Focused ion beams (FIB) were developed more than 20 years ago for fine cross-sectioning in materials science and for semiconductor chip analysis. A fine atomic beam with <10-nm diameter mills out trenches or polishes walls. These newly exposed surfaces, such as a cross section of a transistor, can then be imaged with a scanning electron microscope (SEM) that detects the scattered electrons. This is now being applied to biological samples, particularly neural circuits. A sequence of fine polishing steps of 10 nm or less, each followed by imaging of each new surface, can give a stack of 3D data with isotropic resolution. Such continuous milling/imaging also gives excellent registration and avoids many of the defects, such as tears and folds associated with cutting the thin sections required for transmission microscopy. However, this comes at the cost of slower imaging speeds and lack of long-term system stability, which in turn limit the maximum acquisition volume.
Full neuronal circuit reconstruction demands high resolution and high throughput in all three dimensions. With tremendous integration effort, here we developed customized solutions overcoming these barriers and paving the way of 3D FIB-SEM to become the mainstream imaging solution for connectomics. We have successfully sped up the image acquisition and improved system reliability with numerous stabilizing control, monitoring, and automation techniques. Our improvement made in the past 8 years highlighted in Figure 2A, the speed improvement and system integration optimization lead to over 3370 times of volume expansion since 2009.
Figure 2A: Chronological FIB-SEM volume capability improvement since 2009. 2B: Examples of volume capability in 2011, 2012, and 2013.
These improvements have enabled the extension of the continuous imaged volume to more than 100x100x100 µm3 while maintaining 8x8x8 nm voxel resolution, with the system operating for several months seamlessly. Watch a movie of Drosophila Optic Lobe 3 Month Run by 3D FIB-SEM.
. A few exemplary data sets of neural tissue and chlamydomonas illustrate the unique implications to cell biology, which can be seen from the movies below. The extended regime of fine resolution in all three dimensions combined with larger total volume will empower researchers with new techniques to accelerate discoveries in connectomics and cell biology.
Alternatively, image volume can be traded off for even finer resolution, for example a continuous volume of ~ 30x30x30 µm3 at 4x4x4 nm voxel resolution can be easily accommodated. The greater ease of automated processing of such isotropic voxel data can improve the imbalance in connectomics studies pipeline among image acquisition, analysis, and the large human proof reading effort. We note that the ultimate volume size could be further extended with the aid of