Plastination versus Cryonics

Break down survival as Drake equation, see how plastination differs from cryonics, try to calculate advantage
biology, transhumanism, survey
2011-07-242014-10-22 in progress certainty: likely importance: 9

“No man is an iland, intire of it selfe; every man is a peece of the Con­ti­nent, a part of the maine; if a clod bee washed away by the Sea, Europe is the lesse, as well as if a Promon­to­rie were, as well as if a Man­nor of thy friends or of thine owne were; any mans death dimin­ishes me, because I am involved in Mankinde; And there­fore never send to know for whom the bell tolls; It tolls for thee….”

, Med­i­ta­tion 17

The for is just a num­ber of sequen­tial steps with inde­pen­dent prob­a­bil­i­ties, all of which must suc­ceed but oth­er­wise none more impor­tant than the oth­ers. (One soft­ware ver­sion is the Cry­on­ics Cal­cu­la­tor.) Web devel­op­ers call it a . Spe­cific equa­tions and val­ues have been pro­posed, usu­ally yield­ing prob­a­bil­ity of suc­cess 0<x < 10%. For exam­ple, Steven Har­ris in 1989 esti­mated 0.2-15%, R. Mike Perry in the same arti­cle runs a dif­fer­ent analy­sis to arrive at 13-77%, Ralph Merkle sug­gests >85% (con­di­tional on things like good preser­va­tion, no dystopia, and nan­otech); Robin Han­son cal­cu­lated in 2009 a ~6% chance, Roko gave 23%; Mike Dar­win in 2011 (per­sonal com­mu­ni­ca­tion) put the odds at <10%; an infor­mal sur­vey of >6 peo­ple (LW dis­cus­sion) aver­aged ~17% suc­cess rate; Jeff Kauf­man in 2011 pro­vides a cal­cu­la­tor with sug­gested val­ues yield­ing 0.2%; The 2012 Less­Wrong sur­vey yields a mean esti­mate of cry­on­ics work­ing of 18% (n = 1100) and among ‘vet­er­ans’ the esti­mate is a lower 12% (n = 59) - but inter­est­ing­ly, they seem to be more likely to be signed up for cry­on­ics.


One such Drake equa­tion might break out the steps as fol­lows:

  1. Like­li­hood of get­ting pre­served
  2. * preser­va­tion con­tains needed infor­ma­tion
  3. * infor­ma­tion’s sur­vival over the cen­turies until revival pos­si­ble
  4. * exis­tence of orga­ni­za­tions or enti­ties arrang­ing revival
  5. * the actual revival

With those 5 val­ues, one mul­ti­plies to get the final prob­a­bil­ity of each step com­ing true and hence of a suc­cess­ful revival. Because each step is mul­ti­plied together with no weights, improve­ments are equal - an improve­ment in one fac­tor is as good as the same improve­ment in another fac­tor: a 10% improve­ment in orga­ni­za­tional con­ti­nu­ity is as good as a 10% improve­ment in the odds that the vit­ri­fi­ca­tion pre­serves nec­es­sary infor­ma­tion, which is as good as a 10% improve­ment in odds that revival tech will be devel­oped. This also holds for bal­anc­ing profit and loss (it’s all the same). A tech­nol­ogy that increases the orga­ni­za­tional para­me­ter by 30% and decreases the infor­ma­tion preser­va­tion para­me­ter by 10% would be a net gain, because the gain in one step out­weighs the loss in anoth­er, regard­less of what con­crete val­ues one assigns. (For exam­ple, if X was 50% and Y was 60% for a final chance of 30%, then you would be bet­ter off if you could do some­thing dif­fer­ent where X was 80% and Y was 40% because that yields a final chance of 32%. This would be eas­ier to see in dif­fer­ent nota­tion like .)


Bio­log­i­cal sam­ples have been acci­den­tally pre­served from the deep past through dehy­dra­tion, freez­ing, anox­ia, and chem­i­cal preser­va­tion; has (pos­si­bly) been recov­ered from 250 mil­lion year old salt crys­tals, 23 mil­lion year old insects are clas­si­fi­able and pre­served in high fideli­ty, and ice sam­ples have pre­served 800,000 year old and 400,000 year old DNA. Some recov­ery has been accom­plished of 400,000 year old hominid DNA, 45,000 year old human and 38,000 year old Nean­derthal DNA has been , as has 28,000 year old woolly mam­moth DNA, 80,000 year old hominid DNA, and DNA. 30,000 year old frozen plant tis­sue has been grown into healthy adult plants. One 4000 year old human genome was sequenced. A 2700 year old human brain has been recov­ered from a water­logged Eng­lish pit, heav­ily dam­aged but vis­i­bly still a brain; it is one of hun­dreds of brains recov­ered from watery envi­ron­ments. Many of the sam­ples chem­i­cally pre­served in turned out to be , but nev­er­the­less, the is very good and down to the cel­lu­lar level:

Exam­i­na­tion of the ultra­struc­ture of pre­served tis­sue in the abdomen of a fos­sil fly (Myce­tophil­i­dae Diptera) entombed in Baltic amber revealed rec­og­niz­able cell organelles. Struc­tures that cor­re­sponded to mus­cle fibers, nuclei, ribo­somes, lipid droplets, endo­plas­mic retic­u­lum, and mito­chon­dria were iden­ti­fied with the trans­mis­sion elec­tron micro­scope. Preser­va­tion was attrib­uted to inert dehy­dra­tion as well as the pres­ence of com­pounds in the orig­i­nal sap which func­tioned as nat­ural fix­a­tives. This evi­dence of cell organelles in fos­silized soft tis­sues rep­re­sent an extreme form of mum­mi­fi­ca­tion since Baltic amber is con­sid­ered to have formed about 40 mil­lion years ago.

(Even the color in dinosaur feath­ers has been pre­served in amber.) Ben Best describes amber’s preser­v­a­tive mech­a­nism in “Ancient DNA & Preser­va­tion in Amber”:

“Tree sap (resin) con­tains sug­ars as well as alco­hols & alde­hy­des (in­clud­ing ter­pe­nes), which are dehy­drat­ing & antibi­otic as well as pro­vid­ing an air-tight seal to pre­vent fur­ther entry of oxy­gen. Myrrh is a mix­ture of resin, gum and essen­tial oils from the Com­miphora plant that was used by the ancient Egyp­tians for embalm­ing (by pour­ing it into the cra­nial, chest, abdom­i­nal and pelvic cav­i­ties) and mum­mi­fi­ca­tion (by soak­ing the wrap­ping ban­dages in it)….Am­ber, as a sticky pitch from cer­tain trees, can trap insects when fresh from a tree-­wound. The sug­ars, alco­hols & ter­pene-alde­hy­des dif­fuse into the insect to dehy­drate & pre­serve. The amber sur­rounds the insect, pro­vid­ing an air-tight seal. Fur­ther oxi­da­tion & poly­mer­iza­tion of the ter­penes pro­tect the insect from fur­ther dam­age. The con­tin­ued poly­mer­iza­tion of the amber ter­penes even­tu­ally results in an insol­u­ble gem­stone-qual­ity glass that pre­serves the insect in a strong encase­ment. Although such for­tu­itous com­bi­na­tion of chem­i­cal preser­va­tion and oxy­gen-tight encase­ment should not be expected for preser­va­tion of large spec­i­mens (like humans or dinosaurs), the use of some hard­ened plas­tic or resin encase­ment could assist chem­i­cal and/or dehy­dra­tion preser­va­tion.”

and chem­i­cal brain preser­va­tion have been seri­ously pro­posed1 as an alter­na­tive to cry­on­ics, appar­ently first by Charles Olson in “A Pos­si­ble Cure for Death”. R. Mike Perry dis­cusses it favor­ably in “The Road Less Trav­eled: Alter­na­tives to Cry­on­ics”. Greg Jor­dan says no con­vinc­ing coun­ter-ar­gu­ments have been raised since Olson and strongly approves of it in his post “Biosta­sis through chemo­p­reser­va­tion”.

Is plas­ti­na­tion a net gain?


Advan­tage for plas­ti­na­tion:

  1. Improves sur­vival para­me­ter #3: It is prob­a­ble that scan­ning tech­nol­ogy will out­strip upload tech­nol­o­gy. In many fields, the abil­ity to gather data exceeds the abil­ity to process or under­stand it. Hence, it is pos­si­ble and quite likely that dur­ing the long wait for revival, it will become pos­si­ble to scan a plas­ti­nated brain in suf­fi­cient res­o­lu­tion to even­tu­ally upload it.

    Even if the scan were destruc­tive, such a scan would make it pos­si­ble to dras­ti­cally increase sur­vival odds by copy­ing the dig­i­tal data to many archives and for­mats online and offline. No such option is avail­able to a cry­on­ics brain unless it aban­dons cry­on­ics entire­ly, in which case why did it take the risk of the cry­on­ics fail­ing & it warm­ing up rather than be plas­ti­nated from the begin­ning? It’s hard to imag­ine the ben­e­fits being so equally bal­anced that the actu­al­iza­tion of bet­ter scan­ning would is enough to change the plans - given how many para­me­ters there are, a ‘pure’ strat­egy of 100% cry­on­ics or 100% plas­ti­na­tion will win. (In­deed, one might won­der how one would know that a plas­ti­na­tion+s­can­ning pro­ce­dure was good enough for upload­ing in the absence of a suc­cess­ful human upload. Human biol­ogy often diverges from even close ani­mal mod­els, and should­n’t we expect things like con­scious­ness to be even less reli­ably mod­eled by those ani­mal mod­els? The win­dow between the first suc­cess­ful upload and wide­spread upload­ing will be short com­pared to the time between now and then, even if you assume no Sin­gu­lar­ity of any kind, not even Robin Han­son’s Crack of a Future Dawn, and a slowed-­down Moore’s law.)

  2. Improves orga­ni­za­tional para­me­ter #4: Plas­ti­na­tion may be such a tech­nol­o­gy. It does not require orga­ni­za­tional con­ti­nu­ity; one rough year and your brain is a pile of rot­ting mag­gots with cry­on­ics. one rough year with plas­ti­na­tion, and your brain is a bit dusty2. A plas­ti­nated brain does­n’t even need an orga­ni­za­tion: it may be pre­served as a time cap­sule, a fam­ily heir­loom, a curios­i­ty, or per­haps just buried some­where; but a cryo­geni­cally stored brain must have a sophis­ti­cated sup­port sys­tem which will sup­ply it reg­u­larly with liq­uid nitro­gen, and that rules out pretty much every­one but a cry­on­ics orga­ni­za­tion. Mike Dar­win has been a real wake up call - the Out­side View says and are much more risky than usu­ally assumed3 - and indeed, one cry­on­ics orga­ni­za­tion has already failed with the loss of patients. Past the cen­tury mark, a few per­cent­age points is the opti­mistic esti­mate! Cry­on­ics orga­ni­za­tions have done rea­son­ably well so far, but ALCOR con­sis­tently runs at a loss and if mem­ber­ship does not fol­low an expo­nen­tial growth (as it does not), then rel­a­tively soon the ratio of dead mem­bers to live mem­bers will start get­ting worse.

  3. Improves like­li­hood of preser­va­tion #1: Much cheaper than vit­ri­fi­ca­tion; while cryo­genic stor­age is very cheap in scale the cost is still non-triv­ial for the fore­see­able future.

  4. Improves revival para­me­ter #5:

    1. despite being a rel­a­tively young field (al­beit respectable & well-­fund­ed), plas­ti­na­tion & scan­ning has made tremen­dous progress and is slowly being auto­mated, with one human brain sliced at 70 micro thick­ness and pho­tographed4, or pro­duc­ing par­tial 5 of brains. One might char­ac­ter­ize the two fields as: con­nec­tome:u­pload­::re­vived-rab­bit-kid­ney:­func­tion­ing-brain, and pon­der the fol­low­ing pos­si­bil­i­ties:

      • (=) If one regards the ‘dis­tance’ between the state of the art and the goal as equal, then plas­ti­na­tion’s faster progress is a win
      • (<) If one regards the dis­tance as smaller for plas­ti­na­tion than cry­on­ics, then plas­ti­na­tion wins both on faster progress and how much is left to do
      • (>) Only if one regards the kid­ney as being much closer to a reviv­ing a func­tion­ing brain can it be pos­si­ble for cry­on­ics revival to beat plas­ti­na­tion revival.

      Pon­der­ing the Roadmap and the Blue Brain pro­ject, I strongly doubt the kid­ney-brain is much closer together than con­nec­tome-u­pload, and sus­pect that the lat­ter is clos­er.6

    2. If plas­ti­na­tion turns out to be the ‘right’ start­ing point for an upload and cry­on­ics brains must be plas­ti­nated first, we might expect the cry­on­ic­s->­plas­ti­na­tion process to be more lossy than recent­ly-de­ceased-brain->­plas­ti­na­tion process. It could be that warm­ing the brain up enough to plas­ti­nate does dam­age, or that the cracks caused by vit­ri­fi­ca­tion are not repara­ble and degrade the plas­ti­nated result.



  1. Threat­ens infor­ma­tion preser­va­tion para­me­ter #2 in sev­eral ways:

    1. can plas­ti­na­tion pre­serve the level of detail required for recon­struc­tion? Unknown.7 The Brain Preser­va­tion Tech­nol­ogy Prize (to which I have donat­ed) is attempt­ing to spark research.

      Cry­on­ics assumes, based on anal­o­gous near-death expe­ri­ences, that many things like dynamic elec­tri­cal activ­i­ty, can be dis­re­garded for the pur­pose of per­sonal iden­tity. Plas­ti­na­tion is known to pre­serve over­all neural struc­ture in high res­o­lu­tion, as evi­denced by cur­rent plas­ti­na­tion tech­niques suf­fic­ing to cre­ate con­nec­tomes, but what does it miss? It misses the dynamic activ­i­ty, like cry­on­ics, but cry­on­ics pre­serves things plas­ti­na­tion may not. Does plas­ti­na­tion pre­serve neu­ro­trans­mit­ter lev­els? (It seems incon­sis­tent with the gen­eral idea of plas­ti­na­tion.) Neu­ro­trans­mit­ter lev­els change end­less­ly, but lev­els of neu­ro­trans­mit­ters can be the dif­fer­ence between san­ity and insan­ity in the liv­ing; on the other hand, per­sonal iden­ti­ties per­sist even through careers of mas­sive head trauma like box­ing or foot­ball, which affect neu­ro­trans­mit­ters (see ). What might we be miss­ing?

    2. are the meth­ods well-s­tud­ied and imple­mented even if they are capa­ble in prin­ci­ple of pre­serv­ing the nec­es­sary infor­ma­tion? They have been widely used in neu­ro­science, but there are no checks or ‘round trips’ show­ing that infor­ma­tion and func­tion­al­ity is pre­served with nor­mally exe­cuted tech­niques - at least cry­on­ics has frozen rab­bit kid­neys to test itself on, what does plas­ti­na­tion have?

      Coun­ter-­point: brain scan­ning and the asso­ci­ated plas­ti­na­tion tech­niques are an extremely hot field of research, which is improv­ing at an amaz­ing clip akin to DNA sequenc­ing. This ought to give us con­sid­er­able con­fi­dence in its cur­rent and future tech­niques. (This also raises an inter­est­ing point that any­one not dying in the next decade or two is wast­ing their time by inves­ti­gat­ing plas­ti­na­tion. It’s entirely pos­si­ble that for a young or mid­dle-aged per­son, the field will either have suc­ceeded in plas­ti­nat­ing an ani­mal or human brain and then upload­ing it, or will have dead­-ended and the fun­da­men­tal lim­its dis­cov­ered, by the time they truly need to choose between cry­on­ics and plas­ti­na­tion.)

    3. Are the plas­ti­na­tion processes fast enough? Nor­mal brains are pre­served over weeks to years, which is strictly worse than a hypo­thet­i­cal equally good process which requires hours. Cryo­genic cool­ing appears to be intrin­si­cally faster than chem­i­cal dif­fu­sion and action. How much dam­age does the extra time required do? (There’s some weak evi­dence that the rate of degra­da­tion is some­what con­stant and hence the dam­age lin­ear over time.)

  2. Threat­ens revival para­me­ter #5: a vit­ri­fied brain can, pre­sum­ably, be plas­ti­cized if nec­es­sary. How­ev­er, a plas­ti­cized brain is per­ma­nently plas­ti­cized. The plas­ti­cized brain has only 1 option. A vit­ri­fied brain has 2 options: nor­mal freez­ing and repair (what­ever that will be), and the plas­ti­cized route (scan­ning and upload, like­ly). A dis­junc­tion of two prob­a­bil­i­ties is at least as likely as either dis­junct. Ease of revival also affects how long stor­age must suc­ceed - if revival is fea­si­ble for both, but cry­on­ics is eas­ier, the cry­on­ics brain will have to last a shorter period than the plas­ti­nated brains. (This cuts both ways: if plas­ti­nated brains are eas­ier to revive or upload, then it will be the cry­on­ics brains which lose some prob­a­bil­ity due to the increased wait­-­time.)

    This may not be a large advan­tage for cry­on­ics. Most cry­on­ics advo­cates seem to expect upload­ing will be the ulti­mate solu­tion, inas­much as brain scan­ning is advanc­ing a lot faster than med­ical nan­otech­nol­ogy (see the Whole Brain Emu­la­tion Roadmap), but there’s still a small prob­a­bil­ity that a non-u­pload organic solu­tion will be used, and this small prob­a­bil­ity is for­feited in the plas­ti­na­tion route.


Count­ing the dis­crete items, we found 4 for plas­ti­na­tion and 2 against (yes, one point is counted twice). This is a use­less count, of course. Of those favor­able 5, 2 seem to me to be prob­a­bil­ity dif­fer­en­tials of mag­ni­tude. Of the unfa­vor­able 3, 1 seems to be of mag­ni­tude. This count favors plas­ti­na­tion as well.

I believe the above fairly sets out the signs of all the rela­tion­ships, but it is dif­fi­cult to fill in spe­cific num­bers for one­self, and even more dif­fi­cult to defend those num­bers.

The fun­da­men­tal ques­tion is, does the rapid advance of scan­ning and the robust­ness against orga­ni­za­tional fail­ure of plas­ti­na­tion out­weigh the risk that cry­on­ics uniquely pre­serves key infor­ma­tion?



Aschwin de Wolf 2013:

Plas­ti­na­tion is one-way, while with proper tech­niques, the brain can be cry­on­i­cally stored such that it can later be plas­ti­nated (in case of an extended emer­gency eg.); Mike Dar­win did some pre­lim­i­nary exper­i­ments in this area and fore­casts what such tech­niques might one day look like:

One of most dif­fi­cult prob­lems to be over­come when apply­ing this tech­nique to a whole organ the size of a human brain is, how do you keep the cir­cu­la­tory sys­tem acces­si­ble to allow for the replace­ment of the water in the tis­sue with the monomer that will sub­se­quently be poly­mer­ized into a solid plas­tic, and to remove the truly enor­mous amount of heat lib­er­ated by the exother­mic poly­mer­iza­tion reac­tion?

[Fig­ure 14: A cor­ro­sion cast of the cir­cu­la­tory sys­tem of the human brain. The exten­sive vas­cu­lar­iza­tion of the brain allows for use of the cir­cu­la­tory sys­tem as both a mass and heat exchang­er. Gas per­fu­sion of the cir­cu­la­tory sys­tem prior to cool­ing to vit­ri­fi­ca­tion tem­per­a­tures leave it acces­si­ble dur­ing cryo­genic stor­age should fix­a­tion and plas­ti­na­tion become nec­es­sary as a fall­back posi­tion to cry­op­reser­va­tion.]

This slide (Fig­ure 14) shows the cir­cu­la­tory sys­tem of a human brain. This is the real deal, not a mod­el. What you are look­ing at is some­thing called a cor­ro­sion cast. In this case, the arte­r­ial cir­cu­la­tion of a human brain was injected with a red-t­inted plas­tic mate­r­ial and the brain was then immersed in a strong base, such as a con­cen­trated solu­tion of sodium hydrox­ide. The base dis­solves or cor­rodes the tis­sue away, leav­ing behind the red plas­tic frame­work of the arte­r­ial cir­cu­la­tion. Its easy to see that the human brain is a strongly cir­cu­lated organ in fact, the brain nor­mally received 1/3rd of the rest­ing car­diac out­put about 1.5 liters of blood per minute. The FFP researchers decided that the best way to achieve both heat and mass exchange was to keep the brains cir­cu­la­tory open and acces­si­ble through­out the pro­ce­dure. In order to achieve this dur­ing solid­i­fi­ca­tion of the brain, they turned to gas per­fu­sion replac­ing the liq­uid in the cir­cu­la­tory sys­tem with gas.

One of the inves­ti­ga­tors (Mike Dar­win) real­ized that if the cir­cu­la­tory sys­tem of human cry­on­ics patients was sim­i­larly per­fused with gas dur­ing cool­ing to vit­ri­fi­ca­tion, not only would cool­ing be has­tened, thus reduc­ing the risk of freez­ing, but the cir­cu­la­tory sys­tem of the patient would remain acces­si­ble, even dur­ing stor­age at -150C. What this meant was that it would thus be the­o­ret­i­cally pos­si­ble fix and plas­ti­nate cry­on­ics patients in the event that cry­op­reser­va­tion was no longer pos­si­ble.

In this sce­nar­io, a patient would be removed from stor­age to a spe­cial appa­ra­tus, the Final Fall­back Posi­tion Sys­tem (FFPS), where his arte­r­ial cir­cu­la­tion would be con­nected to a recir­cu­lat­ing sys­tem of sol­vent chilled to -100C. This sol­vent would be pumped through the patient and would begin dis­solv­ing the vis­cous cry­opro­tec­tan­t-wa­ter solu­tion in the patients tis­sues. The sol­vent would also con­tain fix­a­tive ini­tially formalde­hyde to fix the pro­teins and, final­ly, a highly reac­tive met­al, osmium tetrox­ide, that is nec­es­sary to fix the lipids; which com­prise both the cel­lu­lar and the intra­cel­lu­lar mem­branes. Once the patient had been sol­vent sub­sti­tuted and fixed in this fash­ion, it would then be pos­si­ble to safely warm him up to room tem­per­a­ture and intro­duce the monomer required for plas­ti­na­tion. In fact, if nec­es­sary, this could be done by immer­sion, rather by per­fu­sion (though this would neces­si­tate removal of the brain from the head).




“Elec­tron imag­ing tech­nol­ogy for whole brain neural cir­cuit map­ping”, Hay­worth 2012:

As men­tioned above, the diam­e­ter of neu­ronal processes rou­tinely shrink to less than 100 nm; for exam­ple, den­dritic spine necks and fine axons can shrink down to ~40nm. With stan­dard tis­sue fix­a­tion and embed­ding pro­to­cols the mem­branes of these tubu­lar struc­tures are made elec­tron dense, hence to resolve the tubu­lar nature of these neu­ronal processes one requires res­o­lu­tions on the order of 10 nm or less. Today both the trans­mis­sion elec­tron micro­scope (TEM) and the scan­ning elec­tron micro­scope (SEM) can eas­ily achieve such res­o­lu­tion while pro­vid­ing a sig­nal to noise ratio suf­fi­cient to trace the finest neu­ropil in osmium fixed, heavy metal stained tis­sue. How­ev­er, until rel­a­tively recently only the TEM was used for trac­ing neu­ronal cir­cuits. This reliance on TEM has been a major road­block to attempts at large-s­cale automa­tion since TEM requires that sec­tions be phys­i­cally cut thin enough (<100nm) for elec­trons to pass through and that they be mounted on gos­samer thin plas­tic films through­out the imag­ing process. If scan­ning, as opposed to trans­mis­sion, elec­tron microscopy could be used it would open up many more pos­si­bil­i­ties for robust automa­tion since then at least the thin sec­tions could be col­lected and imaged on a thick sturdy sub­strate [Hay­worth, 2008], but before the wide­spread intro­duc­tion of high­-bright­ness field emis­sion elec­tron sources (as opposed to tung­sten thermionic sources) one sim­ply could not achieve SEM elec­tron probes of small enough diam­e­ter (5 nm) and high enough cur­rent to allow qual­ity imag­ing of neural tis­sue [Bogner et al., 2007; Joy, 1991].

In 2004, Denk and Horstman showed that this reliance on TEM for trac­ing neural cir­cuits could in fact be over­come. They pub­lished a sem­i­nal paper demon­strat­ing the SBFSEM (Se­r­ial Block Face Scan­ning Elec­tron Microscopy) method which could robustly auto­mate the process of obtain­ing a series of elec­tron micro­graphs from a block of neural tis­sue [Denk and Horstman, 2004]. Using a high­-bright­ness field emis­sion SEM equipped with a low-en­ergy backscat­ter elec­tron detec­tor they first showed that one could obtain high­-res­o­lu­tion images directly from the face of the tis­sue block (thus elim­i­nat­ing the need to col­lect ultra­-thin sec­tion­s). Then, fol­low­ing an orig­i­nal design from Leighton [1981], they built an ultra­mi­cro­tome into the vac­uum cham­ber of the SEM which would repeat­edly scrape (us­ing an extremely sharp dia­mond knife) 50nm lay­ers of mate­r­ial of the sur­face of the tis­sue block while the SEM was used to image each freshly revealed block face at high res­o­lu­tion. The result was a fully auto­mated method to vol­ume image a block of neural tis­sue. They and oth­ers have con­tin­ued to re ne this tech­nique so that now it can now achieve 23nm sec­tion thick­ness and have suc­cess­fully applied the tech­nique to a recon­struc­tion of the direc­tion selec­tive cir­cuitry of the mam­malian retina [Brig­gman et al., 2011].

…In 2008, Gra­ham Knott et al. intro­duced the FIBSEM (Fo­cused Ion Beam Scan­ning Elec­tron Microscopy) tech­nique for trac­ing neural cir­cuits [Knott et al., 2008]. This tech­nique is also known as Ion Abra­sion Scan­ning Elec­tron Microscopy [Hey­mann et al., 2009]. FIBSEM works sim­i­larly to SBFSEM, but instead of phys­i­cally scrap­ing a thin layer of mate­r­ial off the block face with a dia­mond knife, the mate­r­ial is instead ablated away using a focused beam of gal­lium ions. This change over­comes the lat­eral res­o­lu­tion lim­i­ta­tions of the SBFSEM since the FIB abla­tion process is rel­a­tively insen­si­tive to the mate­r­ial prop­er­ties of the embed­ding plas­tic and there­for a much larger elec­tron dose can be used dur­ing imag­ing. What’s more, ion beams can be tightly focused achiev­ing spot sizes in the 10 nm range. Align­ing the ion beam par­al­lel to the block face so that it just grazes the sur­face allows reli­able and rapid removal of extremely thin lay­ers less than 10 nm in thick­ness. What actu­ally lim­its the res­o­lu­tion of the FIBSEM (for opti­mally sized blocks) is the depth of pen­e­tra­tion of the imag­ing elec­trons into the sur­face of the block [Muller-Re­ichert et al., 2010] and by using very low volt­ages (< 2 kV), along with sen­si­tive low-en­ergy backscat­ter elec­tron detec­tors with energy fil­ter­ing, recent reports [Knott and Can­toni, 2011] have demon­strated depth res­o­lu­tions in the range of ~10nm with lat­eral imag­ing res­o­lu­tions of 5x5 nm. This FIBSEM voxel res­o­lu­tion of 5x5x10nm is more than suf­fi­cient to reli­ably resolve the finest neu­ronal processes and synaps­es, and should be suf­fi­cient to allow for extremely reli­able auto­mated recon­struc­tion algo­rithms [Luc­chi et al., 2010]. Recently Knott has pub­lished a Jour­nal of Visu­al­ized Exper­i­ments online video arti­cle cov­er­ing the entire FIBSEM pro­ce­dure from tis­sue pro­cess­ing to auto­mated FIBSEM milling and imag­ing, demon­strat­ing a final vol­ume image of a piece of cor­tex imaged with 5x5x5 nm vox­els [Knott et al., 2011]. It is stun­ning how clearly demar­cated the neu­ronal processes and synapses are in the result­ing FIBSEM vol­ume video.

…In sum­ma­ry, the FIBSEM tech­nique rep­re­sents a truly enabling tech­nol­ogy for map­ping neural cir­cuits with 100% reli­a­bil­i­ty. It can achieve voxel res­o­lu­tions of at least 5x5x10 nm, suf­fi­cient to allow straight­for­ward algo­rithms for com­puter auto­mated trac­ing of all neu­ronal processes and iden­ti­fi­ca­tion of all synap­tic con­nec­tions [Mer­ch­n-Prez et al., 2009] along with the mor­pho­log­i­cal para­me­ters which are cor­re­lated with their strength (e.g., area of con­tact and size of post synap­tic den­si­ty). Fur­ther­more, its use of a focused ion beam (as opposed to tra­di­tional phys­i­cal sec­tion­ing with a dia­mond knife) gives it the poten­tial to achieve the very high reli­a­bil­ity lev­els needed for large-s­cale automa­tion. A seri­ous lim­i­ta­tion of today’s FIBSEM sys­tems is that they can achieve such high­-res­o­lu­tion images only over tiny vol­umes - typ­i­cally on the order of a few tens of microns across. How­ev­er, this lim­i­ta­tion can be rel­a­tively eas­ily over­come with the use of a loss­less sub­di­vi­sion tech­nique like the one described above. And the avail­abil­ity of such a loss­less sub­-­di­vi­sion tech­nique opens up the pos­si­bil­ity of mas­sively par­al­lel FIBSEM imag­ing, some­thing that would, in any case, be nec­es­sary in order to image any rel­a­tively large vol­ume of tis­sue in a rea­son­able amount of time.

…To­day’s most typ­i­cally used tis­sue prepa­ra­tion pro­to­cols for elec­tron microscopy are only able to pre­pare vol­umes of less than 1 mm3. An ani­mal’s vas­cu­lar sys­tem is per­fused through the heart with a mix­ture of paraformalde­hyde and glu­taralde­hyde. The paraformalde­hyde quickly stops cel­lu­lar degra­da­tion and, at a slightly slower rate, the glu­taralde­hyde pro­vides stronger crosslinks to fix pro­teins in place. If per­formed care­ful­ly, such per­fu­sion through the vas­cu­lar sys­tem is able to quickly fix the entire ner­vous sys­tem of the ani­mal since every cell is within a few tens of microns of a cap­il­lary. How­ev­er, fol­low­ing this fix­a­tive per­fu­sion step the brain of the ani­mal is removed and a very small piece dis­sected to undergo the remain­ing tis­sue prepa­ra­tion steps - which are typ­i­cally all per­formed by sim­ple immer­sion in chem­i­cals. These steps include immer­sion in osmium tetrox­ide (to fix mem­brane lipid mol­e­cules in place), immer­sion in heavy metal stain­ing solu­tions (e.g., uranyl acetate), immer­sion in a graded series of alco­hols (to remove water from the tis­sue), and finally immer­sion in a plas­tic resin dis­solved in an organic sol­vent (to com­pletely infil­trate the tis­sue with the heat-cur­able resin). Because these steps are per­formed by sim­ple immer­sion (and thus dif­fu­sion) the process fails if attempted on blocks larger than 1 mm3. The result is destruc­tion of tis­sue ultra­struc­ture and poor stain­ing in the depths of the block.

Obvi­ously such vol­ume lim­i­ta­tions must be over­come before human mind upload­ing can be attempt­ed. Although it has never been demon­strated that a whole mam­malian brain can be pre­served at the ultra­struc­ture level for elec­tron micro­scopic imag­ing, there are many results that sug­gest that a pro­to­col could be devel­oped to do just that. Per­fu­sion fix­a­tion with osmium tetrox­ide has been demon­strated on whole brains [Palay et al., 1962], and ser­ial vas­cu­lar per­fu­sion first with glu­taralde­hy­de, fol­lowed by osmium tetrox­ide and uranyl acetate, and finally by an alco­hol dehy­dra­tion series has been demon­strated on whole organs [Ba­chofen et al., 1982; Old­mixon et al., 1985] demon­strat­ing suf­fi­cient preser­va­tion to allow ultra­struc­ture stud­ies. Plas­tic infil­tra­tion of whole mouse brains has also been demon­strated [May­erich et al., 2008], and there are recent reports of the devel­op­ment of a full ultra­struc­ture fix­a­tion, stain­ing, and embed­ding pro­to­col for the mouse brain for use in the map­ping of long dis­tance axon tra­jec­to­ries via ser­ial block face SEM [Mikula et al., 2011]. There is even a chal­lenge prize being offered for the first demon­stra­tion of such ultra­struc­ture preser­va­tion across an entire large mam­malian brain [Hay­worth, 2011]. Given these results and the fact that the bur­geon­ing field of Con­nec­tomics will require larger and larger vol­umes of high­-qual­ity pre­pared tis­sue, it is likely that a pro­to­col to pre­serve and plas­tic-em­bed an entire human brain at the ultra­struc­ture level will be per­fected rel­a­tively soon.


  1. As far as I can tell, pretty much every pro-­con for plas­ti­na­tion vs cry­on­ics applies to chem­i­cal fix­a­tion with the excep­tion of the lipids, so in the fol­low­ing I treat them as syn­ony­mous.↩︎

  2. Appar­ently it’s best to store even a plas­ti­cized brain in cryo­genic stor­age; Jor­dan Sparks says “If they don’t tran­si­tion to cry­op­reser­va­tion, dam­age will be ongo­ing for decades.”↩︎

  3. “The Armories of the Lat­ter Day Laputas, Part 5” has the sta­tis­tics. Besides 98% of star­tups dying, estab­lished com­pa­nies die fre­quent­ly: “…if we re-set the graph at 5 years, and then fol­low the remain­ing cohort of enter­prises out to the 10 year mark, the mor­tal­ity rate is still quite high with only 29% of busi­nesses sur­viv­ing.” The total mor­tal­ity is con­sid­er­able: “Thus, the chances of a busi­ness entity (ex­clud­ing reli­gious and aca­d­e­mic insti­tu­tions) sur­viv­ing for >100 years is 1.096%” One some­times see peo­ple pro­vide their own val­ues for a cry­on­ics Drake equa­tion - often the result is a com­fort­ing 1-5%. This shows they expect to be revived very soon, think cry­on­ics orga­ni­za­tions are exempt from these sta­tis­tics, or are unaware. Being non-profit helps only a lit­tle: “How­ev­er, by the 30 year mark, ~95% of NPOs have failed.”↩︎

  4. The brain of the , processed by The Brain Obser­va­tory of UC San Diego.↩︎

  5. Although hope­fully in ways more effi­cient than a few pro­fes­sors and tech­ni­cians labor­ing together or yok­ing dozens of stu­dents! In this vein, the Mouse Brain Archi­tec­ture Project is some­thing to watch - what can be done to a mouse brain may, a few gen­er­a­tions lat­er, be done to a human brain.↩︎

  6. I have a gen­eral skep­ti­cism about applied med­i­cine and biol­ogy (and reviv­i­fi­ca­tion is very applied), from a life­time of bro­ken promises and failed pre­dic­tions about the com­ing fruits of med­i­cine and biol­o­gy. I recently ran across an exam­ple in a by Don­ald Brown, then an accom­plished pro­fes­sor of biol­ogy at Johns Hop­kins:

    We don’t know yet how these genes work, but tech­niques of mod­ern genet­ics which led to the dis­cov­ery of these genes in the first place will pro­vide these insights. In the next ten years we are going to under­stand bet­ter and per­haps even cure some of the most seri­ous dis­eases that afflict mankind, such as dia­betes, arte­rioscle­ro­sis, par­a­sitic dis­eases, the com­mon cold, cys­tic fibro­sis, cer­tain kinds of arthri­tis, immune dis­eases, and infec­tious dis­eases, just to name a few. A mol­e­c­u­lar basis for at least some kinds of schiz­o­phre­nia will be found. We will learn about the bio­chem­istry of the aging process, which also has a strong genetic com­po­nent. This does­n’t guar­an­tee pro­lon­ga­tion of life, but rather an improve­ment of the qual­ity of life in old age. We need sen­si­tive assays for the effects of chem­i­cals, pol­lu­tants, and drugs as causative agents of birth defects like those devel­oped to deter­mine car­cino­genic poten­tial. There have yet to be devel­oped sim­ple, safe, and reversible con­tra­cep­tives for males.

    This reads like it was writ­ten yes­ter­day, and not in 1984 - more than 27 years ago. The pre­dic­tions about the Human Genome Project have fared lit­tle bet­ter. My rule of thumb is that in the future, our under­stand­ing and infor­ma­tion will out­strip our lab­o­ra­tory pro­to­types by even more than today, and the pro­to­types as much out­strip the gen­er­ally avail­able prod­ucts; hence, I am unsur­prised by the astound­ing progress in DNA sequenc­ing, and equally unsur­prised by the astound­ing dearth of new drugs and treat­ments. For­tu­nately for plas­ti­nated brains, enough infor­ma­tion and under­stand­ing can make up for sta­sis in other areas; as long as com­put­ers and scan­ning tech­nol­ogy con­tinue to advance, things may yet work out for them.↩︎

  7. From Per­ry’s arti­cle about chem­i­cal fix­a­tion:

    As with cry­on­ics itself, the basic answers are unknown. Some encour­age­ment is pro­vided by the high level of detail seen in pre­served brain sam­ples using, for exam­ple, formalde­hyde fix­a­tion. Ultra­struc­tural details under the high mag­ni­fi­ca­tion of elec­tron microscopy (10,000x plus) are quite clear, though this alone is not a demon­stra­tion that all the details one would like are pre­sent. How­ev­er, the same prob­lem exists with tis­sue pre­served cryo­geni­cally the answer to whether the preser­va­tion cap­tures fine enough details is unknown though there are at least some encour­ag­ing signs along with rea­sons for con­cern.

    He is not so san­guine about plas­ti­na­tion;

    A pos­si­ble draw­back of this approach, from the stand­point of pre­serv­ing the fine struc­tures that are espe­cially impor­tant from a cry­on­ics stand­point, is the rel­a­tively harsh reg­i­men needed to pro­duce the fin­ished prod­uct. Typ­i­cal­ly, the process starts with an alde­hy­de-­fixed spec­i­men in aque­ous solu­tion. The spec­i­men is placed in ace­tone, and suc­ces­sive changes of the bath remove water and fats. Finally the resin monomer is intro­duced, the remain­ing ace­tone is removed by vac­u­um, and induced catal­y­sis yields the desired poly­mer­iza­tion. Con­cerns have been raised about whether defat­ting would oblit­er­ate impor­tant brain infor­ma­tion, though there does not appear to be strong evi­dence of this. (Here it is appro­pri­ate to men­tion that lipids nev­er­the­less could con­tain impor­tant infor­ma­tion; preser­va­tion of lipids is a dif­fi­cult process that has not been cov­ered in this pre­lim­i­nary sur­vey but deserves con­sid­er­a­tion.)

    Olson men­tions lipids in pass­ing:

    A sec­ond exam­ple of redun­dancy involves the loca­tions of the neu­ronal mem­branes (i.e. the neu­ronal con­fig­u­ra­tion). The infor­ma­tion of the mem­branes’ posi­tions is con­tained not only in the phys­i­cal posi­tions of the mem­brane lipids, but also in the cel­lu­lar cytoskele­ton (which is made of pro­teins) whose pur­pose is, among other things, to hold the mem­brane in its con­fig­u­ra­tion (15). Thus, even if a sub­stan­tial pro­por­tion of lipids were extracted in the course of chem­i­cal preser­va­tion of a brain (as is the case with some preser­va­tion tech­niques), it is plau­si­ble that the infor­ma­tion of the neu­ronal con­fig­u­ra­tion would still exist in the crosslinked cytoskele­tons of the neu­rons.