Rudolf Alexander Steinbrecht

Cryotechniques in biological electron microscopy

I Fundamentals.- 1 Physics of Water and Ice: Implications for Cryofixation.- 1 Introduction.- 2 Functions of Water in Cryospecimens.- 3 Water Below Room Temperature.- 4 Aqueous Solutions Below Room Temperature.- 5 Specimen Cooling.- References.- 2 The Response of Biological Macromolecules and Supramolecular Structures to the Physics of Specimen Cryopreparation.- 1 Introduction.- 2 The Hydration Shells of Biological Macromolecules and Supramolecular Structures.- 3 Phases and Interface Phenomena.- 4 The Particular Case of the Biological Membrane.- 5 A Potpourri of Collapses.- References.- 3 Electron Beam Radiation Damage to Organic and Biological Cryospecimens.- 1 Introduction.- 2 Electron Beam/Specimen Interaction.- 3 Radiation Damage to Organic Materials at Low Temperature.- 4 Radiation Damage to Ice.- 5 Radiation Damage to Frozen-Hydrated and Vitrified-Hydrated Specimens.- 6 Conclusions.- References.- II General Methodology.- 4 Cryofixation Without Pretreatment at Ambient Pressure.- 1 Introduction.- 2 Cryofixation with Liquid Cryogen.- 3 Impact Cryofixation (Slamming).- 4 Discussion.- References.- 5 Cryoeleetron Microscopy of Vitrified Specimens.- 1 Introduction.- 2 Vitrification.- 3 Preparation of Thin Specimens.- 4 Image Formation.- 5 Beam Damage.- References.- 6 Cryoultramicrotomy.- 1 Introduction.- 2 Technical Aspects of Cryoultramicrotomy.- 3 Physical Aspects of Cryoultramicrotomy.- 4 Conclusions.- References.- 7 Freeze-Substitution and Freeze-Drying.- 1 Introduction.- 2 Methodology: Theoretical and Experimental Data.- 3 Procedures and Instrumentation.- 4 Critical Evaluation.- References.- III Special Aspects.- 8 Theory and Practice of High Pressure Freezing.- 1 Introduction.- 2 Freezing Under Atmospheric Pressure.- 3 Freezing Under High Pressure.- 4 The Main Practical Problems of Pressure-Freezing.- 5 The High Pressure Freezing Machine (Balzers HPM 010).- 6 Some Practical Advice.- 7 Discussion of Results.- References.- 9 Freeze-Etching of Dispersions, Emulsions and Macromolecular Solutions of Biological Interest 192.- 1 Introduction.- 2 Specific Problems of Specimen Preparation.- 3 Determination of Particle Concentrations and Molecular Weights.- 4 Measurements of Size and Shape.- 5 Structure of Dispersions in Bulk and at Interfaces.- References.- 10 High Resolution Metal Replication of Freeze-Dried Specimens.- 1 Introduction.- 2 Electron Microscopy and Image Processing.- 3 Characterization of the Test Specimens.- 4 Controlled Freeze-Drying.- 5 High Resolution Shadowing.- References.- 11 Immunogold Labelling of Cryosections and Cryofractures.- 1 Introduction.- 2 Cryoultramicrotomy.- 3 Cryofractures.- 4 Label Efficiency.- 5 Conclusions.- References.- 12 Cryoultramicrotomy for Autoradiography and Enzyme Cytochemistry.- 1 Introduction.- 2 Cryoultramicrotomy for the Autoradiography of Diffusible Substances.- 3 Thin Cryosections in Histochemistry.- 4 Conclusions.- References.- 13 Scanning Electron Microscopy and X-Ray Microanalysis of Frozen-Hydrated Bulk Samples.- 1 Introduction.- 2 Morphology.- 3 Analysis.- 4 Freezing.- 5 Instrumentation.- 6 Electron Interactions.- 7 X-Ray Emission.- 8 The Fracture Surface.- 9 Quantitative Analysis.- 10 Water Content or Dry Weight Fraction.- 11 Beam Damage and Mass Loss.- 12 Detection Limits.- 13 Conclusions.- References.- 14 Cryofixation of Dynamic Processes in Cells and Organelles.- 1 Introduction.- 2 Cooling Rate, Freezing Time, Time Resolution.- 3 Cellular Structures: Rapid Freezing Versus Chemical Fixation.- 4 Membrane Dynamics.- 5 Conclusions and Outlook.- References.- 15 Cryofixation of Diffusible Elements in Cells and Tissues for Electron Probe Microanalysis.- 1 Introduction.- 2 Specimen Preparation.- 3 Some Biological Applications and Results.- 4 Conclusion and Outlook.- References.- IV Appendix.- 16 Safety Rules for Cryopreparation.- 1 Introduction.- 2 Asphyxiation with Gaseous Nitrogen.- 3 Gaseous Propane Explosions.- 4 Burns Caused by Secondary Cryogen Splashing.- 5 Burns Caused by Primary Cryogen Splashing.- 6 Ignition of Combustible Secondary Cryogens.- 7 Bursting of Cryogen Containers.- 8 Transport and Disposal of Cryogens.- 9 Concluding Remarks.- References.

Pore structures in insect olfactory sensilla: A review of data and concepts

Abstract An historical overview is given of the most important discoveries and hypotheses regarding stimulus transport in insect olfaction. The great structural similarity between the pore tubules of olfactory single-walled wall-pore sensilla and the epicuticular filaments of non-olfactory cuticle may reflect not only a similar composition but also a similar transport mechanism. The new “wick concept” of pore tubules comprises 2 developmental periods. First, during ontogeny of the sensillum, pore tubules may be involved in the secretion of the material of the outermost epicuticular layers. The pore tubules may function like the wick in an oil lamp, taking up lipoid molecules from the sensillum lymph for outward transport. During the second period, after the sensillum has been completed, the pore-tubule wick may work as a dispenser of odorant molecules in an inward direction. The large surface of pore tubules as compared with the cross section of the outer pores could facilitate the binding of odorant molecules by the odorant-binding proteins in the sensillum lymph. In double-walled wall-pore sensilla, on the other hand, pore tubules are not involved in stimulus transport. In this class of olfactory sensilla, the dendrites are protected by a palisade of cuticular fingers, and openings between these fingers, the spoke channels, are the stimulus transport pathways. The fundamentally different topology of sensillar wall pores hints at a separate phylogenetic origin of the two categories of insect olfactory sensilla.

Freeze-Substitution and Freeze-Drying

Freeze-substitution (FS) and freeze-drying (FD) are dehydration techniques by which the water is gently removed from a frozen specimen. Both techniques can serve as a link between cryofixation and conventional thin sectioning at room temperature (Fig. 1). They are, therefore, hybrid techniques combining the advantages of the low temperature and the room temperature specimen preparation. With respect to the danger of artefacts, these procedures are much more obscure than “pure” cryotechniques, such as freeze-etching or cryosectioning. Both, FS and FD, are known from light microscopy and have been used in electron microscopy since its early days (for refs. of the older literature see Bullivant 1970; Rebhun 1972; Robards and Sleytr 1985), but only during the last dozen years a breakthrough can be noticed, which is mainly due to improved cryofixation. As for any other cryotechnique in biological electron microscopy, for successful FS and FD the main prerequisite is also good cryofixation with as little freezing damage as possible (see Bachmann and Mayer, Chap. 1; Sitte et al., Chap. 4; Dubochet et al., Chap. 5; Moor, Chap. 8; this Vol.).

Immunolocalization of pheromone-binding protein and general odorant-binding protein in olfactory sensilla of the silk moths Antheraea and Bombyx

The distribution of odorant-binding proteins among olfactory sensilla of three moth species was studied by immuno-electron microscopy. Two polyclonal antisera were used in a post-embedding labelling protocol on sections of cryo-substituted antennae. The first was directed against the pheromone-binding protein (PBP) of Antheraea polyphemus, the second against the general odorant-binding protein (GOBP) of the same species. Immunoblots showed that these antisera were highly specific; both antisera did, however, cross-react with related proteins in the related species A. pernyi, and in the bombycid moth B. mori. PBP and GOBP were localized only in olfactory sensilla trichodea and sensilla basiconica, the principal site being the sensillum lymph surrounding the sensory dendrites. In the males of all three species, the pheromone-sensitive long sensilla trichodea exclusively contained PBP. the majority of the sensilla basiconica in both sexes in these species contained GOBP; these sensilla are known to respond to plant and other ‘general’ odours. Some sensilla were not labelled by either antiserum; presumably, these held an odorantbinding protein of a different subfamily. Never were PBP and GOBP co-localized in the same sensillum. Two observations deserve special attention: (1) PBP was also found in a few sensilla in females, and (2) in B. mori, where the long sensilla trichodea have a different functional specificity in males (pheromone) and females (plant odours), the expression of the odorant-binding protein (males: PBP; females: GOBP) is similarly different. The distinct and complex distribution pattern of odorant-binding proteins supports the notion that these proteins participate in stimulus recognition.

Immunocytochemical localization of pheromone-binding protein in moth antennae

SummaryOdorant-binding proteins are supposed to play an important role in stimulus transport and/or inactivation in olfactory sense organs. In an attempt to precisely localize pheromone-binding protein in the antenna of moths, post-embedding immunocytochemistry was performed using an antiserum against purified pheromone-binding protein of Antheraea polyphemus. In immunoblots of antennal homogenates, the antiserum reacted exclusively with pheromone-binding protein of A. polyphemus, and cross-reacted with homologous proteins of Bombyx mori and Autographa gamma. On sections of antennae of male A. polyphemus and B. mori, exclusively the pheromone-sensitive sensilla trichodea are labelled; in A. gamma, label is restricted to a subpopulation of morphologically similar sensilla trichodea, which indicates that not all pheromone-sensitive sensilla contain the same type of pheromone-binding protein and accounts for a higher specificity of pheromone-binding protein than hitherto assumed. Within the sensilla trichodea, the extracellular sensillum lymph of the hair lumen and of the sensillum-lymph cavities is heavily labelled. Intracellular label is mainly found in the trichogen and tormogen cells: in endoplasmic reticulum, Golgi apparatus, and a variety of dense granules. Endocytotic pits and vesicles, multivesicular bodies and lysosome-like structures are also labelled and can be observed not only in these cells, but also in the thcogen cell and in the receptor cells. Cell membranes are not labelled except the border between thecogen cell and receptor cell and the autojunction of the thecogen cell. The intracellular distribution of label indicates that pheromone-binding protein is synthesized in the tormogen and trichogen cell along typical pathways of protein secretion, whereas its turnover and decomposition does not appear to be restricted to these cells but may also occur in the thecogen and receptor cells. The immunocytochemical findings are discussed with respect to current concepts of the function of pheromone-binding protein.

Der Feinbau olfaktorischer Sensillen des Seidenspinners (Insecta, Lepidoptera)

SummaryThe sensilla (s). trichodea and s. basiconica on the antennae of the silk moth,Bombyx mori, were studied under the transmission electron microscope. Chemical fixation, freeze substitution and freeze etching methods were used. The following results have been obtained: Five sensillum types were distinguished, the olfactory function of which is known from electrophysiological recordings, except for the last one. Thelong s. trichodea and themedium-sized s. trichodea I are innervated by two sensory cells which have essentially unbranched receptor processes (dendrites). Commonly thelarge s. basiconica contain three sensory cells, thesmall s. basiconica only one; the dendrite of these receptor cells branch multiply when entering the hair lumen. For the first time an intermediate type has been described: themedium-sized s. trichodea II, which resemble the s. basiconica in their branching innervation, but must be classified as s. trichodea because of the form and size of the sense hair. For each type, the dimensions of the receptor processes, as well as the number and distribution of the stimulus conducting pores and pore tubules in the hair wall are noted.On the male antenna the long s. trichodea are most abundant; they contain the highly sensitive sex pheromone receptors (mean number per antenna: 17 000 sensilla with 34 000 sense cells). In the female these sensilla are reduced in number to about 35% and supplied with receptor cells of different specificity and lower sensitivity. In both sexes, the two dendrites of the long s. trichodea differ markedly in their mean diameter, and the number of cytoplasmatic microtubules. In the male moth both receptor processes reach the hair tip, whereas in the female the thinner one invades only the proximal third of the sense hair. The cuticle of the hair wall is perforated by pores (♂: 2–7 pores per μ2; ♀: 2–5 pores per μ2), which mostly open to the outside near to characteristic steps in the hair surface. Each pore canal leads into about five pore tubules, which proceed towards the hair lumen, where they end, partly in contact with the receptor membrane of the dendrites. Distal parts of the sense hairs show such tubule-membrane contacts more frequently than proximal regions. The number of contacts counted on the thicker dendrite is about four times greater than on the thinner one. In these sensilla, the two receptor cells constitute functionally different reaction types, which may relate to the observed morphological differences.The s. basiconica have about 20 pores per μ2 of the hair surface, and 12–23 pore tubules per pore: thus, these sensilla have the same or even a greater number of pore tubules per sensillum than the much larger s. trichodea. In the s. trichodea the number of pores per unit surface increases steadily towars the hair tip, while the number of pores per unit length of the hairs soon reaches a constant value. A hypothesis about the morphogenesis of this distribution is given.The functional significance of the epicuticular surface layers and of the pore tubule systems is discussed under the aspect of stimulus conduction. Starting from the site of impact anywhere on the sense hair, odour molecules may diffuse two-dimensionally along the hair surface to the pores, and then proceed by one-dimensional diffusion through pore canals and pore tubules until they eventually reach the receptor membrane at the end of a tubule. The calculated conduction times are shorter than the known receptor latencies; thus, the transport mechanism can be explained by diffusion and does not need a more complex hypothesis.ZusammenfassungDie Sensilla (S.) trichodea und S. basiconica auf den Antennen des Seidenspinners,Bombyx mori, wurden nach chemischer Fixierung, Gefriersubstitution und Gefrierätzung im Transmissionselektronenmikroskop untersucht. Es lassen sich fünf Typen von Sensillen unterscheiden, deren olfaktorische Funktion aus elektrophysiologischen Versuchen bekannt ist, mit Ausnahme des letzten Typs.Lange S. trichodea undhalblange S. trichodea I sind jeweils von zwei Sinneszellen innerviert, deren Rezeptorfortsätze (Dendriten) im wesentlichen unverzweigt bleiben. Diegroßen S. basiconica haben meist drei, diekleinen S. basiconica nur eine Sinneszelle; die Dendriten dieser Rezeptorzellen verzweigen sich büschelförmig beim Eintritt in das Haarlumen. Erstmals wird ein Zwischentyp beschrieben: diehalblangen S. trichodea II ähneln hinsichtlich der Innervation den S. basiconica, sind aber wegen der Form und Größe des Sinneshaars als S. trichodea zu klassifizieren. Für jeden Typ werden die Abmessungen der Rezeptorfortsätze sowie die Zahl und Verteilung der reizleitenden Poren und Porentubuli in der Haarwand angegeben.Auf der männlichen Antenne sind die langen S. trichodea am zahlreichsten; sie enthalten hochempfindliche Sexuallockstoffrezeptoren (mittlere Anzahl pro Antenne: 17 000 Sensillen mit 34 000 Sinneszellen). Beim Weibchen sind diese Sensillen in der Zahl auf etwa 35% reduziert und mit Sinneszellen anderer Spezifität und geringerer Empfindlichkeit ausgerüstet. Die beiden Dendriten der langen S. trichodea unterscheiden sich bei beiden Geschlechtern stark im mittleren Durchmesser und der Anzahl der cytoplasmatischen Mikrotubuli; beim Männchen reichen beide bis zur Haarspitze, beim Weibchen endet der dünnere Fortsatz bereits im proximalen Haardrittel. Die Cuticula der Sinneshaare ist von Poren durchbrochen (♂: 2–7 Poren/μ2; ♀: 2–5 Poren/μ2), die stets in der Nähe von charakteristischen Stufen in der Haaroberfläche münden. Jeder Porenkanal führt in ca. fünf Porentubuli, die bis ins Haarlumen reichen und dort enden, zum Teil in Kontakt mit der Rezeptormembran der Dendriten. Die Häufigkeit solcher Tubulus-Membrankontakte ist in distalen Haarabschnitten größer als in proximalen. Der dickere Dendrit weist etwa viermal so viel Kontakte auf wie der dünnere. Die beiden Rezeptorzellen dieser Sensillen stellen funktionell verschiedene Reaktionstypen dar, was mit den beobachteten morphologischen Unterschieden zusam menhängen dürfte.Die S. basiconica haben ∼20 Poren pro μ2 ihrer Oberfläche und 12–23 Porentubuli pro Pore; dadurch erreichen oder übertreffen sie die viel größeren S. trichodea in der Gesamtzahl der Porentubuli pro Sinneshaar. Auf den S. trichodea steigt die Zahl der Poren pro Oberflächeneinheit zur Spitze hin stetig an, während die Zahl der Poren pro Haarlängeneinheit einen konstanten Wert annimmt. Eine Hypothese über die Morphogenese dieser Verteilung wird aufgestellt.Die funktionelle Bedeutung der äußeren Epicuticulaschichten und der Porentubulussysteme für die Reizleitung wird diskutiert. Ausgehend von beliebigen Orten ihres Auftreffens auf dem Sinneshaar können die Duftmoleküle zunächst durch zweidimensionale Diffusion entlang der Haaroberfläche zu den Poren gelangen und anschließend durch eindimensionale Diffusion über Porenkanäle und Porentubuli die Rezeptormembran erreichen. Die berechneten Diffusionszeiten sind kürzer als die bekannten Rezeptorlatenzen; die Reizleitung kann also durch Diffusion hinreichend erklärt werden und erfordert keine kompliziertere Hypothese.

Cryofixation without cryoprotectants. Freeze substitution and freeze etching of an insect olfactory receptor

Antennae of the silk moth, Bombyx mori, were frozen by immersion into propane at -180 degrees C, and further processed by (a) freeze substitution (FS) or (b) freeze etching (FE). Although no cryoprotectant was used, freezing damage was observed in deeper tissue regions only. Data from FS specimens closely resemble those from FE replicas. Therefore, FS usually does not induce noticeable secondary artefacts by the preparation steps subsequent to freezing. When compared with chemically fixed antennae, the superior quality of cryofixation in this tissue is evident, particularly where cell surfaces and processes border the receptor lymph cavity; membranes are smooth following a steady course; dendrites and axons are round in cross-section with evenly distributed microtubules. The value of cryofixation is discussed with special reference to structures of presumed functional significance (e.g. stimulus conducting pore tubules, intramembrane particles of the receptor membrane, the ciliary segment of the dendrites, intercellular dilations, membrane junctions).

Fine structure of the antennal receptors of the bed bug, Cimex lectularius L.

Sensilla on the antenna of the bed bug, Cimex lectularius, were studied with the scanning and transmission electron microscope. Those which display a tubular body in the dendrite ending are presumed to have a mechanoreceptor function (bristles of type A, flat plate of type B). Bristles of type A1 contain additional dendrites which terminate at the tip of the bristle and may be gustatory receptors. Sensilla with pores in the hair wall are supposed to have an offactory, humidity and/or temperature receptor function (pegs and hairs of types C, D, E). Hairs of type E contain receptors for the alarm pheromones of the bed bug. Special attention has been paid to the pore structures and epicuticular layers of these sensilla. Possible differences in stimulus conduction are discussed between (i) sensilla with a simple wall and pores with pore tubules (types D and E) and (ii) the ribbed pegs (type C), which have a complex wall structure and spoke channels. The immersed cones of type F have a peculiar innervation, which has not been described previously. Two dendrites are held closely together by a third flat dendrite which wraps around them in the region of the outer segment. Coupling structures were found between the central dendrites, and between these and the third enveloping dendrite. Possible functions of this unique innervation are discussed. The dendrites innervating type D are grouped in three to eight bundles by multiple sheaths. The term thecogen cell is introduced to denote the innermost of the three sheath cells of a sensillum (the outer being the tormogen and the trichogen cell) which builds the dendrite sheath during ontogeny. Comparative morphometry revealed type-specific differences in the length and diameter of the dendrites. Some axons were found to lack any glial or perineurial sheath. Microorganisms were observed in the antennal tissue of several animals.

Gustatory organs of Drosophila melanogaster: fine structure and expression of the putative odorant-binding protein PBPRP2.

Abstract. In Drosophila, as in most insects, gustation is mediated by sensory hairs located on the external and internal parts of the proboscis and on the legs and wings. We describe in detail the organization and ultrastructure of the gustatory sensilla on the labellum and legs and the distribution of PBPRP2, a putative odorant-binding protein, in the gustatory organs of Drosophila. The labellum carries two kinds of sensilla: taste bristles and taste pegs. The former have the typical morphology of gustatory sensilla and can be further subdivided into three morphological subtypes, each with a stereotyped distribution and innervation. Taste pegs have a unique morphology and are innervated by two receptor cells: one mechanoreceptor and the other a putative chemoreceptor cell. PBPRP2 is abundantly expressed in all adult gustatory organs on labellum, legs, and wings and in the internal taste organs on the proboscis. In contrast to olfactory organs, where PBPRP2 is expressed in the epidermis, this protein is absent from the epidermis of labial palps and legs. In the taste bristles of the labellum and legs, PBPRP2 is localized in the crescent-shaped lumen of the sensilla, and not in the lumen where the dendrites of the gustatory neurons are found, making a function in stimulus transport unlikely in these sensilla. In contrast, PBPRP2 in peg sensilla is expressed in the inner sensillum-lymph cavity and is in contact with the dendrites. Thus, PBPRP2 could be involved as a carrier for hydrophobic ligands, e.g., bitter tastants, in these sensilla.

Chemo-, Hygro-, and Thermoreceptors

Of all the invertebrate phyla, arthropods have invaded the terrestrial habitat most successfully; with the exception of Crustacea they are predominantly land dwellers. Without doubt, one of the main reasons for this achievement is the evolution of arthropod cuticle which provides mechanical support and protection against water loss at the same time. Sense organs, however, meet a completely new situation, for they must adapt to a rigid exoskeleton which is a barrier between the environment and the milieu interieur. Depending on the stimulus modality, there are various solutions to the contrasting tasks of maintaining the internal milieu and, at the same time, providing optimal accessibility of the sensory structures to environmental stimuli.