Open­ing Win­dows into the Cell

by Kanika Khanna

In the 1940's, elec­tron mi­croscopy (EM) sin­gle hand­edly pro­pelled cell bi­ol­ogy into a new era. The dis­cov­ery of cell or­ganelles and their ul­tra­struc­ture can be at­trib­uted to its ap­pli­ca­tion. The op­por­tu­nity to cross the bar­rier set by the lim­i­ta­tions in the res­o­lu­tion of op­ti­cal mi­croscopy was a cru­cial de­vel­op­ment for many re­searchers. It be­came pos­si­ble not just to see the shapes and forms of cells, but to look in­side them. De­vel­op­ment of cy­to­chem­i­cal tech­niques and im­muno-EM dur­ing the same era al­lowed lo­cal­iza­tion of mol­e­cules within cel­lu­lar con­text at an un­prece­dented res­o­lu­tion. In the words of P. Ball, "...we know about mol­e­cules; we know about cells and or­ganelles; but the stuff in be­tween is messy and mys­te­ri­ous... yet that is the level of mag­ni­fi­ca­tion at which much of the ac­tion takes place: the scale of per­haps a few to sev­eral hun­dred nanome­tres."

Prin­ci­ple of Cryo-Elec­tron To­mog­ra­phy (Steven and Bel­nap, Cur­rent Pro­to­cols in Pro­tein Sci­ence, 2005). A. Cryo-holder with the spec­i­men is ro­tated to suc­ces­sive tilt po­si­tions. B. Pro­jec­tion of the spec­i­men is recorded at each tilt po­si­tion with di­rect detector/CCD cam­eras. C. The 3D-struc­ture is re­con­structed with the help of back-pro­jec­tion of im­ages ob­tained in B.

The field of EM has seen sev­eral ma­jor tech­ni­cal im­prove­ments over the past few decades, par­tic­u­larly in the de­sign of the mi­cro­scope, sam­ple prepa­ra­tion tech­niques, cam­eras for imag­ing, and com­pu­ta­tional tools for im­age pro­cess­ing, all pro­vid­ing in­for­ma­tion at near atomic res­o­lu­tions. In­deed, it wouldn't be wrong to say that this is an era of "Res­o­lu­tion Rev­o­lu­tion." Cryo-EM en­ables bi­o­log­i­cal spec­i­mens (in­clud­ing but not lim­ited to in­tact tis­sues sec­tions, bac­te­ria, viruses and pro­tein mol­e­cules) to be im­aged in their close-to-liv­ing frozen-hy­drated state. This al­lows us to take 'snap­shots' of macro­mol­e­cules un­per­turbed in their na­tive en­vi­ron­ment, that is, the cell. Such an ap­proach would form the ba­sis of 'vi­sual pro­teomics' by en­abling map­ping of mol­e­c­u­lar land­scapes in­side cells com­pre­hen­sively. For all these good rea­sons, cryo-EM was named as the 'Method of the Year 2015' by the jour­nal Na­ture Meth­ods.

A rel­a­tively new field is Cryo-Elec­tron To­mog­ra­phy (Cryo-ET) (Milne, et al. 2013) which has the po­ten­tial to ob­tain 3D im­ages of large ob­jects like cells and or­ganelles and even tis­sues with a res­o­lu­tion of 4–5 nm. These im­ages pro­vide pre­cise in­for­ma­tion about the spa­tial or­ga­ni­za­tion of macro­mol­e­cules in their cel­lu­lar con­text. In this tech­nique, the spec­i­men is tilted in­side the TEM and the sam­ple is im­aged si­mul­ta­ne­ously tak­ing 2D im­ages that can be com­bined to pro­duce a 3D re­con­struc­tion. How­ever, one of the fac­tors that limit the res­o­lu­tion of this tech­nique is the thick­ness of bi­o­log­i­cal spec­i­mens. So, how to make the sam­ples so thin that we can ob­tain beau­ti­ful high res­o­lu­tion im­ages of cells where one can po­ten­tially iden­tify macro­mol­e­cules like ri­bo­somes, mem­brane pro­teins and pro­tein fil­a­ments?

Schematic of FIB Milling. A. Mam­malian cell plunge-frozen on an EM grid. B. Mi­cro-ma­chin­ing of cells at cryo-tem­per­a­tures us­ing FIB. Pat­terns (red boxes) are se­lected de­pend­ing on the milling geom­e­try. C. Thinned sam­ple or the lamella that is used for Cryo-ET by tilt­ing the an­gle around the axis shown re­sult­ing in 3D re­con­struc­tions of cel­lu­lar land­scape (Villa, Schaf­fer, Plitzko, & Baumeis­ter, 2013)

Some of the ways in which sci­en­tists have tried to cir­cum­vent this is­sue in­clude us­ing skinny bac­te­r­ial cells or imag­ing only the thin edges of thick mam­malian cells. One can also use a mi­cro­tome to make thin sec­tions of ma­te­ri­als us­ing steel/glass/diamond blades, de­pend­ing on the sam­ple. This, how­ever, is known to in­tro­duce ar­ti­facts such as com­pres­sions in the sam­ple be­ing im­aged. A re­cently de­vel­oped ap­proach is the use of Fo­cus-Ion-Beam (FIB) milling ma­chine, where frozen-hy­drated spec­i­men can be thinned by sput­ter­ing them with gal­lium ions. This in­stru­ment is now used on a rou­tine ba­sis by ma­te­r­ial sci­en­tists. We have been able to hi­jack it to use it at cryo-tem­per­a­tures to ab­late bi­o­log­i­cal ma­te­r­ial (Villa, et al. 2013). The in­stru­ment is a com­bi­na­tion of a FIB and SEM (Scan­ning Elec­tron Mi­cro­scope) sys­tem in the same cham­ber, the idea be­ing to nav­i­gate and mill the sam­ples us­ing the FIB un­der the vi­sual con­trol of SEM. This re­duces the ra­di­a­tion dam­age to the sam­ple caused by FIB. This leaves 'win­dows' or what we call 'lamel­las' that are thin enough to be elec­tron trans­par­ent in a TEM (Trans­mis­sion Elec­tron Mi­cro­scope).

Ad­vanced tech­nol­ogy like Cryo-elec­tron to­mog­ra­phy com­bined with ge­netic and mol­e­c­u­lar bi­o­log­i­cal tools has pro­vided deep in­sights into sev­eral bi­o­log­i­cal phe­nom­ena. For in­stance, it was be­lieved for a long time that bac­te­ria do not have a cy­toskele­ton. Yet, fil­a­men­tous struc­tures were later vi­su­al­ized in rapidly frozen bac­te­ria and their com­po­si­tion iden­ti­fied via ge­netic stud­ies (Li, et al. 2007). This tech­nol­ogy makes is pos­si­ble to de­ter­mine the struc­tures of un­known mol­e­cules in to­mo­grams. For in­stance, the lo­ca­tion and ori­en­ta­tion of ri­bo­somes were suc­cess­fully de­ter­mined and struc­tural dif­fer­ences be­tween ri­bo­somes in dif­fer­ent func­tional states were also ex­plored (Or­tiz, et al. 2006). Cryo-ET of fla­gella has led to pub­li­ca­tions of de­tailed in situ struc­tures of fla­gel­lar com­po­nents like mi­cro­tubules, dynein, and oth­ers.

In ad­di­tion, it is also pos­si­ble to lo­cal­ize the fea­tures of in­ter­est us­ing Cryo-cor­rel­a­tive flu­o­res­cence mi­croscopy (Cryo-FM) by imag­ing flu­o­res­cently la­beled mol­e­cules on the EM grid seen un­der the op­ti­cal mi­cro­scope (Plitzko, Rig­ort and Leis 2009). Hence, tak­ing ad­van­tage of com­ple­men­tary imag­ing modal­i­ties, we can first iden­tify the fea­tures of in­ter­est us­ing flu­o­res­cence mi­croscopy and cap­ture high-res­o­lu­tion de­tails of cel­lu­lar events us­ing Cryo-ET. In short, one can de­ci­pher the spa­tial and tem­po­ral or­ga­ni­za­tion of pro­teomic net­works. An­other wide­spread tech­nique is the use of clon­able metal la­bels such as met­al­loth­ionein and fer­ritin that al­low di­rect lo­cal­iza­tion of pro­teins within the cell with the EM.

A. Sporu­lat­ing B. sub­tilis cells in a trans­par­ent block of vit­re­ous ice. B. The same block milled to re­veal a thin lamella. CET: cryo-elec­tron to­mo­gram. C. CET of an un­milled cell pro­vides lim­ited res­o­lu­tion, even in ΔponA cells (Tocheva, et al., 2014). D. CET of a milled wild­type cell re­veals many struc­tural de­tails and al­lows quan­ti­ta­tive analy­sis. E. Pre­lim­i­nary 3‑D com­pu­ta­tional model of sporu­lat­ing cell shown in D.

Look­ing at all the ad­vances made in the past few decades and the ones that are yet to come, the fu­ture of Cryo-ET holds sev­eral promises to un­ravel sev­eral un­solved mys­ter­ies in bi­ol­ogy. We have also got­ten to the point where it is pos­si­ble to im­age thick Drosophila brain us­ing a com­bi­na­tion of cryo-X-ray mi­croscopy and Cryo-EM, al­beit at low res­o­lu­tions (Lefor­estier, et al. 2014). The day is not far, my friends, when we may be able to im­age hu­man or­gans and look in­side them! The field of sci­ence has ad­vanced to an ex­tent where sci­en­tists have de­vel­oped origami mi­cro­scopes which cost just 50 cents and are called 'Fold­scopes' (Cy­bul­ski, Clements and Prakash 2014) and fu­ture ef­forts may be to de­velop some­thing sim­i­lar here, which would make this tech­nol­ogy read­ily avail­able to a larger sci­en­tific com­mu­nity, as cost is one of the ma­jor fac­tor lim­it­ing its use. This is a chal­leng­ing task though!

The next fron­tier is cell dy­nam­ics. Right now, we can look at many copies of mol­e­cules in dif­fer­ent con­for­ma­tions and in­fer their dy­namic be­hav­ior, but can we stop peer­ing at frozen mol­e­cules? We would like to see real-time atomic struc­tures go­ing about their day!  There are al­ready in situ EM hold­ers where al­low to see mol­e­cules in liq­uid phase in­ter­act­ing in real time, us­ing a de­vice known as the en­vi­ron­men­tal scan­ning elec­tron mi­cro­scope. The chal­lenge is that in or­der to fol­low mol­e­cules over time we have to con­tin­u­ously ex­pose them to the elec­tron beam, which is the equiv­a­lent to many small atomic bombs. This might be rea­son­able for some hard ma­te­ri­als, but for bi­o­log­i­cal mol­e­cules the ra­di­a­tion dam­age is a show-stop­per (un­less we would be in­ter­ested in study­ing the be­hav­ior of macro­mol­e­cules sub­ject to ex­treme ra­di­a­tion). With cam­eras get­ting faster and more sen­si­tive, one can imag­ine the use of stro­bo­scopic elec­tron mi­croscopy, in which we could cap­ture time-lapse movies of the mol­e­cules as they move in­side cells.  Keep your eyes open – the fu­ture is lu­mi­nous!

 

Ref­er­ences

Cy­bul­ski, James S., James Clements, and Manu Prakash. 2014. "Fold­scope: Origami-Based Pa­per Mi­cro­scope." PloS one 9 (6).

Lefor­estier, Amélie, Pierre Levitz, Thomas Preat , Pe­ter Guttmann , Lau­rent J. Mi­chot , and Paul Tchénio . 2014. "Imag­ing Drosophila brain by com­bin­ing cryo-soft X‑ray mi­croscopy of thick vit­re­ous sec­tions and cryo-elec­tron mi­croscopy of ul­tra­thin vit­re­ous sec­tions." Jour­nal of Struc­tural Bi­ol­ogy 188 (2): 177–182.

Li, Zhuo, Michael J Trim­ble, Yves V Brun, and Grant J Jensen. 2007. "The struc­ture of FtsZ fil­a­ments in vivo sug­gests a force-generating role in cell di­vi­sion." EMBO Jour­nal 26 (22): 4694–4708.

Ma­hamid, Ju­lia, Ste­fan Pf­ef­fer, Miroslava Schaf­fer, Eliz­a­beth Villa , Ra­dostin Danev, Luis Kahn Cuel­lar, Friedrich Förster, An­thony A. Hy­man, Jür­gen M. Plitzko, and Wolf­gang Baumeis­ter. 2016. "Vi­su­al­iz­ing the mol­e­c­u­lar so­ci­ol­ogy at the HeLa cell nu­clear pe­riph­ery." Sci­ence 351 (6276): 969–972.

Milne, Jacque­line L. S., Mario J. Borg­nia, Al­berto Barte­saghi, Erin E. H. Tran, Les­ley A. Earl, David M. Schauder, Jef­frey Lengyel, Ja­son Pier­son, Ar­dan Pat­ward­han, and Sri­ram Sub­ra­ma­niam. 2013. "Cryo-elec­tron mi­croscopy: A primer for the non-mi­cro­scopist." FEBS Jour­nal 280 (1): 28–45.

Or­tiz, Julio O., Friedrich Forster, Ju­lia Kurner, Alexan­dros A. Linaroudis, and Wolf­gang Baumeis­ter. 2006. "Map­ping 70S ri­bo­somes in in­tact cells by cry­o­elec­tron to­mog­ra­phy and pat­tern recog­ni­tion." Jour­nal of Struc­tural Bi­ol­ogy 156 (2): 334–341.

Plitzko, Jür­gen M, Alexan­der Rig­ort, and An­drew Leis. 2009. "Cor­rel­a­tive cryo-light mi­croscopy and cryo-elec­tron to­mog­ra­phy: from cel­lu­lar ter­ri­to­ries to mol­e­c­u­lar land­scapes." Cur­rent Opin­ion in Biotech­nol­ogy 20 (1): 83–89.

Tocheva, Elitza I., Javier López-Gar­rido, H. Ve­loc­ity Hughes, Jen­nifer Freud­lund, Erkin Kuru, Michael S. Van­Nieuwen­hze, Yves V. Brun, Kit Pogliano , and Grant Jensen. 2014. "Pep­ti­do­gly­can trans­for­ma­tions dur­ing Bacil­lus sub­tilis sporu­la­tion." Mol­e­c­u­lar Mi­cro­bi­ol­ogy 88 (4): 673–686.

Villa, Eliz­a­beth, Miroslava Schaf­fer, Ju­r­gen M Plitzko, and Wolf­gang Baumeis­ter. 2013. "Open­ing win­dows into the cell: fo­cused-ion-beam milling for cryo-elec­tron to­mog­ra­phy." Cur­rent Opin­ion in Struc­tural Bi­ol­ogy 23 (5): 771–777.

 

Kanika Khanna is a Grad­u­ate stu­dent in the Di­vi­sion of Bi­o­log­i­cal Sci­ences, Uni­ver­sity of Cal­i­for­nia, San Diego.