Knife-Edge Scanning Microscopy: High-Throughput Imaging and Analysis of Massive Volumes of Biological Microstructures - High-Throughput Image Reconstruction and Analysis - page 12

in the resolution along the x - y plane ( 250 nm) but in that of the z direction

∼

( 700 nm) [2]. Although the resolution and imaging depth can be improved using

more advanced schemes such as multiphoton microscopy [3], optical sectioning
techniques are limited to the tissue surface and have limited z -axis resolution. Slow
imaging speed is another issue, for both confocal and multiphoton, such that even
in enhanced two-photon microscopy, the data rate is less than 8 MB/s (512

∼

484

×

at 30 frames/s reported in [4], and about 1 frame/s in [3]).
Physical sectioning combined with microscopy is one alternative to overcome
the above issues, since z -axis resolution depends only on how thin the tissue can
be sectioned (it can go down to 30 nm using a vibrating microtome), and there

is virtually no depth limit on the tissue thickness.
The five most notable approaches in this direction are listed here:

∼

1. All-optical histology [5];
2. Knife-edge scanning microscopy (KESM) [6--10];
3. Array tomography [11];
4. Serial-block-face scanning electron microscopy (SBF-SEM) [12];
5. Automatic tape-collecting lathe ultramicrotome (ATLUM) [13].

All-Optical Histology
There are some efforts to eliminate the depth limit in optical sectioning microscopy.
For example, all-optical histology combines multiphoton microscopy with tissue
ablation to allow deeper imaging [5]. Imaging is performed using standard opti-
cal sectioning. Next, femtosecond laser pulses are used to ablate 150-mm-thick

sections on the top of the tissue block, exposing new tissue to be imaged. The
main advantage of all-optical histology is that it overcomes the tissue thickness
limit in confocal and multiphoton microscopy by ablating thick chunks of tissue.
However, since multiphoton microscope is used for imaging, it suffers from the
same vertical resolution limit and slow imaging speed.

∼

Knife-Edge Scanning Microscopy
The KESM (U.S. patent #6,744,572) has been designed at Texas A&M University
(TAMU) in recent years. The instrument, shown in Figure 2.1, is capable of scan-
ning a complete mouse brain ( 310 mm 3 ) at 300-nm sampling resolution within

100 hours when scanning in full production mode. The basic idea is to simultane-
ously cut and image thin sections of embedded biological tissue. We will discuss
KESM in more technical detail in the following section (Section 2.2).

∼

Array Tomography
Array tomography uses an ultramicrotome to manually section embedded tissue.
Adhesives on the edge of the embedded tissue block allow successive sections to
stick to each other. As sequential sections are cut, this forms an array of ultrathin
sections. The resulting tissue array is placed on a glass slide and can be repeatedly
washed, stained, and imaged using fluorescence microscopes and scanning electron
microscopes. Volume data are obtained by combining together the imaged sections.
The main advantages of array tomography is that it enables the imaging of multiple