Cryo-fixation and associated developments in transmission electron microscopy: a cool future for nematology

in Nematology
Restricted Access
Get Access to Full Text
Rent on DeepDyve

Have an Access Token?



Enter your access token to activate and access content online.

Please login and go to your personal user account to enter your access token.



Help

Have Institutional Access?



Access content through your institution. Any other coaching guidance?



Connect

At present, the importance of sample preparation equipment for electron microscopy represents the driving force behind major breakthroughs in microscopy and cell biology. In this paper we present an introduction to the most commonly used cryo-fixation techniques, with special attention paid towards high-pressure freezing followed by freeze substitution. Techniques associated with cryo-fixation, such as immunolocalisation, cryo-sectioning, and correlative light and electron microscopy, are also highlighted. For studies that do not require high resolution, high quality results, or the immediate arrest of certain processes, conventional methods will provide answers to many questions. For some applications, such as immunocytochemistry, three-dimensional reconstruction of serial sections or electron tomography, improved preservation of the ultrastructure is required. This review of nematode cryo-fixation highlights that cryo-fixation not only results in a superior preservation of fine structural details, but also underlines the fact that some observations based on results solely obtained through conventional fixation approaches were either incorrect, or otherwise had severe limitations. Although the use of cryo-fixation has hitherto been largely restricted to model organisms, the advantages of cryo-fixation are sufficiently self-evident that we must conclude that the cryo-fixation method is highly likely to become the standard for nematode fixation in the near future.

Cryo-fixation and associated developments in transmission electron microscopy: a cool future for nematology

in Nematology

Sections

References

AdlerC.E.FetterR.D.BargmannC.I. (2006). UNC-6/Netrin induces neuronal asymmetry and defines the site of axon formation. Nature Neuroscience 9511-518.

BosherJ.M.HahnB.-S.LegouisR.SookhareeaS.WeimerR.M.GansmullerA.ChisholmA.D.RoseA.M.BessereauJ.-L.LabouesseM. (2003). The Caenorhabditis elegans vab-10 spectraplakin isoforms protect the epidermis against internal and external forces. Journal of Cell Biology 161757-768.

BumbargerD.J.CrumJ.EllismanM.H.BaldwinJ.G. (2006). Three-dimensional reconstruction of the nose epidermal cells in the microbial feeding nematode, Acrobeles complexus (Nematoda: Rhabditida). Journal of Morphology 2671257-1272.

BumbargerD.J.WijeratneS.CarterC.CrumJ.EllismanM.H.BaldwinJ.G. (2009). Three-dimensional reconstruction of the amphid sensilla in the microbial feeding nematode, Acrobeles complexus (Nematoda: Rhabditida). The Journal of Comparative Neurology 512271-281.

CavalierA.SpehnerD.HumbelB. (2009). Handbook of cryo-preparation methods for electron microscopy. Boca Raton, Florida, USACRC Press Inc.

ClaeysM.VanheckeD.CouvreurM.TytgatT.CoomansA.BorgonieG. (2004). High-pressure freezing and freeze substitution of gravid Caenorhabditis elegans (Nematoda: Rhabditida) for transmission electron microscopy. Nematology 6319-327.

CuevaJ.G.MulhollandA.GoodmanM.B. (2007). Nanoscale organization of the MEC-4 DEG/ENaC sensory mechanotransduction channel in Caenorhabditis elegans touch receptor neurons. Journal of Neuroscience 2714089-14098.

DernburgA.F.McDonaldK.MoulderG.BarsteadR.DresserM.VilleneuveA.M. (1998). Meiotic recombination in C. elegans initiates by a conserved mechanism and is dispensable for homologous chromosome synapsis. Cell 94387-398.

DesaiA.RybinaS.Müller-ReichertT.ShevchenkoA.ShevchenkoA.HymanA.OegemaK. (2003). KNL-1 directs assembly of the microtubule-binding interface of the kinetochore in C. elegans. Genes & Development 172421-2435.

DubochetJ. (2012). Cryo-EM – the first thirty years. Journal of Microscopy 245221-224.

DubochetJ.McdowallA.W. (1981). Vitrification of pure water electron-microscopy. Journal of Microscopy 1243-4.

EscaigJ. (1982). New instruments which facilitate rapid freezing at AT 83-K and 6-K. Journal of Microscopy 126221-229.

EvansJ.E.SnowJ.J.GunnarsonA.L.OuG.StahlbergH.McDonaldK.L.ScholeyJ.M. (2006). Functional modulation of IFT kinesins extends the sensory repertoire of ciliated neurons in Caenorhabditis elegans. Journal of Cell Biology 172663-669.

FavreR.HermannR.CermolaM.HohenbergH.MullerM.BazzicalupoP. (1995). Immuno-gold-labelling of CUT-1, CUT-2 and cuticlin epitopes in Caenorhabditis elegans and Heterorhabditis sp. processed by high pressure freezing and freeze-substitution. Journal of Submicroscopic Cytology and Pathology 27341-347.

FavreR.CermolaM.NunesC.P.HermannR.MüllerM.BazzicalupoP. (1998). Immuno-cross-reactivity of CUT-1 and cuticlin epitopes between Ascaris lumbricoides, Caenorhabditis elegans, and Heterorhabditis. Journal of Structural Biology 1231-7.

FélixM.A.HillR.J.SchwarzH.SternbergP.W.SudhausW.SommerR.J. (1999). Pristionchus pacificus, a nematode with only three juvenile stages, displays major heterochronic changes relative to Caenorhabditis elegans. Proceedings of the Royal Society B: Biological Sciences 2661617-1621.

FischerK.BeattyW.L.WeilG.J.FischerP.U. (2014). High pressure freezing/freeze substitution fixation improves the ultrastructural assessment of Wolbachia endosymbiont – filarial nematode host interaction. PLoS ONE 9e86383.

GilkeyJ.C.StaehelinL.A. (1986). Advances in ultra-rapid freezing for the preservation of cellular structure. Journal of Electron Microscopy Technique 3177-210.

GrabenbauerM.HanH.HuebingerJ. (2014). Cryo-fixation by self-pressurized rapid freezing. In: KuoJ. (Ed.). Electron microscopy: methods in molecular biology. New York, USASpringer Science + Business Media pp.  173-191.

HallD.H.HartweigE.NguyenK.C.Q. (2012). Modern electron microscopy methods for C. elegans. In: RothmanJ.H.SingsonA. (Eds). Caenorhabditis elegans: cell biology and physiology. Waltham, MA, USAElsevier pp.  93-149.

HallD.H.HartweigE.NguyenK.C.Q. (2015). High pressure freeze and freeze substitution. Wormatlas. Available online at http://www.wormatlas.org/EMmethods/HPF.htm.

HayatM. (1981). Fixation for electron microscopy. London, UKAcademic Press.

HohenbergH.MannweilerK.MüllerM. (1994). High-pressure freezing of cell suspensions in cellulose capillary tubes. Journal of Microscopy 17534-43.

HoweM.McDonaldK.L.AlbertsonD.G.MeyerB.J. (2001). Him-10 is required for kinetochore structure and function on Caenorhabditis elegans holocentric chromosomes. Journal of Cell Biology 1531227-1238.

HurbainI.SachseM. (2011). The future is cold: cryo-preparation methods for transmission electron microscopy of cells. Biology of the Cell 103405-420.

KnottG.GenoudC. (2013). Is EM dead? Journal of Cell Science 1264545-4552.

KosinskiM.McDonaldK.SchwartzJ.YamamotoI.GreensteinD. (2005). C. elegans sperm bud vesicles to deliver a meiotic maturation signal to distant oocytes. Development 1323357-3369.

KosterA.J.KlumpermanJ. (2003). Electron microscopy in cell biology: integrating structure and function. Nature Reviews Molecular Cell Biology 4SS6-SS10.

LakB.YushinV.V.SlosD.ClaeysM.DecraemerW.BertW. (2015). High-pressure freezing and freeze-substitution fixation reveal the ultrastructure of immature and mature spermatozoa of the plant-parasitic nematode Trichodorus similis (Nematoda; Triplonchida; Trichodoridae). Micron 2725-31.

LeeR.M. (1984). A critical appraisal of the effects of fixation, dehydration and embedding on cell volume. In: RevelJ.P.BarnardT.HaggisG.H. (Eds). The science of biological specimen preparation. Chicago, USASEM Inc. pp.  61-70.

LeunissenJ.L.M.YiH. (2009). Self-pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy. Journal of Microscopy 23525-35.

LonsdaleJ.E.McDonaldK.L.JonesR.L. (1999). High pressure freezing and freeze substitution reveal new aspects of fine structure and maintain protein antigenicity in barley aleurone cells. Plant Journal 17221-229.

MacQueenA.J.ColaiácovoM.P.McDonaldK.VilleneuveA.M. (2002). Synapsis-dependent and -independent mechanisms stabilize homolog pairing during meiotic prophase in C. elegans. Genes & Development 162428-2442.

Manzanilla-LópezR.H.DevonshireJ.WardE.HirschP.R. (2014). A combined cryo-scanning electron microscopy/cryoplaning approach to study the infection of Meloidogyne incognita eggs by Pochonia chlamydosporia. Nematology 161059-1067.

MartellJ.D.DeerinckT.J.SancakY.PoulosT.L.MoothaV.K.SosinskyG.E.EllismanM.H.TingA.Y. (2012). Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nature Biotechnology 301143-1148.

McDonaldK.L. (2007). Cryopreparation methods for electron microscopy of selected model systems. In: McIntoshJ.R. (Ed.). Cellular electron microscopy. Munich, GermanyElsevier pp.  23-56.

McDonaldK.L. (2009). A review of high-pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues. Journal of Microscopy 235273-281.

McDonaldK.L. (2014a). Out with the old and in with the new: rapid specimen preparation procedures for electron microscopy of sectioned biological material. Protoplasma 251429-448.

McDonaldK.L. (2014b). Rapid embedding methods into epoxy and LR White resins for morphological and immunological analysis of cryofixed biological specimens. Microscopy & Microanalysis 20152-163.

McDonaldK.L.WebbR.I. (2011). Freeze substitution in 3 hours or less. Journal of Microscopy 243227-233.

McDonaldK.L.MorphewM.VerkadeP.Müller-ReichertT. (2007). Recent advances in high-pressure freezing. Equipment- and specimen-loading methods. In: KuoJ. (Ed.). Electron microscopy: methods and protocols2nd edition. Methods in molecular biology Vol. 369. Totowa, NJ, USAHumana Press pp.  143-173.

MielanczykL.MatysiakN.MichalskiM.BuldakR.WojniczR. (2014). Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy: preparation of biological samples. Folia Histochemica et Cytobiologica 521-17.

MöbiusW. (2009). Cryopreparation of biological specimens for immunoelectron microscopy. Annals of Anatomy-Anatomischer Anzeiger 191231-247.

MoorH.RiehleU. (1968). Snap-freezing under high pressure: a new fixation technique for freeze-etching. In: Bocciarelli D.S. (Ed.). Proceedings of the Fourth European Regional Conference on Electron Microscopy Rome Italy Tipografia Poliglotta Vaticana 2 33-34.

Müller-ReichertT.HohenbergH.O’TooleE.T.McDonaldK.L. (2003). Cryoimmobilization and three-dimensional visualization of C. elegans ultrastructure. Journal of Microscopy 21271-80.

Müller-ReichertT.SraykoM.HymanA.A.O’TooleE.T.McDonaldK. (2007). Correlative light and electron microscopy of early Caenorhabditis elegans embryos in mitosis. Methods Cell Biology 79101-119.

Müller-ReichertT.MancusoJ.LichB.McDonaldK. (2010). Three-dimensional reconstruction methods for Caenorhabditis elegans ultrastructure. Methods in Cell Biology 96331-361.

PenkovS.OgawaA.SchmidtU.TateD.ZagoriyV.BolandS.GrunerM.VorkelD.VerbavatzJ.-M.SommerR.J. (2014). A wax ester promotes collective host finding in the nematode Pristionchus pacificus. Nature Chemical Biology 10281-285.

RagsdaleE.J.CrumJ.EllismanM.H.BaldwinJ.G. (2008). Three-dimensional reconstruction of the stomatostylet and anterior epidermis in the nematode Aphelenchus avenae (Nematoda: Aphelenchidae) with implications for the evolution of plant parasitism. Journal of Morphology 2691181-1196.

RagsdaleE.J.NgoP.T.CrumJ.EllismanM.H.BaldwinJ.G. (2009). Comparative, three-dimensional anterior sensory reconstruction of Aphelenchus avenae (Nematoda: Tylenchomorpha). Journal of Comparative Neurology 517616-632.

RagsdaleE.J.NgoP.T.CrumJ.EllismanM.H.BaldwinJ.G. (2011). Reconstruction of the pharyngeal corpus of Aphelenchus avenae (Nematoda: Tylenchomorpha), with implications for phylogenetic congruence. Zoological Journal of the Linnean Society 1611-30.

RappleyeC.A.ParedezA.R.SmithC.W.McdonaldK.L.AroianR.V. (1999). The coronin-like protein POD-1 is required for anterior-posterior axis formation and cellular architecture in the nematode Caenorhabditis elegans. Genes & Development 132838-2851.

RipperD.SchwarzH.StierhofY.D. (2008). Cryo-section immunolabelling of difficult to preserve specimens: advantages of cryofixation, freeze-substitution and rehydration. Biology of the Cell 100109-123.

RobinsonJ.M.TakizawaT.PomboA.CookP.R. (2001). Correlative fluorescence and electron microscopy on ultrathin cryosections: bridging the resolution gap. Journal of Histochemistry and Cytochemistry 49803-808.

RostaingP.WeimerR.L.JorgensenE.M.TrillerA.BessereauJ.L. (2004). Preservation of immunoreactivity and fine structure of adult C. elegans tissues using high-pressure freezing. Journal of Histochemistry and Cytochemistry 521-12.

RudelD.RiebesellM.SommerR.J. (2005). Gonadogenesis in Pristionchus pacificus and organ evolution: development, adult morphology and cell-cell interactions in the hermaphrodite gonad. Developmental Biology 277200-221.

ShuX.K.Lev-RamV.DeerinckT.J.QiY.C.RamkoE.B.DavidsonM.W.JinY.S.EllismanM.H.TsienR.Y. (2011). A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biologye1001041.

SkepperJ.N. (2000). Immunocytochemical strategies for electron microscopy: choice or compromise. Journal of Microscopy 1991-36.

TokuyasuK.T. (1973). A technique for ultracyotomy of cell suspensions and tissues. Journal of Cell Biology 57551-565.

Van DonselaarE.PosthumaG.ZeuschnerD.HumbelB.M.SlotJ.W. (2007). Immunogold labeling of cryosections from high-pressure frozen cells. Traffic 8471-485.

Van RijnsoeverC.OorschotV.KlumpermanJ. (2008). Correlative light-electron microscopy (CLEM) combining live-cell imaging and immunolabeling of ultrathin cryosections. Nature Methods 5973-980.

VanheckeD.BellmannR.BaumO.GraberW.EggliP.KellerH.StuderD. (2008). Pseudovacuoles – immobilized by high-pressure freezing – are associated with blebbing in walker carcinosarcoma cells. Methods in Cell Biology 230253-262.

VerkadeP. (2008). Moving EM: the Rapid Transfer System as a new tool for correlative light and electron microscopy and high throughput for high-pressure freezing. Journal of Microscopy 230317-328.

WoogI.WhiteS.BüchnerM.SraykoM.Müller-ReichertT. (2012). Correlative light and electron microscopy of intermediate stages of meiotic spindle assembly in the early C. elegans embryo. In: Müller-ReichertT.VerkadeP. (Eds). Methods in cell biology. Waltham, MA, USAElsevier pp.  223-234.

Figures

  • View in gallery

    Main transmission electron microscopy preparation pathways, with focus on cryo-fixation and related applications.

  • View in gallery

    A-C: Examples of cryo-fixation (high pressure freezing followed by freeze substitution) vs chemical fixation (A′, B′, C′). A: longitudinal section of the cuticle of Acrobeles complexus; B: Detail of two abutting membranes of A. complexus sperm, the phospholipid bilayer is visible after cryo-fixation; C: Immature spermatozoa in testis of Trichodorus similis (am = amorphous, fb = fibrous bodies, n = nucleus). D, E: Examples of typical cryo-fixation applications; D: Black dots show immunolocalisation of major sperm protein in sperm of Diplolaimella sp.; E: Fertilisation of egg in Pontonema vulgaris, capturing an instantaneous process. Abbreviations: ba = basal zone; med = median zone; cor = cortical zone; ep = epicuticle; n sp = nucleus sperm.

Information

Content Metrics

Content Metrics

All Time Past Year Past 30 Days
Abstract Views 42 42 14
Full Text Views 126 126 85
PDF Downloads 9 9 3
EPUB Downloads 1 1 0