Cell Biosciences CELL BIOSCIENCES,INC. FluorChem HD2 and FC2 User Guide Copyright2010 Cell Biosciences,Inc.All rights reserved. 9692 FlorChem HD2 and FC2 User Guide
page 1 FluorChem HD2 and FC2 User Guide CELL BIOSCIENCES, INC. FluorChem HD2 and FC2 User Guide Copyright © 2010 Cell Biosciences, Inc. All rights reserved. Cell Biosciences, Inc. 3040 Oakmead Village Drive Santa Clara, CA 95051 Toll-free: (888) 607-9692 Tel: (408) 510-5500 Fax: (408) 510-5599 email: support@cellbiosciences.com web: www.cellbiosciences.com
FluorChem HD2 and FC2 User Guide P/N94-15068-00 Revision 2,June 2010 For research use only.Not for use in diagnostic procedures Patents and Trademarks Automatic Image Capture (AIC)and digital ProteomeChip technology are covered by U.S.Patent Nos 6.995,901,6.909,459,6.853,454.6.271,042,7,166.202,and other issued and pending patents in the U.S.and tein Forest logo,the AIC logo,AlphaCal Alphalmager.AlphaPartIIView,AlphaQuant AlphaSnap.AlphaSpec. AlphaUV.AlphaView,ChemiGlow,Chromalight dPC.digital ProteomeChip,FluorChem,MSRAT.Multilmage. NanoPro,the intertwined helix design,iWB,ProteomeChip.red,SpectraPlex,Xpedition,XplorBright,and XplorUV are trademarks or registered trademarks of Cell Biosciences,Inc.Other marks appearing in these materials are marks of their respective owners FluorChem HD2 and FC2User Guid
FluorChem HD2 and FC2 User Guide FluorChem HD2 and FC2 User Guide P/N 94-15068-00 Revision 2, June 2010 For research use only. Not for use in diagnostic procedures Patents and Trademarks Automatic Image Capture (AIC) and digital ProteomeChip technology are covered by U.S. Patent Nos 6,995,901, 6,909,459, 6,853,454, 6,271,042, 7,166,202, and other issued and pending patents in the U.S. and other countries. Cell Biosciences, the Cell Biosciences logo, the Alpha Innotech logo, Protein Forest, the Protein Forest logo, the AIC logo, AlphaCal, AlphaImager, AlphaPart11View, AlphaQuant, AlphaSnap, AlphaSpec, AlphaUV, AlphaView, ChemiGlow, Chromalight, dPC, digital ProteomeChip, FluorChem, MSRAT, MultiImage, NanoPro, the intertwined helix design, iWB, ProteomeChip, red, SpectraPlex, Xpedition, XplorBright, and XplorUV are trademarks or registered trademarks of Cell Biosciences, Inc. Other marks appearing in these materials are marks of their respective owners
Table of Contents Chapter l: Contrast Adjustments.........................44 Introduction and Setup....................I Using the Contrast Adjustments Tools for Grayscale Introduction to the FluorChem Systems and AlphaView m0ges.............45 Software.... …2 Automatic Enhancement...................51 Mouse Functions......... Imaging Tools....... 5 Starting AlphaView Software................. 2 Tool Bar..........5 About This Manual....... 3 Compare View....53 Customer Service and Technical Support...........3 Tool Box..56 FluorChem System Setup.......... Status Bar...... System Components....... .56 .4 System Placement........ .4 Chapter3: Cable Connections..................4 Drop-Down Menus.....................57 Hardware Installation.....5 Main Menu.… .58 Power Up Sequence........ …14 File Menu.. .58 System Information........................5 File Open Menu..................... .58 File Save/Load Analysis.................. File>Close Menu. 60 File Save,Save As,Save Modified,and Save All Basic lmaging Functions...8 Menus..................................60 Acquiring an Image Using the Navigator........8 Acquiring an Image Using the Acquire Button...24 Printer Setup........ .62 Automatic Image Capture (AIC Software-Optional.38 Exit Function............................63 Designing a User Protocol..................39 Edit Menu............ .64 Auto Image Capture (AIC)..................40 Edit Activation...............64 AlphaView System Quick Guide. .42 Copy and Crop...... 65 FlorChem HD2 and FC2 User Guide
page i FluorChem HD2 and FC2 User Guide Chapter 1: Introduction and Setup. . . . . . . . . . . . . . . . . . . . 1 Introduction to the FluorChem Systems and AlphaView Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Mouse Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Starting AlphaView Software. . . . . . . . . . . . . . . . . . 2 About This Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Customer Service and Technical Support. . . . . . . . . . . . 3 FluorChem System Setup . . . . . . . . . . . . . . . . . . . . . . . . 4 System Components . . . . . . . . . . . . . . . . . . . . . . . . 4 System Placement . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Cable Connections. . . . . . . . . . . . . . . . . . . . . . . . . . 4 Hardware Installation . . . . . . . . . . . . . . . . . . . . . . . 5 Power Up Sequence . . . . . . . . . . . . . . . . . . . . . . .14 System Information . . . . . . . . . . . . . . . . . . . . . . . .15 Chapter 2: Getting Started—Basic Imaging Functions . 17 Basic Imaging Functions . . . . . . . . . . . . . . . . . . . . . . . .18 Acquiring an Image Using the Navigator. . . . . . .18 Acquiring an Image Using the Acquire Button. . .24 Automatic Image Capture (AIC) Software–Optional .38 Designing a User Protocol. . . . . . . . . . . . . . . . . . .39 Auto Image Capture (AIC) . . . . . . . . . . . . . . . . . .40 AlphaView System Quick Guide . . . . . . . . . . . . . . . . . .42 Contrast Adjustments . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Using the Contrast Adjustments Tools for Grayscale Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Automatic Enhancement . . . . . . . . . . . . . . . . . . . 51 Imaging Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Tool Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Compare View. . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Tool Box. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Status Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Chapter 3: Drop-Down Menus . . . . . . . . . . . . . . . . . . . . . . . 57 Main Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 File Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 File > Open Menu . . . . . . . . . . . . . . . . . . . . . . . . 58 File > Save/Load Analysis. . . . . . . . . . . . . . . . . . . 59 File > Close Menu. . . . . . . . . . . . . . . . . . . . . . . . . 60 File Save, Save As, Save Modified, and Save All Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Print . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Printer Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Exit Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Edit Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Edit Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Copy and Crop . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Table of Contents
page ii Reset and Clear Options...................65 Chapter 4: m0 ge Menu..........65 Image Enhancement Tools...............85 0vey....66 Tool Box Enhancement Tab.....................8 Equqlize.................................66 Z00m. .86 Arithmetic................. .....66 Hist0gam..............87 Conversion............ ..67 Rotate-Fip. 89 Flat Field Calibrate (Manua). 68 Annotgte.............................. 90 Resize...................................69 Object Attributes... .91 Image Information......... ...69 Drawing Tools....... .95 Setup Menu..............................0 Editing Too水..97 Prnt info................................. False Color............ ....99 Print Mode..................... Gray Scale (Palette 0)... ….100 Print Date......... 72 High-Low (Palette I)..................00 Preferences...........................2 Other Palettes..... System Calibration.........................6 Filter............. .102 0 verlay Menu.............76 General Information. .103 Sharpening Filters. .104 Save Overlay.....7 Noise Filters....... 104 Despeckle Filters.. 104 Show Annotation..........................78 3-D (Contour)Filters........5 Utilities Menu...... .78 Smoothing Filters...............06 Notepad. Edge Filters. ..106 Explorer....... ..79 Horizontal Edge Filter. .06 View Menu.. .80 Vertical Edge Filter 106 Custom Filter. Default Tools Position.... .107 ……82 Undo Button..... ..107 Zoom Functions.. Window Menu. .82 Examples of Filter Results 107 Help Menu.… .83 Movie Mode.......... .107 FlorChem HD2 and FC2 User Guide
page ii FluorChem HD2 and FC2 User Guide Reset and Clear Options. . . . . . . . . . . . . . . . . . . .65 Image Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .65 Overlay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .66 Equalize . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .66 Arithmetic. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .66 Conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67 Flat Field Calibrate (Manual) . . . . . . . . . . . . . . . .68 Resize. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69 Image Information . . . . . . . . . . . . . . . . . . . . . . . . .69 Setup Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .70 Print Info. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71 Print Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71 Print Date. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .72 Preferences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .72 System Calibration. . . . . . . . . . . . . . . . . . . . . . . . .76 Overlay Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76 Load Overlay . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77 Save Overlay . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77 Overlay Libraries . . . . . . . . . . . . . . . . . . . . . . . . . .78 Show Annotation . . . . . . . . . . . . . . . . . . . . . . . . . .78 Utilities Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78 Notepad. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79 Explorer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79 View Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .80 Default Tools Position . . . . . . . . . . . . . . . . . . . . . .81 Zoom Functions. . . . . . . . . . . . . . . . . . . . . . . . . . .82 Window Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82 Help Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83 Chapter 4: Image Enhancement Tools . . . . . . . . . . . . . . . 85 Tool Box Enhancement Tab. . . . . . . . . . . . . . . . . . . . . 86 Zoom. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Histogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Rotate-Flip. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Annotate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Object Attributes . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Drawing Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Editing Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 False Color. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Gray Scale (Palette 0). . . . . . . . . . . . . . . . . . . . .100 High–Low (Palette 1) . . . . . . . . . . . . . . . . . . . . .100 Other Palettes . . . . . . . . . . . . . . . . . . . . . . . . . . .100 Filter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .102 General Information . . . . . . . . . . . . . . . . . . . . . .103 Sharpening Filters . . . . . . . . . . . . . . . . . . . . . . . .104 Noise Filters. . . . . . . . . . . . . . . . . . . . . . . . . . . . .104 Despeckle Filters . . . . . . . . . . . . . . . . . . . . . . . . .104 3-D (Contour) Filters. . . . . . . . . . . . . . . . . . . . . .105 Smoothing Filters . . . . . . . . . . . . . . . . . . . . . . . . .106 Edge Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . .106 Horizontal Edge Filter. . . . . . . . . . . . . . . . . . . . .106 Vertical Edge Filter . . . . . . . . . . . . . . . . . . . . . . .106 Custom Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . .107 Undo Button . . . . . . . . . . . . . . . . . . . . . . . . . . . .107 Examples of Filter Results . . . . . . . . . . . . . . . . .107 Movie Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .107
Chapter 5: Mass Standards..... .165 Image Analysis Tools.. …109 Advanced Lane Profile Options............70 Tool Box Analysis Tools Tab..... …110 Common Features. .180 Molecular Weight Detemmination. .110 Additional Analysis Tools .184 Entering Known Molecular Weights for Markers… 112 The Ruler Function .85 Determining Molecular Weights of Unknown The Scoring Function 186 Band5...... 113 Manual Count...... 88 Using the Molecular Weight Standards Library 115 Analyzing Amays..... …190 Special Functions....17 Lane Profile-Advanced. .194 The Groph Tool.........................18 Common Export Result Feature .194 Colony Count....................119 Editing Toos..............123 Appendix A: Opening AlphaView Files in Other Software Spot Count Data......125 Programs...............................197 Band Analysis Tools............ 126 Viewing AlphaView Images......................98 Creating an Object Area of Interest.........126 Using ResEdit..............................98 Magic Wand and AutoSpot..8 Manipulating Objects...... 132 Appendix B: AlphaView Molecular Weight Library Files 201 Band Analysis Measurements .133 List of Standards..02 Background Tab-Calculating Background Values....... ..135 Mass Standard Calibration Curves for Quantitative Appendix C: Data Description Tables..................205 PCR.. 37 Single Channel Columns.....................206 Lane Profile 140 Profle Path Width.. Appendix D: Auto Lane Detection Using an Area of Interest14 Bias and Dark Master Utility..............09 Manual Lane Detection.............42 Bias and Darkmaster File Generation............210 Bands Darker than Background Option......144 Appendix E: Image Analysis-Detect Bands …144 Flat Field Calibration..............3 Viewing Results..........................44 Gel Smiling Correction.....................57 Calibration Procedure. .214 Molecular Weight Tab..............6 FlorChem HD2 and FC2 User Guide
page iii FluorChem HD2 and FC2 User Guide Chapter 5: Image Analysis Tools. . . . . . . . . . . . . . . . . . . . 109 Tool Box Analysis Tools Tab . . . . . . . . . . . . . . . . . . . 110 Molecular Weight Determination. . . . . . . . . . . . . . . 110 Entering Known Molecular Weights for Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Determining Molecular Weights of Unknown Bands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Using the Molecular Weight Standards Library 115 Special Functions . . . . . . . . . . . . . . . . . . . . . . . . 117 The Graph Tool . . . . . . . . . . . . . . . . . . . . . . . . . 118 Colony Count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Editing Tools. . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Spot Count Data . . . . . . . . . . . . . . . . . . . . . . . . 125 Band Analysis Tools. . . . . . . . . . . . . . . . . . . . . . . . . . 126 Creating an Object Area of Interest . . . . . . . . . 126 Magic Wand and AutoSpot . . . . . . . . . . . . . . . 128 Manipulating Objects . . . . . . . . . . . . . . . . . . . . 132 Band Analysis Measurements . . . . . . . . . . . . . 133 Background Tab–Calculating Background Values. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 Mass Standard Calibration Curves for Quantitative PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Lane Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 Profile Path Width . . . . . . . . . . . . . . . . . . . . . . . 141 Auto Lane Detection Using an Area of Interest141 Manual Lane Detection . . . . . . . . . . . . . . . . . . 142 Bands Darker than Background Option. . . . . . 144 Image Analysis–Detect Bands . . . . . . . . . . . . . 144 Viewing Results . . . . . . . . . . . . . . . . . . . . . . . . . 144 Gel Smiling Correction . . . . . . . . . . . . . . . . . . . . 157 Molecular Weight Tab . . . . . . . . . . . . . . . . . . . 161 Mass Standards. . . . . . . . . . . . . . . . . . . . . . . . . .165 Advanced Lane Profile Options . . . . . . . . . . . . .170 Common Features. . . . . . . . . . . . . . . . . . . . . . . .180 Additional Analysis Tools . . . . . . . . . . . . . . . . . . . . . .184 The Ruler Function . . . . . . . . . . . . . . . . . . . . . . .185 The Scoring Function . . . . . . . . . . . . . . . . . . . . . .186 Manual Count . . . . . . . . . . . . . . . . . . . . . . . . . . .188 Analyzing Arrays . . . . . . . . . . . . . . . . . . . . . . . . .190 Lane Profile - Advanced . . . . . . . . . . . . . . . . . . .194 Common Export Result Feature . . . . . . . . . . . .194 Appendix A: Opening AlphaView Files in Other Software Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .197 Viewing AlphaView Images. . . . . . . . . . . . . . . . . . . . .198 Using ResEdit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .198 Appendix B: AlphaView Molecular Weight Library Files 201 List of Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . .202 Appendix C: Data Description Tables . . . . . . . . . . . . . . . . . .205 Single Channel Columns. . . . . . . . . . . . . . . . . . . . . . .206 Appendix D: Bias and Dark Master Utility . . . . . . . . . . . . . .209 Bias and Darkmaster File Generation. . . . . . . . . . . .210 Appendix E: Flat Field Calibration . . . . . . . . . . . . . . . . . . . . .213 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .214 Calibration Procedure . . . . . . . . . . . . . . . . . . . . . . . . .214