Preface |
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xix | |
Acknowledgments |
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xxiii | |
The Editors |
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xxv | |
Contributors |
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xxvii | |
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Section 1 Introduction, Microscopy, Fluorophores |
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Fluorescence Lifetime-Resolved Imaging: What, Why, How---A Prologue |
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3 | (32) |
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3 | (1) |
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4 | (1) |
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Why Measure Fluorescence Lifetimes? |
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4 | (3) |
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Why Measure Lifetime-Resolved Images? |
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7 | (1) |
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Specific Features of the Different Pathways and Rates of De-Excitation |
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8 | (10) |
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Intrinsic Rate of Emission (Fluorescence) |
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8 | (2) |
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Thermal Relaxation (Internal Conversion) |
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10 | (1) |
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Molecular Relaxation of the Solvent or Molecular Matrix Environment |
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11 | (1) |
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12 | (1) |
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12 | (1) |
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Forster Resonance Energy Transfer (FRET) |
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13 | (2) |
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Intersystem Crossing and Delayed Emission |
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15 | (1) |
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Slow Luminescence without Intersystem Crossing |
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16 | (1) |
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Photolysis (Process and Interpretation of Its Measurement) |
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17 | (1) |
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The Unifying Feature of Extracting Information from Excited-State Pathways |
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18 | (1) |
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Other Parameters Related to Lifetime-Resolved Fluorescence---Dynamic and Steady-State Measurements |
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18 | (3) |
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18 | (3) |
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Steady-State Quenching (Dynamic) Measurement |
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21 | (1) |
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21 | (6) |
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21 | (1) |
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22 | (1) |
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23 | (1) |
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Time and Frequency Domains |
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23 | (1) |
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24 | (1) |
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25 | (1) |
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Equivalence of Time and Frequency Domains |
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26 | (1) |
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Performance Goals and Comparisons |
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26 | (1) |
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27 | (1) |
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Display of Lifetime-Resolved Images |
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28 | (1) |
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28 | (1) |
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29 | (6) |
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Principles of Fluorescence for Quantitative Fluorescence Microscopy |
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35 | (30) |
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35 | (1) |
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35 | (1) |
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36 | (9) |
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Molecular Excitation Rates |
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38 | (1) |
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39 | (2) |
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41 | (4) |
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Fluorescence and Molecular Relaxation Pathways |
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45 | (9) |
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45 | (1) |
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45 | (2) |
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47 | (1) |
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47 | (2) |
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Fluorescence Emission Spectra |
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49 | (1) |
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Nonradiative Relaxation Pathways |
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50 | (1) |
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51 | (1) |
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Intersystem Crossing and Phosphorescence |
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52 | (1) |
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Photoselection and Anisotropy |
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52 | (2) |
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Measuring Fluorescence in the Microscope |
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54 | (5) |
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Sensitivity of Fluorescence Measurements |
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54 | (2) |
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56 | (1) |
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Observation Volumes and Molecular Brightness |
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56 | (1) |
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57 | (1) |
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58 | (1) |
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59 | (1) |
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59 | (6) |
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Visible Fluorescent Proteins for FRET-FLIM |
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65 | (28) |
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65 | (1) |
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66 | (6) |
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Overview of the Fluorescent Proteins |
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66 | (2) |
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Spectral Variants from the Aequorea GFP |
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68 | (2) |
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Aequorea Fluorescent Proteins and Dimer Formation |
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70 | (1) |
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New Fluorescent Proteins from Corals |
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70 | (2) |
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72 | (9) |
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Visible Fluorescent Proteins for FRET Measurements |
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72 | (1) |
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Standards for Live-Cell FRET Imaging |
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73 | (2) |
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Using FRET-FLIM to Detect Protein Interactions in Living Cells |
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75 | (2) |
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Verifying Protein Interactions Using Acceptor Photobleaching FRET |
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77 | (1) |
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Alternative Fluorophore Pairs for FRET-FLIM |
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77 | (2) |
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Fluorescent Proteins Designed Specifically for FLIM Applications |
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79 | (2) |
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81 | (3) |
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General Considerations and Limitations |
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81 | (1) |
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82 | (1) |
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Factors Limiting FRET-FLIM |
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82 | (1) |
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False Positives and False Negatives |
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83 | (1) |
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Analysis in the Cell Population |
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83 | (1) |
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84 | (1) |
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84 | (1) |
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84 | (9) |
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Section 2 Instrumentation |
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Wide-Field Fluorescence Lifetime Imaging Microscopy Using a Gated Image Intensifier Camera |
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93 | (22) |
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93 | (2) |
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95 | (1) |
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95 | (11) |
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Theory behind the RLD Method |
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95 | (1) |
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96 | (1) |
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97 | (2) |
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Components Required for RLD-Based Lifetime Imaging |
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99 | (1) |
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How Data Were Acquired Using the RLD Method |
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100 | (1) |
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Calibration of the System with a Known Fluorophore (Single-Exponential Decay) |
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100 | (2) |
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Double-Exponential Decays: Biological Examples |
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102 | (4) |
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106 | (1) |
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107 | (3) |
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110 | (1) |
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110 | (1) |
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111 | (1) |
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112 | (3) |
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115 | (28) |
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Introduction to Frequency-Domain Methods |
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115 | (2) |
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115 | (1) |
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Heterodyne and Homodyne Methods for Measuring Fluorescence Lifetimes |
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116 | (1) |
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A Few Preliminary Comments |
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117 | (1) |
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Relationship Between Observables and Fluorescence Lifetimes |
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117 | (9) |
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A Primer in Complex Analysis |
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117 | (1) |
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A General Expression for the Fluorescence Signal |
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117 | (4) |
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A General Expression for the Measured Homo-/Heterodyne Signal |
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121 | (1) |
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Single- Versus Multifrequency FLIM |
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122 | (1) |
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122 | (1) |
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123 | (1) |
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Homodyne Multifrequency FLIM |
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124 | (1) |
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Heterodyne Multifrequency FLIM |
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125 | (1) |
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Extracting the Demodulation and Phase Shift Values Using a Digital Fourier Transform |
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126 | (1) |
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127 | (6) |
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127 | (1) |
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127 | (1) |
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Illumination Sources and Electro-optics for Modulated Excitation Light |
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127 | (2) |
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Gain-Modulated Image Intensifiers |
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129 | (1) |
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Optical Setup and Electronics |
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130 | (1) |
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Corrections for Random Noise and Systematic Errors |
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131 | (1) |
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Correcting for Laser Fluctuations and Dark Current |
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131 | (1) |
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Gain-Modulated Image Intensifier Performance |
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131 | (2) |
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133 | (3) |
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133 | (1) |
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134 | (2) |
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136 | (3) |
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136 | (1) |
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Dual-Layer Fractional Concentration Images |
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137 | (2) |
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139 | (1) |
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139 | (4) |
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Laser Scanning Confocal FLIM Microscopy |
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143 | (22) |
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143 | (3) |
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145 | (1) |
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Lifetime Detection Methods In Scanning Microscopy |
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146 | (5) |
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Time-Correlated Single-Photon Counting (TCSPC) |
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146 | (3) |
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149 | (2) |
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Detectors and Electronics |
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151 | (3) |
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151 | (2) |
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153 | (1) |
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Count Rate and Acquisition Time |
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154 | (4) |
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Detector and Electronics Limitations |
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155 | (1) |
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Efficiency of Time-Domain Lifetime Detection Methods |
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156 | (2) |
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158 | (2) |
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160 | (1) |
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160 | (1) |
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161 | (4) |
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Multiphoton Fluorescence Lifetime Imaging at the Dawn of Clinical Application |
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165 | (24) |
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165 | (2) |
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Principle of Multiphoton Imaging |
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167 | (2) |
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Clinical Multiphoton Tomography |
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169 | (1) |
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Multiphoton Flim Technique |
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170 | (4) |
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174 | (9) |
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174 | (3) |
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Two-Photon FLIM Imaging of Stem Cells |
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177 | (6) |
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183 | (1) |
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184 | (1) |
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184 | (5) |
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Flim Microscopy with a Streak Camera: Monitoring Metabolic Processes in Living Cells and Tissues |
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189 | (22) |
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189 | (1) |
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Streakflim: System Integration |
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190 | (5) |
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Step-by-Step Demonstration of StreakFLIM System Application |
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193 | (1) |
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193 | (2) |
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195 | (1) |
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195 | (5) |
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200 | (7) |
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Imaging Cancer Cells in Three-Dimensional Architecture |
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200 | (3) |
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Kinetic Imaging of pH Transients during Glucose Metabolism |
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203 | (2) |
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FLIM-Based Enzyme Activity Assays In Vivo |
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205 | (2) |
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Summary and Future Perspective |
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207 | (1) |
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208 | (3) |
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Spectrally Resolved Fluorescence Lifetime Imaging Microscopy: SLIM/mwFLIM |
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211 | (34) |
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211 | (3) |
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214 | (10) |
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The Spectral Axis of the Fluorescence Decay Surface |
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214 | (2) |
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The Time Axis of the Fluorescence Decay Surface |
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216 | (1) |
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217 | (1) |
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A Special Case: Global Analysis of FRET Measurements |
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218 | (6) |
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224 | (15) |
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224 | (2) |
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Operation Principle of the Streak Camera |
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226 | (1) |
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Operation Principle of the mwFLIM/SLIM Setup |
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226 | (2) |
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Benefits of the Techniques |
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228 | (1) |
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229 | (1) |
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Calibration of the Spectral Axis |
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229 | (1) |
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Calibration of the Time Axis |
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230 | (1) |
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Calibration of the Intensity Axis |
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230 | (1) |
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231 | (1) |
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The Instrument Response Function |
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231 | (1) |
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Deconvolution and Data Fitting |
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232 | (2) |
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234 | (1) |
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Functional Staining of Cell Structures |
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234 | (1) |
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Photodynamic Therapy (PDT) |
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234 | (3) |
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Forster Resonance Energy Transfer |
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237 | (2) |
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239 | (2) |
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241 | (1) |
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241 | (4) |
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Time-Resolved Fluorescence Anisotropy |
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245 | (46) |
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245 | (1) |
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246 | (1) |
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247 | (1) |
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248 | (7) |
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Photoselection of a Randomly Oriented Population of Fluorophores |
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251 | (4) |
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How Do We Detect Polarized Emissions? |
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255 | (6) |
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How Do We Quantify Polarized Emissions? |
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261 | (3) |
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The Anisotropy of Randomly Oriented Populations of Fluorophores |
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264 | (1) |
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Depolarization Factors and Soleillet's Rule |
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265 | (17) |
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Instrumental Depolarization |
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268 | (2) |
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Depolarization Caused by Absorption and Emission Dipole Orientation |
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270 | (1) |
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Timescale of Depolarization |
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270 | (1) |
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Depolarization Caused by Molecular Rotation |
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271 | (4) |
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Depolarization Caused by FRET |
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275 | (7) |
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Fluorescence Anisotropy Applications |
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282 | (3) |
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283 | (1) |
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Putting Limits on the Value of κ2 |
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284 | (1) |
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Differentiating between Directly Excited Acceptors and FRET |
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285 | (1) |
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285 | (1) |
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286 | (1) |
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286 | (5) |
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General Concerns of FLIM Data Representation and Analysis: Frequency-Domain Model-Free Analysis |
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291 | (50) |
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291 | (2) |
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Time Domain Assuming Very Short Excitation Pulses |
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293 | (3) |
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296 | (6) |
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Calculating F(t)meas Directly from the Convolution Integral |
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296 | (3) |
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Calculating F(t)meas from the Finite Fourier Transform of the Repetitive δ-Pulse Result |
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299 | (2) |
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Calculating the Frequency Response from the Convolution Theorem of Fourier Transforms |
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301 | (1) |
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Analysis of the Measured Data, F(T)meas, At Every Pixel |
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302 | (1) |
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Remarks About Signal-To-Noise Characteristics of Time-And Frequency-Domain Signals: Comparison To Single-Channel Experiments |
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303 | (2) |
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Flim Experiments: Challenges, Advantages, and Solutions |
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305 | (1) |
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How Flim Circumvents The Data Deluge |
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306 | (14) |
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Polar Plots of Frequency-Domain Data (Model-Free Analysis) |
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307 | (1) |
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Polar Plot Description of Fluorescence Directly Excited by Light Pulses |
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307 | (4) |
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Polar Plot of Fluorescence from a Product Species of an Excited-State Reaction |
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311 | (3) |
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Combining Spectra and Polar Plots |
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314 | (1) |
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Two Different Noninteracting Fluorophores |
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315 | (3) |
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FRET: Observing Donor and Acceptor Fluorescence Simultaneously |
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318 | (2) |
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320 | (10) |
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Why Use This Image Analysis? |
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320 | (1) |
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Wavelet Transforms for Discriminating Fluorescence Lifetimes Based on Spatial Morphology |
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321 | (1) |
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What Is a Wavelet Transform? |
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321 | (3) |
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Applications of Wavelets to Homodyne FLIM |
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324 | (1) |
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Denoising Homodyne FLIM Data |
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325 | (1) |
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Sources of Noise for Homodyne FLIM |
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325 | (1) |
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Removal of Signal-Dependent Noise: TI-Haar Denoising |
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325 | (1) |
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TI-Haar Denoising Improves Homodyne FLIM Accuracy |
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326 | (2) |
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The Future of Wavelet and Denoising Image Analysis for Homodyne FLIM |
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328 | (2) |
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Noniterative Data Regression (Chebyshev and Laguerre Polynomials) |
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330 | (5) |
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Noniterative Data Regression |
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330 | (1) |
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Convexity in Modeling and Multiple Solutions |
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330 | (2) |
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Formulation of Modeling as a Dynamic System |
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332 | (1) |
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Solution to Convexity in a Hilbert Space |
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332 | (2) |
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334 | (1) |
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335 | (6) |
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Nonlinear Curve-Fitting Methods for Time-Resolved Data Analysis |
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341 | (30) |
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341 | (1) |
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342 | (1) |
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343 | (8) |
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Basic Terminology and Assumptions |
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343 | (2) |
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345 | (1) |
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346 | (2) |
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348 | (1) |
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Least-Squares Parameter Estimation |
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349 | (1) |
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Diagnostics for Quality of Curve-Fitting Results |
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350 | (1) |
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Uncertainty of Curve-Fitting Procedures |
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350 | (1) |
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351 | (16) |
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How to Analyze Experimental Data |
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351 | (1) |
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352 | (1) |
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352 | (1) |
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Color Effect in the Detector |
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353 | (2) |
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355 | (1) |
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356 | (1) |
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357 | (1) |
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Analysis of Multiexponential Decays |
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358 | (1) |
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Effect of the Signal Level |
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359 | (2) |
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Two and Three Components of Intensity Decays |
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361 | (3) |
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Fluorescence Lifetime Distribution: Biological Examples |
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364 | (3) |
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367 | (1) |
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368 | (3) |
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Global Analysis of Frequency Domain FLIM Data |
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371 | (14) |
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371 | (1) |
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Fourier Description of Flim Data |
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372 | (2) |
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Global Analysis of Flim Data |
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374 | (1) |
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Application To Fret-Flim Data |
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375 | (1) |
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375 | (5) |
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380 | (1) |
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380 | (1) |
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380 | (1) |
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Fluorescence Lifetime Imaging Microscopy |
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381 | (1) |
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381 | (4) |
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FLIM Applications in the Biomedical Sciences |
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385 | (16) |
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385 | (1) |
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A Brief Historical Journey Through The Development of Lifetime-Resolved Imaging |
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386 | (2) |
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Autofluorescence Lifetime Imaging of Cells |
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388 | (2) |
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Pap Smear Detection Using Time-Gated Lifetime Imaging Microscopy |
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390 | (4) |
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FLIM in Alzheimer's Disease |
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394 | (1) |
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Optical Projection of Flim Images of Mouse Embryo |
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394 | (1) |
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Full-Field Flim With Quadrant Detector |
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395 | (1) |
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396 | (2) |
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398 | (3) |
Index |
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401 | |