Handbook of Biological Confocal Microscopy

cover-large......Page 1
HANDBOOK OF
BIOLOGICAL
CONFOCAL MICROSCOPY
THIRD EDITION......Page 2
Title Page
......Page 3
Copyright Page
......Page 4
Preface to the Third Edition......Page 6
Preface to the Second Edition......Page 7
Table of Contents
......Page 9
Contributors......Page 23
Lateral Resolution1......Page 27
Axial Resolution......Page 29
CONFOCAL IMAGING......Page 30
Nipkow Disk......Page 31
Electron-Beam-Scanning Television......Page 32
Holography......Page 33
Aperture Scanning......Page 34
CONFOCAL LASER-SCANNING MICROSCOPE......Page 35
TWO- AND MULTI-PHOTON MICROSCOPY......Page 36
Speed of Image or Data Acquisition......Page 37
Yokogawa Disk-Scanning Confocal System......Page 38
Lens Aberration......Page 39
Unintentional Beam Deviation......Page 41
SUMMARY......Page 42
REFERENCES......Page 43
Counting Statistics: The Importance of n......Page 46
Effects of Satu ration......Page 47
PRACTICAL PHOTON EFFICIENCY......Page 50
Objectives......Page 51
Is the Confocal Pinhole a "Good Thing"?......Page 52
The Photomultiplier Tube......Page 54
The Cooled Charge-Coupled Device......Page 56
Digitization......Page 57
Photon Counting......Page 58
Measuring Photon Efficiency......Page 59
Can Resolution Be Too High?......Page 62
Visibility, Resolution, and the Rose Criterion......Page 63
Digitization and the Nyquist Criterion......Page 64
Practical Considerations Relating Resolution to Distortion......Page 65
REFERENCES......Page 67
WAVELENGTH SELECTIVE FILTERING DEVICES......Page 69
Separating the Light Paths......Page 70
Interference Filters......Page 71
Types of Interference Filters......Page 72
Dichroic and Polarizing Beam-Splitters......Page 76
MECHANICAL SCANNERS......Page 77
Galvanometer Scanners......Page 78
ACOUSTO-OPTICAL COMPONENTS......Page 80
Acousto-Optical Beam-Splitters......Page 82
POLARIZING ELEMENTS......Page 83
REFERENCES......Page 84
Pixels, Images, and the Contrast Transfer Function......Page 85
Digitization of Images......Page 88
HOW BIG SHOULD A PIXEL BE? SAMPLING AND QUANTUM NOISE......Page 89
THE NYQUIST CRITERION......Page 90
Estimating the Expected Resolution of an Image......Page 91
NYQUIST RECONSTRUCTION:IIDECONVOLUTION LITE"......Page 94
Some Special Cases......Page 96
The Zone System: Quantified Photography......Page 97
Problems Posed by Non-Linearity of the Visual System and Image Display Devices......Page 98
Matching the Gray levels to the Information Content of the I mage Data......Page 99
What Counts as Noise?......Page 100
Calibrating a Charge-Coupled Device to Measure the ISF......Page 101
GAIN-REGISTER CHARGE-COUPLED DEVICES......Page 102
Multiplicative Noise......Page 103
REFERENCES......Page 105
LASER POWER REQUIREMENTS......Page 106
THE BASIC LASER......Page 107
Laser Modes: longitudinal (Axial) and Transverse......Page 108
Coherent Properties of laser light......Page 109
Heat Removal......Page 110
Sources of Noise in lasers......Page 111
Laser Beam Intensity Stabilization in Current- or Power-Control Mode......Page 112
Fiber-optic Coupling......Page 113
Polarization of the Laser Light......Page 114
Total Internal Reflection Microscopy......Page 115
Argon-ion......Page 116
Helium-Neon......Page 128
Solid-State Lasers......Page 129
Semiconductor or Diode Injection Lasers......Page 131
Violet and Deep Blue Diode Lasers......Page 133
Continuous Wave Fiber Lasers and Up-Conversion......Page 135
PULSED lASERS......Page 136
Cavity-Dumped Lasers......Page 137
Titanium-Sapphire and Related Ultrafast Lasers......Page 138
Ultrafast Fiber Lasers......Page 139
Optical Parametric Oscillators and Optical Parametric Amplifiers......Page 140
What the User Can Do......Page 141
Cooling Water......Page 142
SAFETY PRECAUTIONS......Page 143
CONCLUSION......Page 144
REFERENCES......Page 146
Pulse Length Measurement by Optical Means......Page 149
Expert level......Page 150
LIST OF ABBREVIATIONS......Page 151
Brightness......Page 152
Uniformity......Page 153
Wavelength......Page 155
Coherence......Page 156
Scrambling and Filtering the light......Page 157
The Actual Source of the Light......Page 158
How to Cope with the Heat?......Page 159
Source Alignment......Page 160
Wavelength......Page 161
Ramp-Up and Short-Time Stability......Page 162
Radiance......Page 163
Control......Page 164
The Bare Minimum......Page 165
EXPOSURE TIME AND SOURCE BRIGHTNESS......Page 167
FUTURE TRENDS......Page 169
REFERENCES......Page 170
INTRODUCTION......Page 171
Defocusing......Page 172
Spherical Aberration......Page 173
Flatness of Field......Page 177
Longitudinal Chromatic Aberration......Page 178
Lateral Chromatic Aberration or Chromatic Magnification Difference......Page 181
FINITE VERSUS INFINITY OPTICS......Page 182
WORKING DISTANCE......Page 183
Transmission of Microscope Objectives......Page 184
APPENDIX: LIGHT TRANSMISSION SPECIFICATIONS FOR A NUMBER OF MODERN OBJECTIVES MADE BY DIFFERENT MANUFACTURERS.4......Page 186
INTRODUCTION......Page 188
Absorption Contrast......Page 189
Scattering and Reflection Contrast......Page 193
Phase Contrast......Page 197
Fluorescence Contrast......Page 198
Negative Contrast......Page 199
Special Concerns in Ultraviolet and Near-Infrared Range Confocal Microscopy......Page 200
Total Internal Reflection Contrast......Page 203
Harmonic Generation Contrast......Page 205
Total Internal Refraction Fluorescence Contrast......Page 206
Fluorescence Resonant Energy Transfer......Page 210
Fluorescence Recovery After Photobleaching (FRAP and FLIP)......Page 213
DERIVED CONTRAST (SYNTHETIC CONTRAST)......Page 214
Deconvol ution......Page 215
Spectral Unmixing and Color Reassignment......Page 216
Effects of the Specimen: Spherical Aberration and Optical Heterogeneity......Page 218
Mounting Medium Selection......Page 224
Background level and Ghost Images from the Transmission Illuminator......Page 227
Contrast Resulting from Differences InPhotobleach ing Dynamics......Page 228
Effect of Spectral Leakage and Signal Imbalance Between Different Channels......Page 229
SUMMARY......Page 230
REFERENCES......Page 231
Telecentricity......Page 233
The Scanning System......Page 234
Detection......Page 236
Polarization......Page 237
Class 2......Page 238
Evaluation of Scanner Arrangements......Page 239
Center Pivot/Off-Axis Pivot......Page 240
Disk Scanners......Page 241
Object Scanners......Page 242
MULTI-FLUORESCENCE......Page 243
Setups for the Integration of Laser Cutters......Page 244
CONCLUSIONS AND FUTURE PROSPECTS......Page 245
REFERENCES......Page 246
live Cell Imaging: Probing the Future......Page 247
Advantages and Limitations of Confocal Laser- ScanningMicroscopes......Page 248
A Renaissance - Advantages of Disk-Scanning Confocal Imaging......Page 249
Fill Factor and Spacing Interval F......Page 250
Axial Resolution......Page 251
The Tandem-Scanning Confocal Microscope......Page 254
Confocal Microscopes......Page 255
New Fast Line Scanner - Zeiss LSM510 LIVE......Page 257
Image Intensifiers......Page 258
On-Chip Electron Multiplying Charge-Coupled Device......Page 259
Electron Multiplication Charge-Coupled Devices and Disk Scanners24......Page 260
Blazingly Fast Confocal Imaging......Page 261
FUTURE DEVELOPMENTS?......Page 262
SUMMARY......Page 263
REFERENCES......Page 264
INTRODUCTION......Page 265
Fiber-Optic Interferometer......Page 266
Point Spread Function Measurements......Page 267
CHROMATIC ABERRATIONS......Page 268
Axial Shift......Page 269
Zernike Polynomial Fit......Page 271
Restoration of a 3D Point Spread Function......Page 273
Temperature Variations......Page 274
Polarization Effects......Page 275
REFERENCES......Page 276
THE QUANTAL NATURE OF LIGHT......Page 277
Photovoltaic......Page 278
Image Dissector......Page 280
Comparison of Detectors......Page 281
Noise in Photoemissive Devices......Page 282
Statistics of Photon Flux and Detectors......Page 283
Representing the Pixel Value......Page 284
CONVERSION TECHNIQUES......Page 285
Point Detection Assessment and Optimization......Page 286
Field Detection Assessment and Optimization......Page 287
DETECTORS PRESENT AND FUTURE......Page 288
REFERENCES......Page 290
EXPERIMENTAL CONSIDERATIONS......Page 291
Pattern Generation......Page 292
COMPUTING OPTICAL SECTIONS FROM STRUCTURED-ILLUMINATION DATA......Page 294
RESOLUTION IMPROVEMENT BY STRUCTURED ILLUMINATION......Page 296
REFERENCES......Page 302
APPENDIX: IMAGING THICK SPECIMEN WITH STRUCTURED IllUMINATION......Page 304
Dependence of the Maximal Sample Thickness on the Number of Collected Photons......Page 305
What Is the Microscopist Trying to Achieve?......Page 306
Identifying Unknown Structures......Page 307
Visualization for Multi-Dimensional Measurements......Page 310
What Dimensions Can the Images and Views Have?......Page 312
Calibrating the Image Space......Page 313
Processing Image Data......Page 314
Processor Performance: How Fast Will My Computer Process Images?......Page 315
Loading the Image Subregion......Page 316
True Color......Page 317
Animations......Page 318
Optimal Use of the 5D Display Space......Page 319
Rotations......Page 320
Stereoscopic Views......Page 322
Temporal Coding and z Depth......Page 325
Choosing the Data Objects......Page 326
Scan Conversion......Page 327
Projection Rules......Page 328
Z-Buffering......Page 330
Local Projections......Page 331
Artificial Lighting: Reflection Models......Page 332
Phong Shading......Page 334
Artificial Lighting: Absorption and Transparency......Page 335
CONCLUSION......Page 338
REFERENCES......Page 340
INTRODUCTION......Page 342
TYPES OF AUTOMATED IMAGE ANAlYSJS STUDIES......Page 344
Data Collection Guidelines for Image Analysis Purposes......Page 345
Image Preprocessing Methods......Page 346
Region-Based Segmentation Methods......Page 347
EXAMPLE ILLUSTRATING BLOB SEGMENTATION......Page 348
Model-Based Object Merging......Page 349
Vectorization Methods......Page 350
REGISTRATION AND MONTAGE SYNTHESIS METHODS......Page 354
METHODS FOR QUANTITATIVE MORPHOMETRY......Page 357
METHODS FOR VALIDATING THE SEGMENTATION AND MAKING CORRECTIONS......Page 359
ANALYSIS OF MORPHOMETRIC DATA......Page 360
REFERENCES......Page 361
Singlet State Saturation......Page 364
Autofluorescence from Endogenous Fluorophore......Page 365
Theory......Page 366
Protective Agents......Page 367
Fluorescent Organic Dyes......Page 368
Luminescent Nanocrystals......Page 369
Fluorescent Lanthanide Chelates......Page 371
Ca2+ Indicators......Page 372
Other Forms of Ratioing......Page 373
FUTURE DEVElOPMENTS......Page 374
REFERENCES......Page 375
Organic Dyes......Page 379
Fluorescent Proteins: Green Fluorescent Protein and Phycobiliproteins......Page 382
Multi-Photon Excitation......Page 383
Loading Methods......Page 384
Target Abundance and Autofluorescence Considerations......Page 386
Localization and Metabolism......Page 387
Photobleaching......Page 388
Phototoxicity......Page 389
REFERENCES......Page 390
CHARACTERISTICS OF FIXATIVES......Page 394
Formaldehyde......Page 395
Preparation of the Stock Solutions......Page 396
CRITICAL EVALUATION OF LIGHT MICROSCOPY FIXATION AND MOUNTING METHODS......Page 397
Use of the Cell Height to Evaluate the Fixation Method......Page 398
Well-Defined Structures Can Be Used to Evaluate Fixation Methods......Page 399
GENERAL NOTES......Page 400
Triple Labeling......Page 401
Labeling Thick Sections......Page 402
Microwave Fixation......Page 403
REFERENCES......Page 404
INTRODUCTION......Page 407
Fluorescence lifetime Imaging......Page 408
Combining Fluorescence and Other Imaging Modalities......Page 409
GENERAL CONSIDERATIONS FOR CONFOCAL MICROSCOPY OF LIVING CELLS......Page 412
Fluorescent Probes......Page 413
Improving Photon Efficiency......Page 415
Phototoxicity......Page 416
Low-Dose Imaging Conclusion......Page 417
SPECIFIC EXAMPLE II: MULTI-DIMENSIONAL IMAGING OF MICROGLIAL CElL BEHAVIORS IN LIVE RODENT BRAIN SLICES......Page 418
Maintaining Tissue Health on the Microscope Stage......Page 419
Imaging Methods......Page 420
Handling the Data......Page 421
Conclusion......Page 422
FUTURE DIRECTIONS......Page 424
REFERENCES......Page 425
THEORY......Page 430
RESULTS OF THEORETICAL CALCULATIONS......Page 433
EXPERIMENTS......Page 435
Dry Objectives......Page 436
Spherical Aberration Correction......Page 437
Practical Strategies to Reduce Refractive Index Mismatch......Page 438
REFERENCES......Page 439
linear Absorption......Page 440
Nonlinear Absorption......Page 442
Scattering......Page 443
REFRACTIVE INDEX HETEROGENEITY......Page 444
BIREFRINGENT STRUCTURES IN PLANT CELLS......Page 446
Microspectroscopy......Page 447
Light-Specimen Interaction (Fluorescence Emission)......Page 451
THE EFFECT OF FIXATION ON THE OPTICAL PROPERTIES OF PLANTS......Page 454
Suspension-Cultured Cells......Page 455
Stem and Root......Page 456
Microspores and Pollen Grains......Page 457
Pollen Grains......Page 458
Pollen Germination......Page 459
Cuticles, Hairs, and Waxes......Page 460
Storage Structures......Page 461
Mineral Deposits......Page 462
Fungi......Page 464
REFERENCES......Page 465
Shot Noise and Quantum Efficiency......Page 468
Background Noise......Page 469
SIGNAL LEVEL IN CONFOCAL MICROSCOPES......Page 470
SIGNAl-TO-NOISE RATIO FOR CONFOCAL MICROSCOPES......Page 471
N2 and Detectability......Page 472
DESIGNS OF CONFOCAL MICROSCOPES......Page 473
Bleaching-limited Performance......Page 474
Effects of Scanning Speed......Page 476
SUMMARY......Page 477
REFERENCES......Page 478
THE POINT SPREAD FUNCTION: IMAGING AS A CONVOLUTION......Page 479
DECONVOLUTION......Page 483
Combination of Charged-Coupled Device and Confocal Imaging......Page 484
Fluorescent light Detection......Page 485
Gain Register Charge-Coupled Devices......Page 486
The Best Solution: Deconvolving Confocal Data......Page 487
PRACTICAL COMPARISONS......Page 489
CONCLUSION......Page 492
REFERENCES......Page 493
Advantages and limitations......Page 494
Transmitted Light, Bright-field (TLB)......Page 498
Differential Interference Contrast (DIC)......Page 499
3D......Page 501
2D Image Filtering......Page 502
LIGHT SOURCE AND OPTICS ALIGNMENT......Page 503
Subpixel......Page 504
Polarized light......Page 505
Blind Deconvolution and Spherical Aberration......Page 506
Two Photon......Page 507
SPEED......Page 508
ACKNOWLEDGMENTS......Page 509
REFERENCES......Page 510
Convolution: An Introduction For Those Who Don't Remember Second-Year Calculus......Page 511
About Out-of-Focus Light......Page 512
Convolution, Fourier Transforms, and the FFT......Page 513
BACKGROUND......Page 514
Image Formation......Page 515
FORWARDS: CONVOLUTION AND THE IMAGING SYSTEM......Page 516
QUANTIFYING THE POINT SPREAD FUNCTION......Page 518
THE MISSING CONE PROBLEM......Page 520
Nearest-Neighbor Deconvolution......Page 521
Nonlinear Constrained Iterative Deconvolution Algorithms......Page 522
Comparison of Methods......Page 523
REFERENCES......Page 525
Step Index and Gradient Index Optical Fibers......Page 527
Modes in Optical Fibers......Page 528
Fused Biconical Taper Couplers: Fiber-Optic Beam-Splitters......Page 529
Fiber Image Transfer Bundles......Page 530
Optical Fiber for Delivering Light......Page 531
Same Fiber for Both Source and Confocal Detection......Page 532
BENCHTOP SCANNING MICROSCOPES EXPLOITING FIBER COMPONENTS......Page 533
Miniature Confocal Imaging Heads Based on Coherent Imaging Bundles......Page 534
Bundle Imagers for In Vivo Studies in Animals......Page 535
Vibrating the lens and Fiber......Page 536
Scanning with Micromirrors......Page 537
Scanning Fiber Confocal Microscopes for In Vivo Imaging in Animals......Page 538
SUMMARY......Page 539
REFERENCES......Page 541
Fluorescence Lifetime Imaging Applications......Page 542
Fluorescence Resonance Energy Transfer......Page 543
The Phase Fluorometry Method......Page 544
Widefield, Slit-Scanning, and Spinning-Disk Implementations......Page 545
Time Correlated Single-Photon Counting Implementations......Page 546
TCSPC FLIM......Page 548
Multi-Exponential Lifetimes......Page 549
Photon Economy......Page 550
Acquisition Time......Page 551
Detectors for Fluorescence Lifetime Imaging......Page 552
Multi-labeling and Segmentation......Page 553
lon-Concentration Determination......Page 554
pH Imaging......Page 555
Probes for Fluorescence lifetime Microscopy......Page 556
SUMMARY......Page 558
REFERENCES......Page 559
Physics of Multi-Photon Excitation......Page 561
Optical Pulse length......Page 563
Wavelengths......Page 564
Photodamage: Heating and Bleaching......Page 565
Hybrid Mode-Locked Dye laser......Page 566
Whole-Area and External Detection......Page 567
Optical Aberrations......Page 568
Two-Photon Absorption Cross-Sections......Page 569
CEll VIABILITY DURING IMAGING......Page 570
ACKNOWLEDGMENTS......Page 571
REFERENCES......Page 572
Determination of the Optimum Degree of Parallelization......Page 576
A Multi-Focal Multi-Photon Microscopy Setup Using a Nipkow-Type Microlens Array......Page 577
Resolution......Page 578
Time Multiplexing as a Solution to Interfocal Crosstalk......Page 579
Alternative Realizations......Page 580
Fluorescence Lifetime Imaging......Page 581
LIMITATIONS......Page 582
CURRENT DEVELOPMENTS......Page 584
REFERENCES......Page 585
INTRODUCTION......Page 587
The Point Spread Function......Page 588
MULTI-FOCAL MULTI-PHOTON MICROSCOPY-4Pi MICROSCOPY......Page 589
Live Mammalian Cell 4Pi Imaging......Page 590
TYPE C 4Pi MICROSCOPY WITH THELEICA TCS 4PI......Page 591
Resolution......Page 593
SUMMARY AND OUTLOOK......Page 594
I5M-OTF......Page 595
Additional Information from the 4Pi-PSF......Page 596
BREAKING THE DIFFRACTION BARRIER: THE CONCEPT OF REVERSIBLE SATURABLE OPTICAL FLUORESCENCE TRANSITIONS......Page 597
DIFFERENT APPROACHES OF REVERSIBLE SATURABLE OPTICAL FLUORESCENCE TRANSITIONS MICROSCOPY......Page 599
STIMULATED EMISSION DEPLETION MICROSCOPY......Page 600
CHALLENGES AND OUTLOOK......Page 603
REFERENCES......Page 605
Data Compression......Page 606
Sequential Devices......Page 611
Compact Disks......Page 612
Digital Video Disk (DVD)......Page 613
Monitors......Page 614
Liquid Crystal Displays......Page 615
Photographic Systems......Page 616
Digital Printers......Page 617
Bulk Storage......Page 619
REFERENCES......Page 620
INTRODUCTION......Page 621
UNIQUE FEATURES OF COHERENT ANTI-STOKESRAMAN SCATTERING UNDER THE TIGHT-FOCUSING CONDITION......Page 622
FORWARD AND BACKWARD DETECTED COHERENT ANTI -STOKES RAMAN SCATTERING......Page 623
OPTIMAL LASER SOURCES FOR COHERENT ANTI -STOKES RAMAN SCATTERING MICROSCOPY......Page 625
Phase Control of Excitation Pulses......Page 626
COHERENT ANTI-STOKES RAMAN SCATTERING CORRELATION SPECTROSCOPY......Page 628
COHERENT ANTI-STOKES RAMAN SCATTERING IMAGING OF BIOLOGICAL SAMPLES......Page 629
CONCLUSIONS AND PERSPECTIVES......Page 630
REFERENCES......Page 631
SURFACE IMAGING MICROSCOPY AND EPISCOPIC FLUORESCENCE IMAGE CAPTURE......Page 633
OPTICAL COHERENCE TOMOGRAPHY......Page 635
OPTICAL PROJECTION TOMOGRAPHY......Page 636
Optical Setup......Page 639
Contrast and Dose......Page 640
Computed Tomography Scanning Systems......Page 641
Basic Principles of Nuclear MagneticResonance......Page 644
Magnetic Resonance Image Formation......Page 645
Image Contrast in Magnetic Resonance Microscopy......Page 648
Histology......Page 649
CONCLUSION......Page 650
REFERENCES......Page 651
Bleaching - The Only Thing That Really Matters......Page 653
GETTING A GOOD CONFOCAL IMAGE......Page 655
Pinhole Size......Page 657
Stray light......Page 658
Statistical Considerations in Confocal Microscopy......Page 659
The I mportance of Pixel Size......Page 660
Nyquist Reconstruction and Deconvolution......Page 661
Description of the Test Specimen......Page 662
Using the Test Specimen......Page 663
The Diatom: A Natural 3D Test Specimen......Page 664
Aberrations......Page 666
Curvature of Field......Page 667
Dirty Objective......Page 668
Singlet-State Saturation......Page 669
WHICH 3D METHOD IS BEST?......Page 670
MULTI-PHOTON VERSUS SINGLE-PHOTONEXCITATION......Page 672
SUMMARY......Page 673
REFERENCES......Page 674
Power Measurement......Page 676
x and y Galvanometers......Page 677
Optical Performance and Objective Lenses......Page 678
Subresolution Beads......Page 681
Axial Resolution Using a Mirror......Page 682
Lateral Chromatic Registration......Page 683
Field Illumination......Page 684
Photon (Shot) Noise......Page 686
PMT Linearity......Page 687
Spectral Resolution......Page 688
How Much Is Bleed-Through Contributing to the Image?......Page 689
Spectral Unmixing to Separate Overlapping Fluorophores......Page 690
Image Collection for Colocalization......Page 693
Spatial Deconvolution in Colocalization Studies......Page 694
REFERENCES......Page 696
COMBINING LIGHT SHEET ILLUMINATION AND ORTHOGONAL DETECTION......Page 698
SELECTIVE PLANE ILLUMINATION MICROSCOPY SETUP......Page 699
LIGHT SHEET THICKNESS AND AXIAL RESOLUTION......Page 700
PROCESSING SElECTIVE PLANE ILLUMINATION MICROSCOPY IMAGES/MULTI-VIEW RECONSTRUCTION......Page 701
SUMMARY......Page 704
REFERENCES......Page 705
INTRODUCTION......Page 706
Laser Exposure Parameters......Page 708
Evidence for Near Infrared-Induced Reactive  Oxygen Species Formation......Page 709
Photodynamic-Induced Effects......Page 710
MODIFICATIONS OF ULTRASTRUCTURE......Page 711
NANOSURGERY......Page 712
CONCLUSION......Page 713
REFERENCES......Page 714
INTRODUCTION......Page 716
Photobleaching Mechanisms......Page 717
Reducing Photobleaching......Page 719
PHOTOBLEACHING AT THE SINGLE-MOLECULE LEVEL......Page 722
Photobleaching of Single Molecules......Page 723
Other Fluorescent Proteins......Page 724
CONCLUSION......Page 725
REFERENCES......Page 726
INTRODUCTION......Page 729
Second Harmonic Generation......Page 730
Multi-Photon Absorption and Fluorescence......Page 731
LIGHT SOURCES AND DETECTORS FOR SECOND HARMONIC GENERATION AND THIRD HARMONIC GENERATION IMAGING......Page 732
NONLINEAR OPTICAL MICROSCOPY SETUP......Page 734
Optically Active Structures in Plants......Page 736
Optically Active Structures in Animal Tissues......Page 740
Polarization Dependence of Second Harmonic Generation......Page 743
SUMMARY......Page 745
REFERENCES......Page 746
Acute Slices......Page 748
Protocol for Acute Neocortical Slices......Page 749
Biolistic Protocol......Page 750
Genetic Manipulation with Dominant-Negativeand Constitutively Active Mutants......Page 751
Slice Loading and "Painting" with Acetoxymethyl Ester Indicators......Page 752
Choice of Objectives......Page 753
Beam Collimation and Pulse Broadening......Page 754
Second Harmonic Imaging......Page 755
Biocytin Protocol......Page 756
Protocol for PCNCA......Page 757
Alignment Based on the Center of Mass......Page 758
Protocol for Center of Mass Alignment......Page 759
REFERENCES......Page 760
The limiting Case......Page 762
CHOICE OF INDICATOR......Page 763
INTRODUCING THE INDICATORS INTO CEllS......Page 764
CARE OF FLUORESCENT PROBES......Page 765
INTERPRETATION OF MEASUREMENTS......Page 766
KINETICS......Page 767
CALIBRATION......Page 768
REFERENCES......Page 771
Imaging Embryos Often Requires1/4D" Imaging......Page 772
Imaging Embryos Involves Inherent Trade-Offs......Page 773
Up from the Deep: Explants Can Reduce theThickness of Specimens Dramatically......Page 774
Multi-Photon Microscopy Can Penetrate More Deeply into Specimens......Page 775
Deconvolution and OtherPost-Acquisition Processing......Page 777
Simple Solutions: Reducing Image Dimensions,Increasing Slice Spacing, and Scan Speed......Page 779
Additional Hardware Improvements Can Increase Acquisition Speed......Page 780
LOCALIZING LABEL: STRATEGIES FOR INCREASING EFFECTIVE CONTRAST IN THICK SPECIMENS......Page 781
Translational Fusions Allow Analysis of the Subcellular Distribution of Specific Proteins......Page 782
Quantum Dots......Page 783
Photo-Activatable Dyes and Photo-Activable GFP......Page 785
Bulk Vital Labeling Can Enhance Contrast......Page 786
SEEING IN SPACE: STRATEGIES FOR 4D VISUALIZATION......Page 787
Depicting Embryos in Time and Space:2D + Time Versus 3D + Time......Page 788
Fluorescence Resonance Energy Transfer......Page 790
REFERENCES......Page 792
INTRODUCTION......Page 795
THE EVER PRESENT PROBLEM OF AUTOFLUORESCENCE......Page 796
SINGLE-PHOTON CONFOCAL MICROSCOPY......Page 798
Clearing Intact Plant Material......Page 800
3D Reconstruction......Page 801
3D Segmentation......Page 802
Improved Signal-to-Noise Ratio and Dynamic Range......Page 804
Fading, Vital Imaging, and Cell Viability......Page 805
Two-Photon Excitation Imaging of Green Fluorescent Protein......Page 808
DYNAMIC IMAGING......Page 809
DECONVOLUTION......Page 810
REFERENCES......Page 811
INTRODUCTION......Page 814
FUTURE PERSPECTIVES: 3D MICROSCOPY,BIOLOGICAL COMPLEXITY, AND IN VIVO MOLECULAR IMAGING......Page 830
Microinjection......Page 829
FLUORESCENCE RESONANCE ENERGY TRANSFER THEORY......Page 816
FLUORESCENT PROTEINS AND FLUORESCENCERESONANCE ENERGY TRANSFER......Page 820
Sensitized Emission of Acceptor......Page 821
Donor Fluorescence......Page 822
Acceptor Bleach......Page 823
Fluorescence Resonance Energy Transfer-Based Sensors......Page 824
Fluorescence Resonance Energy Transfer and Fluorescence Lifetime Imaging Microscope......Page 825
Cloning of Fluorescent Chimeras......Page 827
Expression and Over-Expression......Page 828
ACKNOWLEDGEMENTS......Page 832
REFERENCES......Page 833
INTRODUCTION......Page 835
PLATFORMS USED FOR AUTOMATED CONFOCAL IMAGING......Page 836
TYPES OF ASSAYS......Page 837
3D CEll MICROARRAY ASSAYS......Page 841
DATA MANAGEMENT AND IMAGE IN FORMATICS......Page 842
REFERENCES......Page 843
From 2DCHO to 2DHela......Page 844
2D Subcellular Location Features......Page 845
3D3T3......Page 846
Image Acquisition Considerations When Using Automated Analysis......Page 847
3D Subcellular Location Features......Page 848
Texture Features......Page 849
Downsampled Images with Different Gray Scales......Page 850
Determination of Optimal Clustering......Page 851
STATISTICAL COMPARISON OF LOCATION PATTERNS......Page 852
FUTURE DIRECTIONS......Page 853
REFERENCES......Page 854
INTRODUCTION......Page 855
TESTING......Page 856
"Static" Image Performance......Page 857
Resolution: Changing the Display Size of Your Images......Page 858
Compression......Page 861
MOTION PICTURES......Page 862
Up-Sampling or Frame Rate Matching......Page 864
Motion Picture Artifacts......Page 865
MPEG Display Formats......Page 866
Performance Benchmark......Page 867
Storing Your Presentation for Remote Use......Page 868
HELPFUL URlS......Page 870
REFERENCES......Page 871
Light Microscope and Electron Microscope Have Different Requirements......Page 872
Finding the Same Cell Structure in Two Different Types of Microscope: light Microscope/ Scanning Electron Microscope......Page 876
Making LM Labels Visible in the Transmission Electron Microscope......Page 878
Using Phalloidin as a Correlative Marker......Page 880
Cryo-Immobilization Followed by Post-Embedding Confocal laser Scanning Microscopy on Thin Sections......Page 882
Cryopreparation of C. elegans......Page 883
Tiled Montage Transmission Electron Microscope Images Aid Correlation......Page 884
REFERENCES......Page 886
Recent Developments......Page 887
THE AIMS OF MODERN MICROSCOPE SYSTEM DESIGN......Page 888
IMAGE DATABASE MODEl......Page 890
Biolmage......Page 891
Query by Content......Page 892
REFERENCES......Page 893
APPENDIX......Page 894
Flowcells and Other Perfusion Chambers......Page 896
Setup of a Flow Chamber System Setup - A Practical Example......Page 898
Fluorescent Proteins......Page 899
Nucleic Acid Stains......Page 900
Fluorescence In Situ Hybridization......Page 901
General Procedure for Embedding of Flowcell-Grown Biofilms for FluorescenceIn Situ Hybridizatioin......Page 902
Antibodies......Page 903
Preparation of Labeled Primary Antibodies......Page 904
IMAGING EXTRACEllULAR POLYMERICSUBSTANCES IN BIOFILMS......Page 905
APPLICATION OF TWO-PHOTON LASER-SCANNING MICROSCOPY FOR BIOFILM ANALYSIS......Page 908
Limitations of Confocal laser ScanningMicroscopy and Two-Photon laser-Scanning Microscopy in Biofilm Analysis......Page 910
Time-lapse Confocal Imaging......Page 911
REFERENCES......Page 913
B. HISTORICAL INTEREST......Page 915
C. THEORY (MOSTLY)......Page 916
E. GENERAL......Page 917
H. DISPLAY......Page 918
J. INDEX MISMATCH......Page 919
M. POLARIZATION......Page 920
O. POINT SPREAD FUNCTION......Page 921
R. TURBIDITY......Page 922
S. VARIANTS ON THE MAIN THEME......Page 923
TESTING ALIGNMENT AND SYSTEM PERFORMANCE......Page 926
MONITORING LASER PERFORMANCE......Page 927
CONTROLLING LASER POWER......Page 929
SIMULTANEOUS IMAGING OF MULTIPLE LABELS......Page 930
REFERENCES......Page 931
INTRODUCTION......Page 932
Charge Coupling......Page 944
Quantum Efficiency......Page 946
Leakage or "Dark Charge"......Page 947
What Is a Charge Amplifier?......Page 949
Fixed Pattern Noise......Page 950
A NEW IDEA: THE GAIN REGISTER AMPLIFIER!!......Page 951
Of Course, There Is One Snag!......Page 952
1. Quantum Efficiency (QE):......Page 953
4. Array Size:19 The argument for small......Page 954
1. Dynamic Range:......Page 955
D. Intensified Charge-Coupled Devices......Page 956
REFERENCES......Page 957
Index......Page 958