Редактирование: Whole brain emulation (раздел)

== Scanning ==

''(Ordered by increasing resolution)''

=== Optical Procedures ===

Optical microscopy methods are limited by the need for staining tissues to make relevant 
details stand out and the diffraction limits set by the wavelength of light (≈0.2 μm). The main 
benefit is that they go well together with various spectrometric methods (see below) for 
determining the composition of tissues. 
 
Sub‐diffraction optical microscopy is possible, if limited. Various fluorescence‐based methods 
have been developed that could be applicable if fluorophores could be attached to the brain 
tissue in a way that provided the relevant information. Structured illumination techniques 
use patterned illumination and post‐collection analysis of the interference fringes between the 
illumination and sample image together with optical nonlinearities to break the diffraction 
limit. This way, 50 nm resolving power can be achieved in a wide field, at the price of 
photodamage due to the high power levels (Gustafsson, 2005). Near‐field scanning optical 
microscopy (NSOM) uses a multinanometre optic fiber to scan the substrate using near‐field 
optics, gaining resolution (down to the multi‐nanometer scale) and freedom from using 
fluorescent markers at the expense of speed and depth of field. It can also be extended into 
near field spectroscopy.  
 
Confocal microscopy suffers from having to scan through the entire region of interest and 
quality degrades away from the focal plane. Using inverse scattering methods depth‐
independent focus can be achieved (Ralston, Marks et al., 2007).  

==== Optical Histology ====

All‐optical histology uses femtosecond laser pulses to ablate tissue samples, avoiding the 
need for mechanical removal of the surface layer (Tsai, Friedman et al., 2003). This treatment 
appears to change the tissue 2‐10 μm from the surface. However, Tsai et al. were optimistic 
about being able to scan a fluorescence labelled entire mouse brain into 2 terapixels at the 
diffraction limit of spatial resolution.  
 
Another interesting application of femtosecond laser pulses is microdissection (Sakakura, 
Kajiyama et al., 2007; Colombelli, Grill et al., 2004). The laser was able to remove 100 μm 
samples from plant and animal material, modifying a ~10 μm border. This form of optical 
dissection might be an important complement for EM methods, in that, after scanning the 
geometry of the tissue at a high resolution, relevant pieces can be removed and analyzed 
microchemically. This could enable gaining both the EM connectivity data and detailed 
biochemistry information. Platforms already exist that can both inject biomolecules into 
individual cells, perform microdissection, isolate and collect individual cells using laser 
catapulting, and set up complex optical force patterns (Stuhrmann, Jahnke et al., 2006).

=== MRI Microscopy ===

=== X-Ray Microscopy ===

X-ray microscopy also allows spectromicroscopy, which adds additional information to the scan about the chemical environment of the tissue. Different aminoacids can be detected with this method and individual proteins could be classified.

Currently, X-ray microscopy is too slow to be relevant for WBE of mammals: Scanning X-ray microscopes have exposure times measured in minutes, although they deposit five to ten times less radiation in the sample<ref>Jacobsen,1999</ref>.

=== Atomic Beam Microscopy ===

Atomic-beam microscopy consists of using a beam of neutral atoms, instead of electrons of photons, to image tissue. The de Broglie wavelength of thermal atoms is in the subnanometer range, making the resolution match that of the best [[#Electron Microscopy|electron microscopes]]. If uncharged, inert atoms like Helium are used, the beam would not destroy tissue even at such a resolution<ref>Holst and Allison, 1997</ref>. Moreover, Helium atom scattering has a large cross-section with Hydrogen, which might make it possible to detect membranes even in unstained tissue.

High resolution atomic beam microscopy has not been achieved, although low resolution has been<ref>Doak, Grisenti et al., 1999</ref>. Recent developments<ref>Oberst, Kouznetsov et al., 2005</ref><ref>Shimizu and Fujita, 2002</ref> have enabled focusing neutral atom beams to a spot size of tens of nanometers<ref>Kouznetsov, Oberst et al., 2006</ref>, which could be scanned across the tissue to construct the full image.

=== Electron Microscopy ===

==== SSET ====

By tilting the sample relative to the electron beam, the TEM can detect depth and create high-resolution 3D images<ref>Frank, 1992</ref><ref>Penczek, Marko et al., 1995</ref>. Due to the limitations on depth (1µm), it is useful mostly for scanning of 'local' tissue samples, ie the organelles and cellular structures of small volumes of tissue.<ref>Lučić, Förster et al., 2005</ref>

==== SSTEM ====

'''Serial Section Transmission Electron Microscopy'''

By making ultrathin slices, a three-dimensional model can be made. This method has been used to build a model of a neuromuscular juncture (50nm-thick sections)<ref>Tsang, 2005</ref> and to construct the connectome of the ''C. Elegans''<ref>White, Southgate et al., 1986</ref>. However, this process is labor intensive unless it can be automated.

==== SBFSEM ====

One way of reducing the problems of sectioning is to place the microtome inside 
the microscope chamber (Leighton, 1981)

for further contrast, plasma etching was used 
(Kuzirian and Leighton, 1983)

(Denk and Horstmann, 2004) demonstrated that 
backscattering contrast could be used instead in a SEM, simplifying the technique. 

 They 
produced stacks of 50‐70 nm thick sections using an automated microtome in the microscope 
chamber, with lateral jitter less than 10 nm. The resolution and field size was limited by the 
commercially available system. They estimated that tracing of axons with 20 nm resolution 
and S/N ratio of about 10 within a 200 μm cube could take about a day (while 10 nm x 10 nm 
x 50 nm voxels at S/N 100 would require a scan time on the order of a year). 


Reconstructing volumes from ultrathin sections faces many practical challenges. Current 
electron microscopes cannot handle sections wider than 1‐2 mm. Long series of sections are 
needed but the risk of errors or damage increase with the length, and the number of specimen 
holding grids becomes excessive (unless sectioning occurs inside the microscope (Kuzirian 
and Leighton, 1983)). Current state of the art for practical reconstruction from tissue blocks is 
about 0.1 mm3 , containing about 107‐108 synapses (Fiala, 2002).

==== FIBSEM ====

The semiconductor industry has long used focused ion beams to perform failure analysis tests on integrated circuits. [http://www.fei.com/ FEI] researchers have shown that this can be used to image [[#Plastination|plastinated]] neural tissue.

An ion beam ablates the top 30 to 50 nanometers of a 100x100μm tissue sample. The backscatter is imaged by the SEM, and the process is then repeated. It is similar to SBFSEM, but without the problems caused by high beam current.

==== Increasing Speed of SEM ====

From the above discussion it is clear that long imaging times constitute a major barrier to 
whole brain emulation using SEM techniques. However, there is currently a major research 
push toward massively parallel multi‐beam SEMs which has the potential to speed up SEM 
imaging by many orders‐of‐magnitude. This research push is being driven by the 
semiconductor industry as part of its effort to reduce feature sizes on computer chips below 
the level that traditional photolithography can produce.  
 
The circuitry patterns within computer chips are produced through a series of etching and 
doping steps. Each of these steps must affect only selected parts of the chip, so areas to be left 
unaffected are temporally covered by a thin layer of polymer which is patterned in exquisite 
detail to match the sub‐micron features of the desired circuitry. For current mass production 
of chips this polymer layer is patterned by shining ultraviolet light through a mask onto the 
surface of the silicon wafer which has been covered with the photopolymer in liquid form. 
This selectively cures only the desired parts of the photopolymer. To obtain smaller features 
than UV light can allow, electron beams (just as in a SEM) must instead be used to selectively 
cure the photopolymer. This process is called e‐beam lithography. Because the electron beam 
must be rastered across the wafer surface (instead of flood illuminating it as in light 
lithography) the process is currently much too slow for production level runs. 
 
Several research groups and companies are currently addressing this speed problem by 
developing multi‐beam e‐beam lithography systems (Kruit, 1998; van Bruggen, van Someren 
et al., 2005; van Someren, van Bruggen et al., 2006; Arradiance Inc). In these systems, 
hundreds to thousands of electron beams raster across a wafer’s surface simultaneously 
writing the circuitry patterns. These multi‐beam systems are essentially SEMs, and it should 
be a straightforward task to modify them to allow massively parallel scanning as well 


(Pickard, Groves et al., 2003). For backscatter imaging (as in the SBFSEM, FIBSEM, and 
ATLUM technologies) this might involve mounting a scintillator with a grid of holes (one for 
each e‐beam) very close to the surface of the tissue being imaged. In this way the interactions 
of each e‐beam with the tissue can be read off independently and simultaneously. 
 
It is difficult to predict how fast these SEMs may eventually get. A 1,000 beam SEM where 
each individual beam maintains the current 1 MHz acquisition rate for stained sections 
appears reachable within the next ten years. We can very tentatively apply this projected SEM 
speedup to ask how long imaging a human brain would take. First, assume a brain were 
sliced into 50nm sections on ATLUM‐like devices (an enormous feat which would itself take 
approximately 1,000 machines – each operating at 10x the current sectioning rate – a total of 
3.5 years to accomplish). This massive ultrathin section library would contain the equivalent 
of 1.1∙1021 voxels (at 5×5×50 nm per voxel). Assuming judicious use of directed imaging 
within this ultrathin section library only 1/10 may have to be imaged at this extremely high 
resolution (using much lower, and thus faster, imaging on white mater tracts, cell body 
interiors etc.). This leaves roughly 1.1∙1020 voxels to be imaged at high resolution. If 1,000 
SEMs each utilizing 1,000 beamlets were to tackle this imaging job in parallel their combined 
data acquisition rate would be 1∙1012 voxels per second. At this rate the entire imaging task 
could be completed in less than 4 years.

=== Nondestructive Procedures ===

''Main article: [[Non-destructive uploading]]''

=== Moravec Procedure ===

(This totally ruins the otherwise 100% science contents of the article, so please ignore it)

Scanning of the neural structures may take the form of gradual replacement, in which a robot surgeon equipped with a manipulator that subdivies into increasingly smaller branches. While the patient is awake and conscious, this manipulator begins removing cells, clamping blood vessels, exposing synapses for analysis, etc. Once the onboard computing has a good picture of what's going on, it creates a simulation of the specific volume of the brain and replaces the part of the brain with the hardware running this simulation, using magic nanofingers to plug everything together. After a while, the entire brain is composed of this hardware, maintaining the same functionality as before. '''REF MORAVEC 1988'''. While this may help mitigate fears of loss of consciousness in an all-in-one 'kill,cut,scan' approach, it is technically infeasible as the system has to be both seamlessly integrated with living, changing, moving biological tissue.

It is sometimes suggested that the successive aggregation of brain-computer interfaces to a brain will lead to a state where transfer is possible, by reaching a state where most functions are carried out in the external hardware and the brain is no longer necessary, or by reaching a point where the systems are so pervasive that it is possible to scan the whole of the brain with them, destructively or otherwise.

=== Summary ===

* '''Resolution:'''

All forms of Electron Microscopy, except SBFSEM have sufficient resolution to construct a graph of the connectivity of the brain and also inspect the properties of individual synapses (Count the synaptic vesicles). It may be possible, with future modifications, for SBFSEM to reach the necessary resolution.

* '''Reliability:'''

* '''Time:'''

{| border="1" class=wikitable
! style="background-color:#C0C0C0;" | Method || style="background-color:#C0C0C0;" | Resolution || style="background-color:#C0C0C0;" | Notes
|-
| MRI || > 5.5 µm (Non-frozen) || Does not require sectioning, may achieve better resolution on vitrified brains.
|-
| MRI microscopy || 3 µm || None
|-
| NIR microspectroscopy || 1 µm || None
|-
| All‐optical histology  || 0.7 µm || None
|-
| KESM || 0.3 µm x 0.5 µm || None
|-
| X‐ray microtomography || 0.47 µm || None
|-
| MRFM || 80 nm || None
|-
| SI || 50 nm || None
|-
| X‐ray microscopy || 30 nm || Spectromicroscopy possible?
|-
| SBFSEM || 50-70 nm x 1-20 nm || None
|-
| FIBSEM || 30-50 nm x 1-20 nm || None
|-
| ATLUM || 40 nm x 5 nm || None
|-
| SSET || 50 nm x 1 nm || None
|-
| Atomic beam microscopy || 10 nm || Not implemented yet
|-
| NSOM || 5 nm? || Requires fluorescent markers, spectroscopy possible. 
|-
| SEM || 1-20 nm || None
|-
| Array tomography || 1-20 nm SEM, 50x200x200 nm fluorescence stains || Enables multiple staining 
|-
| TEM || <1 nm || Basic 2D method, must be combined with sectioning or tomography for 3D imaging. Damage from high energy electrons at high resolutions.
|}