector When saturation occurs the results of image analysis are likely to be in error Fig 21 If an image is composed of pixels with saturated va
127. nalysis of CyDye labelled PCR products imaged using Typhoon 8600 is illustrated in Figure 44 Bandshift assay The gel mobility shift assay also called the bandshift assay gel shift assay or gel retardation assay is a useful tool for identifying protein DNA interactions that can mediate gene expression DNA repair or DNA packaging 24 It can also be used to determine the affinity abundance binding constants and binding specificity of DNA binding proteins The assay is performed by incubating a labelled DNA fragment containing the test binding sequence with an extract containing one or more binding protein s The mixture is then separated on a non denaturing polyacrylamide gel DNA fragments that are bound by protein migrate more slowly than free fragments and appear as bands that are shifted relative to the bands from the unbound duplexes Traditionally the DNA fragments or oligonucleotides are end labelled with 2P However fluorescent end labelled oligonucleotides are now commonly used and kits such as the 5 Oligolabelling Kit can be used for their rapid preparation The availability of sensitive fluorescence imaging systems makes it practical to perform bandshift assays without radioactivity 25 26 Gels containing bandshift products can also be stained after electrophoresis with Vistra Green or other sensitive DNA intercalating dyes In either case with fluorescent labelling gels can be scanned shortly after electrophores
128. nc microplate strips BSA was used at concentrations of starting at the top left well 10 6 3 1 0 6 0 3 0 1 0 06 0 03 and 0 01 g ml The last two wells in the bottom row contained negative controls which was NanoOrange working solution only tm 63 0035 28 66 Expected results The expected limits of detection and linear ranges for protein quantification in solution are given in Table 15 Quantification of a BSA solution using NanoOrange and Typhoon 8600 is shown in Figure 35 Table 15 Fluorescence based quantification of protein in solution using NanoOrange Typhoon Fluorlmager Storm Dye LOD LDR LOD LDR LOD LDR ug ml fold ug ml fold ug ml fold NanoOrange 1 0 3 10 30 0 5 20 1 10 BSA diluted in 1x TE was used used for analysis Results are expressed as limit of detection LOD and linear detection range LDR First number from assay performed using Costar flat bottomed plate Second number from assay performed using Nunc Separable Strips CHAPTER 5 FLUORESCENCE APPLICATIONS Southern and Northern blotting The transfer of DNA from an electrophoresis gel to a membrane is termed a Southern transfer or blot In this technique a complex mixture usually genomic DNA is probed to detect individual target DNA molecules Similarly in a Northern blot RNA either mRNA or total cellular RNA is transferred from a gel to a membrane and probed for the prese
129. nce of specific mRNA transcripts Both methods permit the sensitive measurement of nucleic acid size and quantity In traditional Southern and Northern procedures probes are labelled with radioactive isotopes e g P for detection With radioactivity however safety issues must be considered In contrast non radioactive detection methods such as fluorescence provide a safe alternative and deliver comparable sensitivity Additionally unlike radioactively labelled probes fluorescent probes are stable for long periods Fluorogenic substrates for Southern and Northern detection Fluorescent Southern and Northern detection chemistries employ enzyme amplified detection schemes using alkaline phosphatase AP enzyme 15 Enzymatic turnover of a fluorogenic substrate gives the highest sensitivity because each enzyme molecule produces multiple fluorescent products ECF reagent and DDAO phosphate are fluorogenic substrates commonly used with the AP enzyme and suitable for Southern and Northern detection Their spectral characteristics are shown in Table 16 Table 16 Fluorogenic substrates for Southern and Northern blots Excitation Emission Fluorescence Substrate max nm max nm emission colour Enzyme DDAO phosphate 646 660 Red Alkaline phosphatase ECE 440 560 Green Alkaline phosphatase FLUORESCENCE IMAGING Fig 36 Schematic showing the indirect detection of a fluorescein labelled DNA probe in a Southern blot The ECF Signal Amp
130. nced to the points on the cutoff edge where the transmission is one half of the maximum transmission a device used to determine the presence direction and strength of electric current in a conductor a vertical gel typically polyacrylamide cast between two supporting glass electrophoresis plates a horizontal glass stage or platform used to support samples i e gels membranes microplates for imaging typically used in imagers with moving head mechanisms the flow of energy per unit area Intensity is a function of the number of photons per unit area and their energy thin adhesive tape that is used to raise a gel sandwich a defined distance above a glass platen an acronym for light amplified stimulated emission of radiation A laser produces highly monochromatic coherent and collimated light light emitting diode a semiconductor device that emits visible light when an electric current passes through it the signal range over which a laser scanner yields a linear response to fluorochrome concentration the smallest amount of a sample that can be reliably detected eiii FLUORESCENCE IMAGING long pass filter monochromatic multichannel image noise numerical aperature optical filter parallax photobleaching PMT photon of light pixel quantification quantum efficiency tm 63 0035 28 112 an optical filter that transmits light of wavelengths longer than a specified cutoff The
131. ndary of each closed object to determine a different background value for each object Fig 29a In global methods a single global background value is applied equally to a group of analysis targets in the same image These correction methods include using a straight baseline below a lane profile i e determined from the minimum signal in the profile or choosing one or more representative site s in the image to generate a background value that is applied to multiple objects Fig 29b 900 800 710 650 600 E a Pixel 900 800 710 650 i fa E 600 b Pixel CHAPTER 4 IMAGE ANALYSIS The type of background pattern apparent in an image will suggest the method of background correction to apply For example if background signal is variable across the image then a local method of correction may be appropriate because it can account for different background counts at each site where quantification is applied Alternatively one global background value for the whole image may be the best choice when background signal is uniform It is also important to select the most appropriate method for calculating the background value s The choice between an average and a median value for background calculation can significantly affect the results of quantification For example if high signal spikes are contributing to the background noise in an area of interest calculation of an average background
132. nergy levels of the two states Fig 2 and that energy difference determines the wavelength of the emitted light Aem hc Esy where E the energy difference between the energy levels of the two states during emission EM of light h Planck s constant c the speed of light A laser scanning instrument or a CCD camera can be used to measure the intensity of the fluorescent light and subsequently create a digital image of the sample Image analysis makes it possible to view measure render and quantify the resulting image Properties of fluorochromes Excitation and emission spectra A fluorescent molecule has two characteristic spectra the excitation spectrum and the emission spectrum Excitation spectrum The relative probability that a fluorochrome will be excited by a given wavelength of incident light is shown in its excitation spectrum This spectrum is a plot of total emitted fluorescence versus excitation wavelength and it is identical or very similar to the absorption spectrum Fig 3a commonly provided by fluorochrome manufacturers FLUORESCENCE IMAGING Vibrational relaxation 8765 43 2 1 123 4 a Excitation Emission Stokes shift 2 a g 8 7 6 5 43 211727 3 4 b Wavelength Fig 4 Diagram of the energy levels of a fluorochrome molecule including superimposed vibrational energy levels a and an example of excitation and fluorescent spectra b Reproduced from reference 3
133. ng a fluorogenic substrate esses 77 Western blotting using a fluorochrome conjugated antibody 80 Expected rr EH Lm ret area 82 tm 63 0035 28 ii FLUORESCENCE IMAGING Using covalent labels for nucleic acid and protein analysis 83 Nucleic acid Lee reete eere 84 Protein labelling 22 2 ea rent ttt RR Eee ie 85 Instrument treo rete enero theta reote apap 86 Applications and reete trt retener ra eod edere ea 86 Differential display analysis eter rire enean 86 In lane PCR product anal ysis 25 eoe eere Feind eei Prae ida 89 Baudshift assay iiie ii ape A ER RE BERE EHE a Aa FERE ERE CE 92 Using naturally occurring fluorescent proteins ssssessssesssrsssrsssrsssresseessessses 96 Green fluorescent protein and its variants essere 96 Instrument 97 Examples of applications using GFP eese 98 Expected results aceti 98 PhycobiliprOt Ims 45 inn iR RR HORE EP Ebr 99 Instrument compatibility Te Eg 100 Chapter 6 Practical recommendations 101 Oi M D 101 Sample pre Parathnecsssesicsiccssssesscssicsivcascceesesidesiaesnces ELE Xo AEE A EEEE EREE 101 Sample placement
134. ng dye to a minimum or load the tracking dye into a separate lane of the gel eo Gel electrophoresis Load the prepared samples into the wells Perform electrophoresis at 5 V cm using the EPS 301 power supply e Gel staining For Vistra Green or the SYBR stains dilute the stains 1 10 000 in 1x TE pH 7 5 For ethidium bromide use a concentration of 0 25 pg ml in 1x TE pH 7 5 Stain the gel in a polypropylene container for 30 min with gentle agitation longer staining times may be needed for gels with high agarose content Cover the staining container with aluminium foil to prevent photobleaching of the stains Gels attached to one of the electrophoresis plates For Vistra and SYBR stains pour enough staining solution on the gel to cover and use a large pipette to distribute liquid If ethidium bromide was used destain the gel for 30 min in water o Imaging Wet gels Place the wet gel directly onto the platen Typhoon and Storm glass tray FluorImager or platform VDS CL of the imager in a small just enough to create a film amount of water Avoid trapping air bubbles between the gel and the glass For Typhoon imaging choose platen for the focal depth setting For thick agarose gels it may be necessary to use the 3 mm focal depth setting Acquire the image according to the recommended instrument set up Fig 31 Detection of DNA in an agarose gel using Vistra Green and Typhoon 8600 Following
135. ns in gels Protein gel stains Conventional colourimetric methods for visualizing proteins in gels include staining with Coomassie Brilliant Blue CBB or silver CBB staining is commonly used even though it has low sensitivity and requires a long processing time and large volumes of organic solvents Though more sensitive than CBB staining with silver is expensive labour intensive and exhibits protein to protein variation SYPRO protein gel stains Table 7 are easy to use fluorescent stains with sensitivities equivalent to those of silver staining 11 After electrophoresis the gel is simply stained destained optional and then imaged Because these stains bind to SDS coated proteins in gels they give more consistent staining between different types of proteins In addition their ability to detect proteins is not affected by the presence of contaminating nucleic acids or lipopolysaccharides SYPRO protein gel stains can be used with both denaturing and native gels and do not interfere with upstream applications such as Western detection or microsequencing SYPRO Orange and Red stains are optimal for rapid and efficient fluorescent staining of one dimensional protein gels However they require acetic acid fixation which interferes with protein transfer to a membrane For blotting techniques SYPRO Tangerine is recommended because no acetic acid fixation is necessary SYPRO Ruby protein gel stain provides sensitive fluorescen
136. orogenic substrates with fluorescence imaging systems Typhoon Fluorlmager Storm VDS CL Substrate Excitation Emission Excitation Emission Fluorescence nm filter nm filter mode Excitation Emission DDAO 633 670BP30 NA NA Red NA NA phosphate ECF 532 526SP 488 570DF30 Blue Reflection UV high NA Not applicable Typical protocol Amersham Pharmacia Biotech products available for this application Product Product number m Hoefer HE 99X Max Submarine Unit 80 6061 57 m Hoefer EPS 301 Power Supply 18 1130 01 Nucleic acid gel stains Ethidium bromide solution 10 mg ml 17 1328 01 Vistra Green nucleic acid gel stain RPN5786 a Hybond N membranes see catalogue m ECF Random Prime Labelling and Detection System RPN5732 m Imaging systems Typhoon 8600 see catalogue Storm 840 860 see catalogue Fluorlmager 595 see catalogue ImageMaster VDS CL see catalogue Other materials required Product Vendor DDAO phosphate Molecular Probes Inc FLUORESCENCE IMAGING m 63 0035 28 70 e Preliminary preparations and general handling instructions m Prepare the probe according to the instructions or directions provided with the labelling kit m Successful fluorescent detection protocols require that background be carefully controlled Special attention to cleanliness is required with alkaline phosphatase based detection m Block the membrane thoroughly by incubating in blocking buffer with agitation on a
137. otector A low fluorescence glass plate can be placed on top of the sample to keep it flat Use thin 0 2 0 4 mm spacers when scanning through another glass plate on a glass platen Sequencing gel spacers Kapton tape supplied with Typhoon or a thin layer of water can be inserted between the glass plate and the glass platen to minimize optical refraction artefacts and interference patterns and to protect the platen from scratching Place one sided opaque samples such as membranes or thin layer chromatographs face down If the sample is physically uneven on one side such as an agarose gel place the smooth side down on the glass surface For opaque samples such as membranes place the side with the nucleic acid or protein face down The sample should be positioned to create a smooth and even surface Avoid trapping air bubbles as they can appear on the scanned image and interfere with quantification CHAPTER 6 PRACTICAL RECOMMENDATIONS Instrument operation The detection and measurement of the emitted fluorescent signal can be enhanced in a number of ways Add optical filters to reduce background fluorescence from the sample matrix When the background signal from the sample matrix e g some gels TLC plates and membranes has a broad flat spectrum a band pass optical filter can be used to remove background signal comprising wavelengths longer or shorter than the fluorochrome emissions This type of filter rejects wave
138. r page protector squeeze out excess substrate and incubate for up to 24 h Make sure the blot is kept wet during the development process Note Signal development can be monitored by periodic imaging DDAO phosphate substrate To prepare the stock solution dissolve the DDAO phosphate in water at a concentration of 1 25 mg ml Dilute the DDAO phosphate stock 1 1000 in 10 mM Tris Cl pH 9 5 1 mM MgCl Add 5 ml of substrate per cm of membrane covering it evenly and incubate for 4 h o Imaging Place the covered developed blot face down onto the glass platen Storm Typhoon or glass tray Fluorlmager or face up on the platform of VDS CL Note Water can be used between the plastic bag and the platform to minimize the occurrence of interference patterns in the image Use a glass plate to hold the blot flat during imaging optional Acquire the image according to the recommended instrument setup The choice of pixel size and PMT voltage settings will depend on the individual experiment Reduce the PMT voltage setting or signal integration time for VDS CL to prevent signal saturation Analysis See Chapter 4 for information concerning image analysis e 7i FLUORESCENCE IMAGING Expected results Typical results from a fluorescent Southern blot of a single copy human gene acquired using the Typhoon 8600 scanner are shown in Figure 37 LI Results that can be expected for other systems and substr
139. reaching the detector General guidelines for selecting fluorochromes and filters Single colour imaging Excitation efficiency is usually highest when the fluorochrome s absorption maximum correlates closely with the excitation wavelength of the imaging system However the absorption profiles of most fluorochromes are rather broad and some fluorochromes have a second or additional absorption peak or a long tail in their spectra It is not mandatory that the fluorochrome s major absorption peak exactly match the available excitation wavelength for efficient excitation For example the absorption maxima of the fluorescein and Cy3 fluorochromes are 490 nm and 552 nm respectively Fig 16 Excitation of either dye using the 532 nm wavelength line of the Nd YAG laser may seem to be inefficient since the laser produces light that is 40 nm above the absorption peak of fluorescein and 20 nm below that of Cy3 In practice however delivery of a high level of excitation energy at 532 nm does efficiently excite both fluorochromes See Appendix 1 for a discussion of fluorescein excitation using 532 nm laser line For emission selecting a filter that transmits a band at or near the emission peak of the fluorochrome generally improves the sensitivity and linear range of the measurement Figure 17 shows collection of Cy3 fluorescence using either a 580BP30 or a 560LP emission filter Please refer to Appendixes 2 and 3 for a list of fluorochrom
140. rinicples of Instrumental Analysis Harcourt Brace Philadelphia pp 307 312 1998 e 135 FLUORESCENCE IMAGING tm 63 0035 28 136 Fluorophores and fluorescent probes Berlman I B Handbook of Fluorescence Spectra of Aromatic Molecules Second Edition Academic Press San Diego 1971 Drexhage K H Structure and Properties of Laser Dyes in Dye Lasers Third Edition Schafer F P ed Springer Verlag Heidelberg pp 155 200 1990 Green F J The Sigma Aldrich Handbook of Stains Dyes and Indicators Aldrich Chemical Company Milwaukee WI 1990 Haugland R P Coupling of Monoclonal Antibodies with Fluorophores Meth Molec Biol 45 205 221 1995 Hermanson G T Bioconjugate Techniques Academic Press San Diego 1996 Johnson I D et al Comparing Fluorescent Organic Dyes for Biomolecular Labeling in Methods in Nonradioactive Detection Howard G C ed Appleton and Lange Publishing Norwalk CT pp 47 68 1993 Kasten F H Introduction to Fluorescent Probes Properties History and Applications in Fluorescent and Luminescent Probes for Biological Activity Mason W T ed Academic Press San Diego pp 12 33 1993 Krasovitskii B M and Bolotin B M Organic Luminescent Materials VCH Publishers New York 1988 Lakowicz J R ed Topics in Fluorescence Spectroscopy Probe Design and Chemical Sensing Vol 4 Plenum Publishing New York 1994 Mason W T ed
141. rising mirrors and lenses to direct the excitation beam to the sample Some filtering of the laser light may also be required before the excitation beam is directed to the sample Light collection optics High quality optical elements such as lenses mirrors and filters are integral components of any efficient imaging system Optical filters generally referred to as interference filters are typically made from laminates of multiple glass elements Filters can be coated to selectively absorb or reflect different wavelengths of light thus creating the best combination of wavelength selection linearity and transmission properties Refer to Chapter 3 for additional information concerning optical filters Filtration of the emitted light Although emitted fluorescent light radiates from a fluorochrome in all directions it is typically collected from only a relatively small cone angle on one side of the sample For this reason light collection optics must be as efficient as possible Any laser light that is reflected or scattered by the sample must be rejected from the collection pathway by a series of optical filters Emitted light can also be filtered to select only the range or band of wavelengths that is of interest to the user Systems that employ more than one detector require additional beamsplitter filters to separate and direct the emitted light along separate paths to the individual detectors CHAPTER 2 FLUORESCENCE IMAGING SYS
142. rlaps that of other fluorochromes Fig 20 When emissions from one fluorochrome contaminate the light collection for other fluorochromes in the sample a process is needed to remove or reduce this cross contamination for accurate quantification of each separated channel Fluorochrome separation uses a mathematical transformation of the original images to create new images that more closely represent light emitted from the different fluorochromes used in the sample Fig 30 Because the original image files are left unchanged the separation process can be undone and repeated using different settings to optimize the results To enhance the quality of the image software filters can also be used to eliminate variation in background without affecting target signal b CHAPTER 4 IMAGE ANALYSIS Image filtering Image artefacts caused by dust or bubbles complicate fluorescence analysis of gels and membranes Removing these artefacts can improve the quality and accuracy of image analysis by reducing background noise without affecting the integrity of the overall image A digital filter can reduce unusually intense or bright single pixel values blending them more evenly into the surrounding image For example the single highest pixel value noise spike in a small group of contiguous pixels can be replaced with a lower value based on an assessment of the neighbouring pixel values Because filtering alters the original data file results fro
143. rm signal across the image m non uniform uneven or patchy regions m noise spikes or small groups of pixels with high counts m high signal within lanes Fig 27 Examples of different types of fluorescent image background CHAPTER 4 IMAGE ANALYSIS e 2 gt _ dil e _ a Uniform luas b Non uniform TU See Go s E Al rM 995 Gib de c Noise spikes d Lane specific e 37 FLUORESCENCE IMAGING Fig 29 Comparison of local and global background correction methods applied to the same image The local method panel a uses different background values at each band in the gel lane with background based on the average signal from the boundary of each band In panel b a single global background value of 500 counts is applied to each band in the analysis tm 63 0035 28 38 A full range of background correction choices includes both local and global methods Local methods account for the local environment at each region of interest that is in the immediate neighbourhood of a band spot or slot target to be quantified Depending on the quantification method used a local method can define background threshold by connecting the low points or valleys in a lane profile or it may use the signal defined by the bou
144. rophoresis SDS PAGE which separates proteins by molecular weight is an established tool for protein analysis Its resolving power is useful for the routine sizing and quantification of proteins from both complex mixtures and purified fractions The denaturing conditions used in SDS PAGE cause the proteins to unfold thus minimizing differences in their molecular shape and providing for more accurate molecular weight determination 12 Using appropriate gel systems proteins can also be studied under non denaturing or native conditions that preserve the higher order structure and even the biological function of some proteins For example native gel electrophoresis is required to preserve the structure and therefore the intrinsic fluorescence of green fluorescent protein GFP Amersham Pharmacia Biotech products available for this application Product Product number m Hoefer EPS 301 Power Supply 18 1130 01 m Hoefer miniVE Vertical Electrophoresis System 80 6418 77 m Bovine serum albumin BSA protein standard 27 8915 01 m Protein gel stains SYPRO Orange protein gel stain RPN5801 SYPRO Red protein gel stain RPN5803 SYPRO Tangerine protein gel stain RPN5805 m Imaging systems Typhoon 8600 see catalogue Storm 840 860 see catalogue Fluorlmager 595 see catalogue ImageMaster VDS CL see catalogue Other materials required Product Vendor Protein samples prepared in appropriate loading buffer m SYPRO Ruby protein gel stain Molecular Pro
145. rs Detection High sensitivity PMT Imaging modes Blue and green excited fluorescence Scanning area 20 x 24 cm Sample types Gels blots microplates and TLC plates IMAGEMAsTER VDS CL Automated CCD camera based system Excitation sources UV white light Filters 2 emission filters up to 6 emission filter positions Detection Cooled CCD Imaging modes Chemiluminescence fluorescence and colourimetric detection Scanning area 21 x 25 cm Sample types Gels blots and TLC plates Focus Automated FLUORESCENCE IMAGING im 63 0035 28 24 100 D 80 2 60 uo cutoff point 40 o Ss S 20 E 0 550 560 570 580 590 600 a Wavelength nm 100 D 80 c 2 60 uo cutoff point 40 20 0 500 510 520 530 540 550 b Wavelength nm Fig 14 Transmission profiles for a 560 nm long pass a and a 526 nm short pass b filter The cutoff points are noted CHAPTER FLUOROCHROME AND FILTER SELECTION Chapter 3 FLUOROCHROME AND FILTER SELECTION Introduction To generate fluorescence excitation light delivered to the sample must be within the absorption spectrum of the fluorochrome Generally the closer the excitation wavelength is to the peak absorption wavelength of the fluorochrome the greater the excitation efficiency Appropriate filters are usually built into scanner instruments for laser line selection and elimination of unwanted background lig
146. s or to reduce the overall file size before archiving Other common software utilities for image manipulation include rotation as well as filtering which reduces undesirable extraneous fluorescent signal caused by sample contamination e g dust or lint Fig 23 Three methods for signal quantification Line profile and integration of area under the curve panel a integration of signal from manually created closed objects panel b software assisted detection and quantification of lane and bands panel c CHAPTER 4 IMAGE ANALYSIS Documentation of image files is facilitated by the use of region of interest tools that allow images to be copied directly to a clipboard and pasted into another type of file such as a word processing or spreadsheet document Images can thus be readily combined with the contents of a relevant analysis sheet or experiment report An image copy paste function is useful in the preparation of presentations as well as the production of publication quality figures and illustrations for papers or journal articles Quantification One dimensional gel blot analysis One dimensional 1 D gel blot analysis is performed by signal integration of either the lane as a whole or of the individual elements within a lane i e bands as separate items Fig 23 Three approaches are commonly used for quantification Although they all calculate integrated fluorescent signal they do so in different
147. s the sensitivity of non fluorescence microplate based methods is typically in the pg ml range fluorescence based methods can detect nucleic acids at concentrations in the ng ml range In assays using the fluorescent dye PicoGreen double stranded DNA can be measured in solution at concentrations as low as 2 5 ng ml The linear detection range of this assay is typically 70 1400 fold depending on which imaging instrument is used See Table 12 The fluorescent detection of nucleic acids in solution can be achieved using PicoGreen for double stranded DNA OliGreen for single stranded DNA and oligonucleotides and RiboGreen and SYBR Green II for RNA However it is recommended that RNA samples be treated with DNase to remove any DNA contamination as no dye is yet available that exhibits fluorescence enhancement specifically by binding to RNA Table 10 Fluorescent dyes for the quantification of nucleic acids in solution Excitation Emission Fluorescence Dye max nm max nm emission colour Application OliGreen 500 523 Green Quantification of ssDNA and oligonucleotides PicoGreen 502 523 Green Quantification of dsDNA RiboGreen 500 525 Green Quantification of RNA FLUORESCENCE IMAGING NR Instrument compatibility Table 11 Instrument settings for use with nucleic acid dyes Typhoon Fluorlmager Storm Dye Excitation Emission Excitation Emission Fluorescence nm filter nm filter mode OliGreen
148. se ECF 440 560 Green Alkaline phosphatase ECL Plus 430 503 Blue Horseradish peroxidase e 73 FLUORESCENCE IMAGING Fig 40 Example of a direct fluorescent Western blot developed using secondary antibody conjugated to Cy5 A purified recombinant protein was resolved at 120 ng 60 ng 30 ng and 15 ng The blot was imaged using Storm 860 The far left lane contains Full Range Rainbow Molecular Weight Markers m 63 0035 28 74 Rfu JZ m 30 000 1 2 15 000 1 0 20 000 0 8 15 000 0 6 10 000 0 4 5 000 0 2 0 0 O 10 20 30 40 50 60 70 80 90 100 O 10 20 30 40 50 60 70 80 90 100 Tubulin ng d Tubulin ng Fig 39 Limits of detection for chemifluorescence a and chemiluminescence b A dilution series of purified tubulin was prepared in duplicate separated by SDS PAGE and transferred to two PVDF membranes Blots were incubated with mouse anti tubulin monoclonal primary antibody followed by incubation with secondary antibody For chemifluorescent detection one blot a was incubated with goat anti mouse IgG alkaline phosphatase followed by ECF substrate The blot was imaged using Fluorlmager with a PMT setting of 500 V For chemiluminescent detection the other blot b was incubated with sheep anti mouse IgG horseradish peroxidase and then developed using ECL Western Blotting Kit The blot was exposed to Hyperfilm ECL for 5 min The developed film was scanned using Personal Densitome
149. sired focal plane the wavelength of light at which transmission through an optical filter is 50 of the maximum transmission the files and folder that make up a multichannel image a coated glass filter used to split light by reflecting one wavelength range and transmitting another range a semiconductor device that produces coherent radiation in the visible or infrared transmission spectrum when current passes through it the amount of time the excitation light illuminates a spot pixel in a sample the range over which a detected signal can be quantified extinction coefficient fluorescence fluorochrome FWHM galvanometer gel sandwich glass platen intensity of light Kapton tape laser LED linearity limit of detection GLOSSARY E the amount of light absorbed The molar extinction coefficient is the optical density of a one molar solution of a compound through a one cm light path The value usually quoted is the molar extinction coefficient at the wavelength of maximum absorption the emission of light or other electromagnetic radiation of longer wavelength by a substance as a result of absorption of other radiation Emission continues only as long as the stimulus producing it continues and persists with a half life of less than 10 second or fluorophore a fluorescent dye full width at half maximum transmission defines the width of the pass band of a band pass filter It is refere
150. specific target molecules even against the background of a complex mixture Unlike general gel stains covalent labels can be used to specifically tag a molecule or class of molecules as with the generation of fluorescently labelled PCR primers fluorochrome conjugated antibodies and recombinant proteins fused with a naturally fluorescent protein marker These tagged molecules are widely used in a variety of applications including PCR based DNA assays e g DNA typing differential display and RT PCR Western blotting and cellular localization of fluorescent fusion proteins Fluorochrome labels are available in a reactive form that is suitable for attachment to the primary amines and thiol groups of biomolecules Although both groups occur naturally in protein molecules as for example at lysine and cysteine side chains nucleic acids must be chemically modified to produce a site that will bind with a reactive dye Additionally one or more fluorochromes can be incorporated during synthesis of DNA oligonucleotides and nucleic acids can be labelled internally by the enzymatic incorporation of fluorochrome linked nucleotides For example fluorescein linked UTP can be added to RNA during in vitro transcription reactions or Cy3 labelled dCTP can be incorporated into newly synthesized DNA fragments during PCR The choices for covalently attaching fluorophores to nucleic acids and proteins present numerous options for matching labels with the capa
151. sualization and interpretation of results Fig 26 a Be S S OF 11 19 t i 148 aus p b c B Fig 26 Display and analysis of array experiments In panel a images from a two channel gene expression array using Cy3 green and Cy5 red labels are overlaid or merged Levels of gene over or under expression are indicated by the relative strength of the green and red colours respectively Software displays yellow when signal from both fluorochromes is equal In panel b only the array elements exhibiting expression above green or below red a defined threshold are shown In panel c a scatter plot presents the normalized signal ratios of each array element e 35 FLUORESCENCE IMAGING Fig 27 2 D gel analysis software Spot borders are identified using spot finding algorithms Background must be removed using a global or a local background correction method tm 63 0035 28 36 Two dimensional protein gel analysis Software packages for two dimensional 2 D protein gel analysis feature specialized algorithms for spot finding and analysis routines for gel to gel comparisons Fig 27 Other important tools in these software packages include data normalization background correction gel matching and grouping and database input of analysis results 7 o s o0 i e tex a e s Ems EN Es
152. t is a sensitive and quantitative method that is widely used in molecular biology and biochemistry laboratories for a variety of experimental analytical and quality control applications Commonly used techniques including total nucleic acid and protein quantification Western Northern and Southern blotting product analysis and DNA sequencing can all benefit from the application of fluorescence based methods for detection Fluorescent detection offers a number of important advantages over other methods several of which are described below Sensitivity Fluorescent probes permit sensitive detection of many biological molecules Fluorescent stains and dyes are frequently the most sensitive option for detection of total DNA RNA and protein compared with traditional colourimetric methods Many fluorescence applications approach the sensitivity afforded by radioisotopes Multiple label possibility With fluorescent labelling two or more fluorochromes can be detected separately using optical filters and a fluorochrome separation algorithm Therefore components can be labelled specifically and identified separately in the same sample or lane of a gel Fig 1 For example standards and unknowns used in PCR can be labelled with different fluorochromes to provide an internal standard for the assay Stability Fluorescently labelled molecules offer several distinct advantages over radiolabelled molecules with respect to stability Where
153. t detection Available fluorescent stains substrates and covalent labels are described along with Amersham Pharmacia Biotech instrument compatibility and recommendations for imaging setup and analysis Detection of nucleic acids in gels Nucleic acid gel stains Fluorescent detection of nucleic acids in gels is used to visualize the results of DNA preparations restriction digests and PCR analyses as well as other more specialized applications Ethidium bromide is a popular fluorescent stain that is used for the routine detection of nucleic acids in gels The dye binds by intercalating between the bases of nucleic acid molecules and its fluorescence is detected by imaging the stained gel using UV or laser illumination More sensitive fluorescent stains such as Vistra Green and SYBR Green are available for nucleic acid applications requiring lower limits of detection in both agarose and polyacrylamide gel formats These stains have a high affinity for their target nucleic acid and upon binding their fluorescence and quantum yield are significantly enhanced Because their background fluorescence is negligible in the absence of nucleic acids gels e 45 FLUORESCENCE IMAGING Table 3 Nucleic acid gel stains Excitation Emission Fluorescence Stain max nm max nm emission colour Application Ethidium 526 605 Red Classic general purpose nucleic bromide acid stain SYBR Gold 495 537 Orange gr
154. t detection for both one and two dimensional protein gels and is compatible with subsequent mass spectrometry and Edman based sequencing Table 7 Fluorescent protein gel stains Excitation Emission Fluorescence Stain max nm max nm emission colour Application SYPRO Orange 300 470 570 Orange Routine SDS PAGE SYPRO Red 300 550 630 Red Routine SDS PAGE SYPRO Ruby 280 450 610 Red 2 D gels SDS PAGE critical sensitivity SYPRO Ruby IEF 280 450 610 Red Isoelectric focusing IEF gels SYPRO Tangerine 300 490 640 Red SDS PAGE followed by immunodetection or zymography FLUORESCENCE IMAGING Instrument compatibility Table 8 Instrument settings for use with protein gel stains Typhoon Fluorlmager Storm VDS CL Stain Excitation Emission Excitation Emission Fluorescence uU 2 nm filter nm filter mode Excitation Emission SYPRO Orange 532 580BP30 488 570DF30 Blue Transmission UV high SYPRO Red 532 610BP30 514 610RG Red Transmission UV high SYPRO Ruby 532 610BP30 488 610RG Blue Transmission UV high SYPRO Ruby IEF 532 610BP30 488 610RG Blue Transmission UV high SYPRO Tangerine 532 610BP30 488 610RG Blue Transmission UV high tm 63 0035 28 52 CHAPTER 5 FLUORESCENCE APPLICATIONS Typical protocols Protein detection in one dimensional gels One dimensional gel electrophoresis is routinely used to study the size or molecular weight amount and purity of proteins SDS polyacrylamide gel elect
155. ted pixels are present the microplate should be rescanned at a lower PMT voltage setting Use the Gray Color Adjust function to adjust the image contrast Ellipse objects can be used to quantify integrated signal from the microplate wells Draw an ellipse object within the inner walls of one well and copy it to the other wells Report the median values with background correction set to None In Microsoft Excel subtract the median value of the negative control well from each of the other wells This is important for good low end linearity Generate a standard curve from the DNA standards used Note For the greatest accuracy the DNA standards should be similar to the unknown DNA i e similar size and source Determine the unknown DNA concentration by extrapolating from the standard curve Expected results The limits of detection and linear detection ranges for quantification of DNA in solution are given in Table 12 Figure 34 is an image from a PicoGreen microplate assay detected using Typhoon 8600 Table 12 Fluorescence based quantification of DNA in solution Typhoon Fluorlmager Storm Dye LOD LDR LOD LDR LOD LDR ng ml fold ng ml fold ng ml fold PicoGreen 10 350 5 700 50 70 2 51 1400 RiboGreen ND ND 1000 10 100 A dilution series of lambda phage DNA prepared in 1x TE was used for the analysis Results are expressed as limit of detection LOD and linear detection rang
156. ter SI For both blots the dilution series from left to right was 1 6 3 12 6 25 12 5 25 50 100 200 300 and 400 ng of tubulin The signals for 1 6 100 ng were quantified using ImageQuant Software The average signals in relative fluorescence units rfu for chemifluorescence c and optical density OD for chemiluminescence d were plotted against the amount of tubulin loaded CHAPTER 5 FLUORESCENCE APPLICATIONS Direct fluorescent detection Direct fluorescent detection of Westerns Fig 40 is an alternative to enzyme amplified fluorescence Because the secondary antibody is conjugated directly with a fluorochrome there is no need for substrate development steps Though simpler than the enzyme method direct fluorescent detection is less sensitive because there is no signal amplifica tion However it is easier to quantify and by using combinations of fluorochromes and or fluorogenic substrates it is possible to detect more than one target on the same Western blot Fig 41 The development of direct fluorescent detection schemes is also facilitated by the wide availability of secondary anti species antibodies conjugated to a variety of different fluorochromes such as fluorescein and the CyDye and Fig 41 A dual target Western blot showing P detection of actin and tubulin Proteins were Alexa Fluor series see Appendix 3 for a list of multipurpose labels serially diluted two fold and resolved by gel elec
157. ter partitions the incident fluorescent light beam into two beams passing one and reflecting the other The reflected light creates a second channel that is filtered independently and detected by a separate detector In this way the fluorescent signal from each label is determined accurately in both spatial and quantitative terms See Chapter 3 for additional information on multichannel experiments Emission filter Mirror PMT Short wavelength Emitted light PMT Beamsplitter Long wavelength Emission filter FLUORESCENCE IMAGING m o Cathode radiant sensitivity mA W o 0 01 100 200 300 400 500 600 700 800 900 1000 Wavelength nm Fig 12 An example of the response of a PMT versus wavelength Copyright O 1994 Hamamatsu Photonics K K Used with permission m 63 0035 28 18 After the fluorescent emission has been filtered and only the desired wavelengths remain the light is detected and quantified Because the intensity of light at this stage is very small a PMT must be used to detect it In the PMT photons of light hit a photocathode and are converted into electrons which are then accelerated in a voltage gradient and multiplied between 10 to 10 times This produces a measurable electrical signal that is proportional to the number of photons detected The response of a PMT is typically useful over a wavelength range of 300 800 nm Fig 12 High performance PMTS exten
158. th W J in Modern Optical Engineering McGraw Hill Boston MA pp 142 145 1990 Skoog D A et al in Principles of Instrumental Analysis Harcourt Brace Philadelphia p 108 1998 Gonzalez R C and Woods R E in Digital Image Processing Addison Wesley Reading MA pp 31 37 1978 Smith W J in Modern Optical Engineering McGraw Hill Boston MA pp 135 139 1993 Application Note 64 Fluorescent DNA Gel Stain Detection Amersham Pharmacia Biotech code number 63 0031 02 2000 Application Note 56 Oncogene mRNA Profiling Using Fluorescent Quantitative PCR Amersham Pharmacia Biotech code number 63 0028 68 1999 Application Note 66 Fluorescent Protein Gel Stains Amersham Pharmacia Biotech code number 63 0031 04 2000 Protein Electrophoresis Amersham Pharmacia Biotech code number 80 6013 88 pp 13 36 1999 2 D Electrophoresis Using Immobilized pH Gradients Principles and Methods Amersham Pharmacia Biotech code number 80 6429 60 1998 Mansfield E S et al Molecular and Cellular Probes 9 145 156 1995 Ausubel F M et al eds Current Protocols in Molecular Biology John Wiley and Sons New York 1998 e 133 FLUORESCENCE IMAGING tm 63 0035 28 134 16 17 18 19 20 2 1 22 23 24 25 26 27 28 29 30 31 Pickett S and McNamara P Amerbsam Life Sciences Editorial Comments 23 2 20 21 1997 Applicat
159. the gel with plastic wrap being careful not to trap air bubbles or create wrinkles Place the gel face down on the glass platen Other options transfer the gel to Whatman 3MM filter paper and dry it Use Bind Silane to fix the gel to one glass electrophoresis plate and dry the gel directly on the glass plate Select the appropriate instrument settings for the fluorochrome label used see Table 24 or Appendix 3 Fluorlmager 595 Position the gel sandwich in the universal tray Select the instrument settings appropriate for the fluorochrome label used see Table 22 or Appendix 3 Analysis Display and analyse the gel image s using ImageQuant software FLUORESCENCE IMAGING Fig 44 Cy3 and Cy5 labelled DNA fragments Cy3 labelled fragments are in green and Cy5 labelled fragments are in red Lane 1 Cy3 labelled fragments 500 bp 365 bp 230 bp 150 bp 88 bp lane 2 Cy5 size ladder 500 bp 450 bp 400 bp 350 bp 300 bp 250 bp 200 bp 150 bp 100 bp with Cy3 labelled fragments 365 bp 268 bp 150 bp lane 3 Cy3 labelled fragments same as in lane 1 with Cy5 size ladder lane 4 Cy5 size ladder The presence of both Cy3 and Cy5 signal in the same region of the gel is displayed as yellow by the ImageQuant software lanes 2 and 3 m 63 0035 28 92 FluorSep software or Fragment Analysis software as appropriate refer to user documentation for details Expected results In lane size a
160. tional to the product of its extinction coefficient and its quantum efficiency as indicated in the following relationship Brightness e The extinction coefficient of a fluorochrome is the amount of light that a fluorochrome absorbs at a particular wavelength The molar extinction coefficient is defined as the optical density of a 1 M solution of the fluorochrome measured through a 1 cm light path For fluorochromes that are useful molecular labels the molar extinction coefficient at peak absorption is in the tens of thousands FLUORESCENCE IMAGING m 63 0035 28 6 6 The probability that an excited fluorochrome will emit light is its quantum efficiency and is given by the following equation number of photons emitted number of photons absorbed Values for range from 0 for nonfluorescent compounds to 1 for 100 efficiency For example fluorescein has a of 0 9 and Cy 5 has a of 0 3 In practice is usually listed as the quantum efficiency at the wavelength of maximum absorption Both fluorescein e 70 000 0 9 and CyS e 200 000 0 3 very bright fluorochromes Although their quantum efficiencies and extinction coefficients are quite different they are similar in brightness This illustrates the importance of considering both extinction coefficient and quantum efficiency when evaluating new fluorochromes Fluorescence intensity is also affected by the intensity of incident
161. trophoresis Tubulin red was detected using anti B tubulin monoclonal antibody and Total protein stains for Western blots Cy5 linked anti mouse IgG Blot stains facilitate the direct comparison of total and target protein Amounts of tubulin from left to right were 31 ng 62 ng 125 ng 250 ng 500 ng and 1000 ng Actin green was detected with rabbit anti from the same blot thus eliminating uncertainty associated with the transfer efficiency Fluorescent blot stains are more sensitive than actin antibody linked anti rabbit IgG common colourimetric stains such as Ponceau S amido black or Amounts of actin from left to right were 640 ng Coomassie Brilliant Blue Properties of commonly used fluorescent blot 320 ng 160 ng 80 ng 40 ng and 20 ng stains are summarized in Table 20 Table 20 Properties of fluorescent blot stains Excitation Emission Fluorescence Stains max nm max nm emission colour Application SYPRO Rose Plus 350 610 Red Blot stain PVDF or nitrocellulose SYPRO Ruby blot 280 450 618 Red Blot stain PVDF or nitrocellulose FLUORESCENCE IMAGING Instrument compatibility Table 21 Instrument settings for fluorescent detection of Western blots Substrates Typhoon Fluorlmager Storm VDS CL Substrate Excitation Emission Excitation Emission Fluorescence nm filter nm filter mode Excitation Emission DDAO 633 6
162. ucing agents Protein detection is much more sensitive using fluorescence Whereas the sensitivity of non fluorescence microplate based detection methods is typically in the pg ml range fluorescence based detection is generally in the ng ml range For example with NanoOrange protein can be measured in solution at concentrations as low as 300 ng ml The linear detection range of this assay is typically 10 30 fold Table 13 Fluorescent dyes for the quantification of proteins in solution Excitation Emission Fluorescence Dye max nm max nm emission colour Application CBQCA 465 550 Orange Protein quantification based on the number of primary amines NanoOrange 470 570 Orange Total protein quantification e 63 FLUORESCENCE IMAGING NN Instrument compatibility Table 14 Instrument settings for use with protein solution dyes Typhoon Fluorlmager Storm Dye Excitation Emission Excitation Emission Fluorescence nm filter nm filter mode CBQCA 532 580BP30 488 570DF30 Blue NanoOrange 532 580BP30 488 570DF30 Blue A 3 mm focal depth setting should be used on Typhoon when imaging microplates Typical protocol Amersham Pharmacia Biotech products available for this application Product Product number m Imaging systems Typhoon 8600 see catalogue Storm 840 860 see catalogue Fluorlmager 595 see catalogue Other materials required Product Vendor NanoOrange protein quantification kit Molec
163. ular Probes Inc m Clear polystyrene 96 well microplate Corning Costar Corp m Protein standards Suitable clear flat bottomed low fluorescence microplates should be used The recommended microplate is manufactured by Corning Costar Image quality and quantification for Storm and Typhoon are improved when using Nalge Nunc 96 well plates with removable strips so that the wells sit flat directly on the platen e Working stain preparation Prepare sufficient working solution of the NanoOrange reagent by diluting the stock solution 1 500 using the 1x diluent prepared according to manufacturer s instructions Note the NanoOrange working solution should be protected from light to prevent photodegradation and should be used within a few hours of its preparation m 63 0035 28 64 CHAPTER 5 FLUORESCENCE APPLICATIONS eo Sample staining Using the NanoOrange working solution from the previous step dilute the protein sample solution in microcentrifuge tubes to a final volume of at least 100 pl for FluorImager and Storm or 160 pl for Typhoon Note Using a higher dilution of the experimental sample ensures that any contaminants are maximally diluted Note NanoOrange is minimally affected by the presence of salts urea detergents DNA and amino acids see manufacturer s literature Heat the sample at 90 96 C for 10 min Cool to room temperature Pipette the samples into the microplate wells e Imagin
164. xposure The VDS CL has a cooled CCD that significantly reduces the noise however exposures longer than 30 minutes do not improve the sensitivity Alexa Fluor 350 346 442 1 1 1 1 1 250 300 350 400 450 500 550 600 Wavelength Alexa Fluor 488 495 519 1 1 350 400 450 500 550 600 650 700 Wavelength nm Alexa Fluor 546 556 573 Fluorescence excitation 350 400 450 500 550 600 650 700 Wavelength nm Fluorescence excitation 650 Fluorescence excitation 750 750 APPENDIX 2 Appendix 2 SPECTRAL CHARACTERISTICS OF COMMONLY USED FLUOROPHORES AND FLUORESCENT PROTEINS Note Gray line excitation blue line emission Alexa Fluor 430 433 539 Fluorescence emission Fluorescence excitation 1 1 1 1 1 1 300 350 400 450 500 550 600 650 Wavelength nm Alexa Fluor 532 532 554 Fluorescence emission Fluorescence excitation 1 1 1 1 1 l 350 400 450 500 550 600 650 700 Wavelength nm Alexa Fluor 568 578 603 5 S 8 E 5 8 8 g c o 8 8 S 3 T T EE T T T T 350 400 450 500 550 600 650 700 Wavelength nm Fluorescence emission 700 Fluorescence emission 750 Fluorescence emission 750 e 119 FLUORESCENCE IMAGING Alexa Fluor 594 Alexa Fluor 633 590 617 632 647 Fluorescence excitation Fluorescence emission Fluorescence excitation Fluorescence emission 400 450 500 550 600 650 700 750 80 400 450 500 550 600 650 700 750 800
165. ymatic reaction on PVDF membrane t Spectra were obtained in the presence of nucleic acids Spectra were obtained in the presence of protein Spectra of Cy2 Cy3 Cy3 5 Cy5 Cy5 5 Cy7 DDAO phosphate ECF ECL Plus and FluorX were 300 400 500 600 7 obtained at Amersham Pharmacia Biotech Wavelength nm DsRed EBFP ECFP EGFP and EYFP spectra are courtesy of Clontech All other spectra are courtesy of Molecular Probes Inc Fluorescence excitation Fluorescence emission o tm 23 4567 01 126 Excitation Fluorophore Nucleic acid gel stains Ethidium bromide SYBR Gold SYBR Green SYBR Green Il Vistra Green Protein gel stains SYPRO Orange 300 SYPRO Red 30 SYPRO Ruby 28 SYPRO Ruby IEF 28 SYPRO 30 Tangerine Nucleic acids solution stains OliGreen PicoGreen RiboGreen Protein solution stains CBQCA NanoOrange 465 470 Emission max nm 570 630 610 610 640 Ww O1 Co 570 Appendix 3 APPENDIX 3 INSTRUMENT COMPATIBILITY AND SETUP WITH COMMON FLUOROPHORES AND FLUORESCENT PROTEINS Typhoon Excitation nm 532 532 532 532 532 532 532 532 532 532 532 532 532 532 532 Substrates for Northern and Southern detection DDAO phosphate ECF 646 440 660 560 633 532 Emission filter 610BP30 526SP 526SP 526SP 526SP 580BP30 610BP30 610BP30 610BP30 610BP30 526SP 526SP 526SP 555BP20 580BP30
166. ysis Automated grid production and alignment Flagging of spots Analysis templates Sample nomenclature import and automatic identification of replicate sets Additional capabilities with database module Multiple array and or ratio experiments Querying on experiments arrays and spots Combining a series of queries Identification of similar expression patterns Organization of data subsets into results sets ARRAYVISION Premier analysis tool for array applications in medium to high throughput environments m Automated template alignment and analysis m Quality metrics and error flagging Up to three levels of template organization spots spot groups and sub arrays Pre configured and user defined protocols Direct comparisons between images and Elemental Display to highlight key targets Batch processing Wizard guides CHAPTER 5 FLUORESCENCE APPLICATIONS Chapter 5 FLUORESCENCE APPLICATIONS USING AMERSHAM PHARMACIA BIOTECH IMAGING SYSTEMS Introduction This chapter provides the basic information necessary for maximizing the fluorescence imaging capabilities of your system The application of fluorescence in standard molecular biology methods such as gel electrophoresis blotting and solution analysis of nucleic acids and proteins is discussed and typical protocols for each application area are included together with materials suggestions and tips for successful implementation of fluorescen
167. ystem typically has a zoom capacity so that different sample sizes can be captured in a single view Some falloff in light intensity detected at the corners and edges of the field can be expected in large field photographic imaging with a lens because light at the corners of the imaging field is farther from the centre of the lens than light on the axis 8 Such aberrations in field uniformity associated with CCD systems can be improved using software flat field corrections Signal detection and amplification An image that is focused on a two dimensional CCD array produces a pattern of charge that is proportional to the total integrated energy flux incident on each pixel The CCD array can be programmed to collect photonic charge over a designated period of time The total charge collected at a given pixel is equal to the product of the photonic charge generation rate and the exposure time Thermal cooling of the CCD can improve detection sensitivity by reducing the level of electronic noise System performance The performance of any CCD camera system is dependent on the system resolution sensitivity linearity and dynamic range Resolution The resolution of a captured image is linked to the geometry of the CCD with the size of each pixel varying from 6 30 pm Currently CCDs with formats from 512 x 512 4096 x 4096 elements are available Image resolution is reduced when charges from adjacent pixels are combined or binned during i

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