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Volume Tools
16-9
16.4 Volume Background Subtraction
When you draw a volume, you will probably include some non-data
background pixels inside the volume. These background pixels will usually
have an intensity value that you do not want to include in your volume
quantitation. There are two ways of calculating this background intensity for
your volumes: local and global.
The background subtraction method is selected in the Volume Report Options
dialog box (section 19.5.a).
Local Background Subtraction
Local background subtraction calculates a separate background intensity for
each unknown and standard volume you create. For each volume, the
intensities of the pixels in a 1-pixel border around the volume are added
together and divided by the total number of border pixels. This gives an
average intensity for the background around each volume, which is then
subtracted from the intensity of each pixel inside the volume.
Fig. 16-7. Local background is calculated from a one-pixel border around the volume
One-pixel border
around the volume
- Quantity One 1
- Table of Contents 3
- Contents 5
- 1. About This Document 9
- 2. Bio-Rad Listens 10
- 1. Introduction 11
- 1.3 About Gel Quality 13
- Acquire Image 14
- Optimize Image 14
- 1.5 Computer Requirements 15
- Macintosh 16
- 1.6 Installing on a PC 17
- 1.7 Installing on a Macintosh 18
- 1.8 Starting the Program 19
- 1.8.a Software License 20
- (If Necessary) 20
- Introduction 21
- 1.8.c Trial Period 22
- 1.8.d Software Registration 24
- Registering by Internet 25
- Registering by Fax or E-mail 26
- Entering a Password 26
- 1.9 Contacting Bio-Rad 27
- 2. General Operation 29
- 2.1.b Main Toolbar 30
- 2.1.c Status Boxes 30
- 2.1.d Secondary Toolbars 31
- 2.1.e Quick Guides 32
- General Operation 33
- 2.2 Mouse-assignable Tools 34
- 2.3 Keyboard Commands 35
- Opening TIFF Images 38
- Close All 39
- Save All 40
- Revert to Saved 40
- Image Info 40
- Change the Image Dimensions 41
- Reduce File Size 42
- 2.5 Preferences 45
- 2.5.a Misc 46
- 2.5.b Toolbar 47
- 2.5.c Application 49
- 2.5.d Display 50
- 2.6 User Settings 51
- 3. Gel Doc 53
- 3.2 Step I. Position Gel 56
- 3.3 Step II. Acquire Image 57
- Manual Expose 58
- Fig. 3-5. Manual Expose 59
- 3.4 Step III. Select Output 60
- 3.5 Image Mode 61
- 3.6 Exposure Status 62
- 3.7 Display 62
- 3.8 Options 64
- DAC Settings 65
- Imaging Area 65
- Auto Exposure Threshold 65
- Reminder 65
- Save Options 66
- 4. Chemi Doc 67
- Chemi Doc 69
- 4.2 Step I. Position Gel 70
- 4.3 Step II. Acquire Image 71
- 4.4 Step III. Select Output 75
- Video Print 76
- Annotate 76
- 4.5 Image Mode 77
- 4.6 Exposure Status 78
- 4.7 Display 78
- 4.8 Options 80
- 5. GS-700 Imaging 83
- 5.1 GS-700 Acquisition Window 84
- GS-700 Imaging Densitometer 85
- 5.3 Step II. Select Scan Area 88
- Selecting an Area 89
- 5.5 Acquire the Image 91
- 5.6 Calibration 92
- 5.6.a Editing the Step Tablet 93
- 5.7 Other Options 96
- 6. GS-710 Imaging 99
- 6.1 GS-710 Acquisition Window 100
- GS-710 Imaging Densitometer 101
- Quantity One User Guide 102
- Choosing Your Own Settings 104
- 6.3 Step II. Select Scan Area 105
- 6.5 Calibration 107
- 6.5.a Editing the Step Tablet 108
- Diffuse Versus Specular O.D 110
- 6.6 Acquire the Image 111
- 6.7 Other Options 112
- Highlight Saturated Pixels 113
- Hide Grid 113
- 7. GS-800 Imaging 115
- Densitometer 115
- 7.1 GS-800 Acquisition Window 116
- GS-800 Imaging Densitometer 117
- 7.3 Step II. Select Scan Area 120
- 7.5 Calibration 122
- 7.5.a Editing the Step Tablet 123
- 7.5.b Calibration Settings 125
- 7.6 Acquire the Image 126
- 7.7 Other Options 126
- Auto Save After Scan 127
- Make Backup Copy 127
- 8. Fluor-S MultiImager 129
- Simulation Mode 130
- Fluor-S MultiImager 131
- Custom Applications 133
- 8.3 Step II. Position/Focus 134
- Position 135
- 8.5 Acquire the Image 138
- 8.6 Options 139
- 8.6.a Dark Subtraction Type 140
- Referenced 141
- Reset Reference 141
- 8.6.b Live Acquire Mode 142
- 8.6.c Save 143
- 8.7 Other Features 144
- File Size of Images 145
- 9. Fluor-S MAX MultiImager 147
- Fluor-S MAX MultiImager 149
- Scan Dimension 152
- 9.3 Step II. Position/Focus 153
- 9.5 Acquire the Image 156
- Live Acquire 157
- 9.6 Options 158
- Reset Reference Button 159
- 9.6.b Live Acquire Mode 160
- 9.6.c Save 161
- 9.6.d Imaging Area Size 162
- 9.7 Other Features 163
- 10.Personal Molecular 165
- Imager FX 165
- Personal Molecular Imager FX 167
- 10.2 Step I. Select Scan Area 168
- 10.4 Acquire the Image 170
- 10.5 Options 171
- 11. Molecular Imager FX 173
- 11.1 FX Acquisition Window 174
- Molecular Imager FX 175
- Standard Applications 176
- Standard FX Applications 176
- Sample Intensity 178
- 11.5 Acquire the Image 183
- 11.6 Options 184
- 12. Viewing and Editing 187
- Zoom Box 188
- Viewing and Editing Images 189
- Zoom In/Zoom Out 189
- Arrow Keys 189
- View Entire Image 190
- Centering an Image 190
- Imitate Zoom 190
- Tiling Windows 190
- 12.2 Density Tools 191
- Density in Box 192
- Plot Density Distribution 192
- Plot Cross-section 192
- Plot Vertical Trace 193
- 12.4 Multi-Channel Viewer 194
- Viewing Options 195
- 12.5 Image Stack Tool 196
- 12.6 Colors 198
- Changing a Color 199
- 12.7 Transform 201
- 12.7.a Transform Subwindows 202
- 12.7.b Transform Controls 203
- Gamma Slider 205
- 12.7.c Other Features 206
- 12.8.a Cropping Images 208
- Flipping 209
- 90° Rotations 210
- Custom Rotation 210
- Auto-scale 214
- Dark Contrast Slider 214
- Background Slider 215
- Background Box 215
- Background Stripe 216
- Completing the Subtraction 216
- 12.10 Filtering Images 217
- 12.10.a Filter Wizard 218
- Step 2: Select Filter Size 219
- Step 3: Begin Filtering 220
- 12.11 Invert Data 222
- 12.12 Text Overlays 223
- Editing a Text Overlay 224
- Line Tool 224
- 12.14 Sort and Recalculate 226
- 13. Lanes 227
- 13.1.a Creating a Lane Frame 228
- Manual Frame Lanes 229
- 13.1.b Editing the Frame 230
- Adjusting the Entire Frame 231
- Adding and Adjusting Anchors 231
- 13.1.c Defining a Single Lane 233
- 13.1.d Adjusting Single Lanes 233
- 13.1.f Deleting Lanes 234
- 13.1.g Lane Width 234
- 13.1.h Creating a Lane Profile 235
- Lanes to Model 237
- Trace Display 237
- When to Apply 237
- Calculation Method 237
- 13.3 Compare Lanes 239
- Magnifying the Graph 240
- Show Gaussian Modeling 240
- Align Band Types 241
- Printing and Exporting 242
- 13.4 Lane-based Arrays 243
- Adjusting the Array Matrix 244
- Analyzing Array Data 246
- 14. Bands 247
- Quantified 248
- 14.2.a Detection Parameters 251
- Shadow Rejection 252
- Band Limit 252
- Sensitivity 252
- Lane Width 253
- Minimum Density 254
- Noise Filter 254
- Shoulder Sensitivity 255
- Size Scale 255
- Saving Parameters 255
- Loading Parameters 256
- Deleting Parameters 256
- Resetting Parameters 256
- In Brackets Mode 257
- In Lines Mode 258
- 14.3.b Adjusting Bands 259
- 14.4 Plotting Traces of Bands 260
- 14.5 Band Attributes 261
- 14.7 Gauss-Modeling Bands 267
- Incorrect Modeling 270
- One pixel enlarged to 271
- 14.8.a Contouring Bands 272
- 14.8.b Drawing Tools 273
- Density in Region 274
- Drawing Band Boundaries 274
- Editing Band Boundaries 276
- 15. Standards and Band 277
- Matching 277
- Standards and Band Matching 279
- Selecting Predefined Standards 279
- Creating New Standards 279
- 15.1.a Standards Form 281
- Entering Standard Values 282
- Showing the Modeling Lines 284
- Standards Regression Models 286
- Display Options 287
- Read-Only Standards 288
- 15.2 Band Matching 289
- 15.2.b Band Set Form 292
- List of Band Types 294
- Band Set Toolbar 295
- Other Band Set Form Functions 296
- 15.2.e Graphs of Match Data 297
- 15.3 Quantity Standards 299
- 15.3.b The Calibration Curve 304
- Checking the Imported Curve 308
- 16. Volume Tools 309
- Volume Contour Tool 310
- Volume Tools 311
- Volume Freehand Tool 311
- Volume Rect Tool 312
- Volume Circle Tool 312
- Volume Labels 313
- Displaying Volumes 314
- 16.3 Volume Standards 315
- Local Background Subtraction 317
- Global Background Subtraction 318
- 16.5 Volume Arrays 319
- What Is a Volume Array? 320
- Creating a Volume Array 320
- Moving an Array 322
- Resizing an Array 322
- Resizing the Array Cells 323
- Copying an Array 323
- Ungrouping an Array 324
- 17. Colony Counting 325
- Colony Counting 327
- 17.2 Counting the Colonies 328
- 17.3 Displaying the Results 329
- Colonies 330
- White and Blue Colonies 331
- 17.8 Saving to a Spreadsheet 334
- Creating/Opening a Batch File 335
- Naming/Saving a Count 336
- Loading Another Image 336
- 18. Differential Display and 337
- Normalization 338
- Displaying the Results 339
- Testing for Ambiguity 341
- 19. Reports 343
- Printing Your Report 345
- Exporting Your Report Data 346
- 19.2 Lane and Match Reports 347
- 19.3 1-D Analysis Report 349
- Comparison Options 350
- Comparison Method 351
- 19.4.a Compare Lane Images 352
- 19.4.b Phylogenetic Tree 354
- Neighbor Joining 356
- Other Methods 356
- Neighbor Joining Only 359
- All Other Methods 359
- 19.4.c Similarity Matrix 360
- 19.5 Volume Analysis Report 361
- 19.5.a Volume Report Options 362
- Background Subtraction Method 363
- 19.6 Volume Regression Curve 364
- 19.7 VNTR Report 366
- 20. Printing and Exporting 369
- 20.1.b Print Actual Size 371
- 20.1.d Video Printing 373
- Analysis Export Mode 374
- Publishing Export Mode 374
- Exporting the Image 375
- Appendix A 377
- Appendix B 379
- B.2 Gel Layout 381
- Appendix B. Other Features 383
- Zoom tools 401
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