Knife-Edge Scanning Microscopy: High-Throughput Imaging and Analysis of Massive Volumes of Biological Microstructures - High-Throughput Image Reconstruction and Analysis - page 13

Figure 2.1 The Knife-Edge Scanning Microscope (KESM). A photo of the KESM is shown with its
major components marked: (1) high-speed line-scan camera, (2) microscope objective, (3) diamond
knife assembly and light collimator, (4) specimen tank (for water immersion imaging), (5) three-axis
precision air-bearing stage, (6) white-light microscope illuminator, (7) water pump (in the back) for
the removal of sectioned tissue, (8) PC server for stage control and image acquisition, (9) granite
base, and (10) granite bridge.

molecular markers from the same slide, thus providing perfect registration across
different data modalities.

Serial Block-Face Scanning Electron Microscopy
Serial block-face scanning electron microscopy (SBF-SEM), is capable of imaging
tissue volumes of approximately 500 m in linear dimension at a resolution on the
order of 10 nm 50 nm (exact values range from 12.7 nm to 13.4 nm

for x and y ; and 55 nm to 63 nm for z ) [12]. The main advantage of SBF-SEM is
its extremely high resolution, where detailed cellular and subcellular ultrastructure
can be imaged. However, the volume is limited to cubes of several hundred mm,
and scanning time can be prohibitive if sufficient signal-to-noise is to be achieved (a
200-mm cube at the above resolution can take up to one year). Improved staining
methods to increase contrast can (theoretically) improve the speed 400-fold. SBF-
SEM development is in a mature stage, where production models are now available
from a commercial vendor (GATAN, Inc.).

× 10 nm ×

Automatic Tape-Collecting Lathe Ultramicrotome
Automatic tape-collecting lathe ultramicrotome (ATLUM) is the latest development
in serial sectioning microscopy [13]. ATLUM is capable of collecting a continuous
ribbon sized 1 mm

50 nm that get automatically pasted on a contin-
uously running tape. The tape is then cut and organized into a ultrathin section
library. The typical volume ATLUM can handle is about 10 mm 3 , which is roughly
a cube with a 2.15-mm linear dimension. One distinction in ATLUM is that imag-
ing (typically with SEM) is done only when needed, thus a digitized library is not

× 5mm ×