5.2.1 Sample Processing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
5.2.2 Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
5.3 Serial Block-Face Scanning Electron Microscopy
(SBF-SEM or SBEM). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
5.3.1 Sample Processing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
5.3.2 Sample Setup and Mounting . . . . . . . . . . . . . . . . . . . . . 129
5.3.3 SBEM Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
5.4 Focused Ion Beam Scanning Electron Microscopy . . . . . . . . . . . 134
5.4.1 Sample Processing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
5.4.2 Sample Setup and Mounting . . . . . . . . . . . . . . . . . . . . . 137
5.4.3 FIB-SEM Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
5.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
6 Conjugate Immunofluorescence—SEM Array Tomography
for Studying Mammalian Synapses and Axons . . . . . . . . . . . . . . . . . 149
Kristina D. Micheva and Kristen D. Phend
6.1 Introduction/Background. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
6.2 Approaches to Large-Scale Molecular and Ultrastructural
Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
6.3 Array Tomography: A Different Kind of Tomography . . . . . . . . 153
6.4 Array Tomography Sample Preparation: Workflow and
General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
6.5 Array Tomography: Computer Assisted Imaging of Serial
Sections on Rigid Substrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
6.6 Application of Conjugate IF-SEM Array Tomography
for the Study of Mammalian Synapses and Axons . . . . . . . . . . . 161
6.7 Variations of the Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
6.8 Detailed Protocol for Conjugate IF-EM AT Using Freeze
Substitution and Embedding in Lowicryl HM-20 . . . . . . . . . . . . 166
6.9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
7 Alignment of Tilt Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
A. Verguet, C. Messaoudi, C. O. S. Sorzano and S. Marco
7.1 Why Do We Need to Align Tilt Series? . . . . . . . . . . . . . . . . . . . 183
7.2 Standard Alignment Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
7.2.1 Precentering the Tilt Series . . . . . . . . . . . . . . . . . . . . . . 186
7.2.2 Determining the Orientation of the Tilt Axis . . . . . . . . . 190
7.3 Advanced Alignment Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 194
7.3.1 Multiscale Registration . . . . . . . . . . . . . . . . . . . . . . . . . 194
7.3.2 Invariant Feature Recognition . . . . . . . . . . . . . . . . . . . . 197
7.4 Extension to Data Other than Tilt Series . . . . . . . . . . . . . . . . . . 203
7.4.1 Data Alignment in Multiple-Axis Tomography . . . . . . . 203
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Contents