Free ebooks ==> www.Ebook777.com www.Ebook777.com Free ebooks ==> www.Ebook777.com Low-Temperature Microscopy and Analysis www.Ebook777.com Low-Temperature Microscopy and Analysis Patrick Echlin University of Cambridge Cambridge, England Springer Science+Business Media, LLC Library of Congress Cataloging-in-Publication Data Echiin , Parr ick Low-temperature microscopy and analysis / Patrick Echlin p cm Includes bibliographical references and index Cryomicroscopy I Title QH225.E34 1992 578'.4—dc20 Cryopreservation of organs, tissues, etc 91-39738 CIP Figures 5.11, 9.4, 9.6, and 9.7 from Journal of Electron Microscopy Technique, reprinted by permission of John Wiley and Sons, Inc ISBN 978-1-4899-2304-2 DOI 10.1007/978-1-4899-2302-8 ISBN 978-1-4899-2302-8 (eBook) © Springer Science+Business Media New York 1992 Originally published by Plenum Press, New York in 1992 Softcover reprint of the hardcover 1st edition 1992 All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher Free ebooks ==> www.Ebook777.com To my wife, Shirley www.Ebook777.com Very high and very low temperatures extinguish all human sympathy and relations It is impossible to feel affection beyond 78 or below 20 of Fahrenheit: human nature is too solid or too liquid beyond these limits -SYDNEY SMITH, 1836 I have gathered a posie of other men's flowers and nothing but the thread that binds them is my own -MICHEL MONTAIGNE, 1592 Foreword The frozen-hydrated specimen is the principal element that unifies the subject of lowtemperature microscopy, and frozen-hydrated specimens are what this book is all about Freezing the sample as quickly as possible and then further preparing the specimen for microscopy or microanalysis, whether still embedded in ice or not: there seem to be as many variations on this theme as there are creative scientists with problems of structure and composition to investigate Yet all share a body of common fact and theory upon which their work must be based Low- Temperature Microscopy and Analysis provides, for the first time, a comprehensive treatment of all the elements to which one needs access What is the appeal behind the use of frozen-hydrated specimens for biological electron microscopy, and why is it so important that such a book should now have been written? If one cannot observe dynamic events as they are in progress, rapid specimen freezing at least offers the possibility to trap structures, organelles, macromolecules, or ions and other solutes in a form that is identical to what the native structure was like at the moment of trapping The pursuit of this ideal becomes all the more necessary in electron microscopy because of the enormous increase in resolution that is available with electron-optical instruments, compared to lightoptical microscopes On the size scale below one micrometer, frozen-hydrated specimens offer the hope of escaping from the dilemma that the "unlimited" resolution of electron optics can, on the one hand, often be wasted by inadequate specimen preparation, while light microscopy can give perfect specimen preparation, but only inadequate resolution In this context, the time has certainly come in which a comprehensive and unified coverage of low-temperature techniques can strongly influence the continued development of biological electron microscopy and microanalysis The pursuit of improved, if not ideal specimen preparation by low-temperature techniques has developed steadily over more than 25 years Methods have been developed at the level of cellular fine structure (notably freeze fracture and freeze substitution), microanalysis of diffusible substances, the structure of macromolecular assemblies (notably freeze-drying and shadowing), and most recently even the internal structure of macromolecules At the highest resolution, low temperature is needed not so much to preserve the native, hydrated state, which it does admirably well (but so other techniques, such as glucose embedment), as for the extra margin of protection which it provides against radiation damage The development of all of these techniques has been aided not just a little in the ix x FOREWORD past by the author of this book, Patrick Echlin, through his effort in organizing a series of four International Meetings on Low-Temperature Microscopy, beginning in 1977, and through the emphasis that he has given to the field in his role as editor of the Journal of Microscopy The next logical step, given the maturity of development of the subject, would have to be nothing else than to write this book It is a volume that will speed access to existing techniques and greatly expand awareness of related work for all who seek a unified presentation of low-temperature methods and their underlying theoretical foundation Publication of this book therefore makes a truly important contribution toward advancing the process of learning what goes on in biology at a level below what can be seen with the light microscope Robert M Glaeser Department of Molecular and Cell Biology Universitv of California at Berkelev Preface Water is the most abundant and most important molecule in the biosphere and outer lithosphere As a vapor it forms a vital envelope around our planet; as a liquid it covers about 75% of the Earth's surface and dissolves almost everything As a solid it is permanently present at the Poles and on many mountain peaks and is a seasonal reminder of the changing climate of our environment Liquid water is vital for living organisms It is both a reactant and the medium in which reactions occur and their products are transported Water is the most abundant and least expensive building block of living matter and when converted to the solid state can provide the perfect matrix in which to study the structure and in situ chemistry of hydrated material This book considers the nature of this solid matrix, its constituent components, and how it may be formed, manipulated, examined, and analyzed This volume has grown from the firm belief that low-temperature microscopy and analysis is the only way we may hope to obtain a true picture of the fine structure and composition of ourselves and our water-filled environment I will discuss the physical basis and the practical aspects of the different procedures we need to use, the problems that occur, and the advantages that accrue The conversion of liquids (primarily water) to their solid phases (primarily ice) forms a central feature of this book The text falls into four unequal parts The first three chapters consider water in the liquid and solid states The next four chapters discuss the various manipulations we may make to the solidified matrix There then follows three chapters that show what we may hope to see in the frozen samples by means of photons and electrons, and another chapter considers the processes we need to use to analyze their constituent elements and molecules The final chapter contains updated information on the whole subject The book provides a number of well-tested procedures that will enable the novice to cryomicroscopy and analysis to get started It also gives a detailed background from which future developments can take place The reader is provided with sufficient general information on how to implement a particular low-temperature process and the reasons why it should be used A comprehensive bibliography at the end of the book provides the provenance and specific details of existing practices Low-temperature microscopy and analysis is not the sole preserve of biologists and those interested in hydrated organic samples-although these types of samples present both the greatest challenge to the existing technologies and the only hope of xi Free ebooks ==> www.Ebook777.com xii PREFACE solving the unresolved questions posed by such samples The processes that will be discussed can be used to study and analyze the solid state of all the liquid and gaseous materials which exist on our planet (with the possible exception of helium) Low temperatures provide an important way to study radiation-sensitive and labile samples, hydrated organic systems, and phenomena that only exist at temperatures at which living processes stop Low-temperature microscopy and analysis is not without potential dangers, and it is important that experimentalists are fully aware of safety issues in the laboratory Cryogenic liquids can cause severe bums, and exposed parts of the body must be protected with the appropriate clothing and face masks when using these materials Liquid nitrogen should only be used in a well-ventilated laboratory, as I liter of the liquid expands to nearly 700 liters of an inert tasteless gas which can cause asphyxia Some secondary organic liquid cryogens have very low flash points and form dangerously explosive mixtures with oxygen, which may condense from the atmosphere Some of the resins used in low-temperature embedding may cause contact dermatitis and, like all laboratory chemicals, should be handled with gloves An electron microscope laboratory is replete with potential hazards associated with vacuum and highpressure equipment and containers, high voltages and ionizing radiation, and toxic and inflammable chemicals Reputable industrial companies provide safety information about their products and supplies; responsible governments issue safety legislation about laboratory practices These warnings should be heeded, because by understanding the potential difficulties and adopting sensible laboratory procedures, low-temperature microscopy and analysis can take place in a safe, productive (and happy) environment The arguments for adopting cryotechniques and low-temperature microscopy and analysis are secure and proven It should come as no surprise that so many people are now using one or more of these methods to examine and analyze hydrated, liquid, and gaseous specimens It is, however, astonishing that in 1991, the onehundredth anniversary of the electron, anyone should continue to use an electron beam instrument at ambient temperatures Patrick Echlin Cambridge www.Ebook777.com REFERENCES 525 Ting-Beall, H P., Burgess, F M., and Robertson, J D (1986) J Microsc 142:311-316 Tobler, M., and Frelburghaus, A U (1990) J Microsc 160:291-298 Tokuyasu, K T (1973) J Cell BioI 57:551-565 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E.M Israel Vol 2, pp 223-225 Zierold, K (1980) Microsc Acta 83:25-37 Zierold, K (1982) Ultramicroscopy 10:45-54 Zierold, K (1984) Ultramicroscopy 14:201-210 Zierold, K (1984) J Phys (Paris) 45:C2447-C2450 Zierold, K (1985) J Microsc 140:65-71 Zierold, K (1986) Scanning Electron Microsc 2:713-724 Zierold, K (1986) In The Science of Biological Specimen Preparation M Muller, R P Becker, A Boyde, and J L Wolosewick, eds Chicago: SEM Inc., pp 119-127 Zierold, K (1987) In Cryotechniques in Biological Electron Microscopy R A Steinbrecht, and K Zierold, eds Berlin: Springer-Verlag, pp 132-148 Zierold, K (1988) J Electron Microsc Tech 9:65-82 Zierold, K (1989) Microbeam Anal 1989:109-111 Zierold, K (1991) J Microsc 161 :357-366 Zierold, K., and Hagler, H (1989) Electron Probe Microanalysis: Applications in Biology and Medicine Heidelberg: Springer-Verlag Zierold, K., and Schafer, D (1987) Verh Dtsch Zool Ges 80:111-118 Zierold, K., and Steil'lbrecht, R A (1987) In Cryotechniques in Biological Electron Microscopy K Zierold, and R A Steinbrecht, eds Berlin: Springer-Verlag, Chap 15 528 REFERENCES Zierold, K., Tobler, M., and Muller, M (1991) J Microsc 161 :RPI Zingsheim, H P (1984) J Microsc 133:307~317 Zs-Nagy, I (1983) Scanning Electron Microsc 3:1255-1268 Zs-Nagy, I (1988) Scanning Microsc 2:301~309 Zs-Nagy, I (1989) Scanning Microsc 3:473~482 Zs-Nagy, I., and Casoli, T (1990) Scanning Microsc 4:419~428 Zs-Nagy, I., Pieri, c., Guili, c., Bertoni-Freddari, C., and Zsa-Nagy, V (1977) J Ultrastruct Res 58:22~ 33 Zs-Nagy, I., Lustylik, G., Zs-Nagy, V., Zarandi, V., and Bertoni-Freddari, C (1981) J Cell Bioi 90:769~ 777 Index Acoustic microscopy, 388 Adiabatic cooling, 150, 352, 356-357 Amorphous ice crystalline structure of, 29 formation, 30 freeze drying of, 195, 207-209 high-pressure form, 30 irradiation, 30 low-pressure form, 30 See also Vitreous ice, 20, 29-32, 165 Anticontamination devices, 148-149, lSI, 162, 310, 313, 319-321, 344, 357-358 Antifreeze agents: see Cryoprotectants Antistatic devices, 122, 130, 139 Antiroll plates, lll, 117, 122 Apolar dehydrating agents, 232-233 Apolar embedding media, 231, 233, 241, 243, 246, 248,256 Apolar solvents, 236-237 Apoplast, 16 Aqueous compartments, 430 Aqueous layers evaporation of, 303-304 production of, 303-305, 307-308 layers for TEM, 303-309 solutions, 42-47 Artifacts freeze-fracture replication, 159, 177-178 freeze substitution, 258-259 low-temperature embedding, 258-259 low-temperature microanalysis, 422, 454-455, 479-487 low-temperature scanning electron microscopy, 397-399 low-temperature transmission electron microscopy, 303, 330-345 Association-induction hypothesis, 15 Atomic force microscopy, 178 Auger electrons, 379, 388 Autoradiography, 179, 231, 338 Backscattered electrons, 379, 381-382, 443-444 Ballistic phonon imaging, 388 Bands in sections, 133 Bare grid preparation method, 305-306 Beam damage, 330-345, 392-397,479-487; see also Radiation damage Beam induced specimen motion, 495 Biochamber, 150-153, 190 Biochemical effects of low temperatures, 49-50 Biot number, 33, 34 Bound water, 9, 10, 243 Boundary cooling, 276 Bubbling artefacts in ice, 336-337, 343 Bulk water, 10, IS Cathodoluminescence, 379, 384-385, 407 Charging, 154, 432, 447 surface, 372, 435, 447, 474 bulk, 372, 434-435, 447 Charge separation, 287 Chatter, 106, 129-130 Chemical dehydration, 223 Chemical fixation ambient temperature, 224, 225, 226 effects on specimens, 61 for freeze-etch replication, 167 for freeze substitution, 224 for isothermal freeze fixation, 257 speed of, 61, 230-231 Chilling injury, 289 Clatbrates, Closed-loop controllers, 270 Collapse phenomena during drying, 194-196, 219 Cold shock, 289 Cold shrouds, 319, 321, 377; see also Anticontamination devices Cold stages comprehensive for light microscopy, 270-278, 494 conductive heat transfer stage, 270, 272-274, 281 529 530 Cold stages (Cont.) comprehensive for light microscopy (Cont.) convective heat transfer, 270, 274-278 geometric arrangement, 275 unidirectional, 276 for cryopreparation units, 144, 146-149, 150-153 general features of, 150 heat pipe, 315 liquid helium cooled, 297, 312-315 liquid nitrogen cooled, 297, 315-317 Peltier, 278-279 simple for light microscopy, 266-270, 278 conductive heat transfer, 270, 274-277, 281 convective heat transfer, 270, 272-273 comparison of types, 278 design of, 267-270 general features of, 266 for microanalysis, 421-422 for scanning electron microscopy, 146-149, 351358 direct supply, 352, 354-355 flexible braid, 353-354 Joule-Thompson, 352, 356-357 stability of, 310-311, 315-316 temperature control, 267, 279-281, 319-322 thermoelectric, 278-279 for transmission electron microscopy, 309-322 basic requirements, 309-311 double tilt, 314 side entry, 313-314, 316, 321 top entry, 312-314, 316, 320, 346 Comparative cooling rates, 71, 77 Comparative radiation doses, 341 Comparative SEM signals, 388 Comparison of coating materials, 448 Comparison of electron doses used in x-ray microanalysis, 485 Comparison of frozen specimen types, 390-392, 397-398, 471-474 Comparison of x-ray detection limits, 461 Comparison of x-ray spatial resolution, 441 Compression in sections, 130-132 Contrast mechanisms atomic number, 381-382 amplitude, 275, 325 bright field, 444-445 dark field, 325, 444-445, 481 diffraction, 328 low, 325 phase, 323-324 topographic, 382, 389, 443-444 z,326 Conductive coatings high resolution, 174, 373 INDEX Conductive coatings (Cant.) procedures, 146-147, 153-155, 170, 174,372379, 448-451 thickness measurement, 175 Conductive paints, 366 Conductive processes cooling, 34 heat transfer, 33 Contamination, 69, 113, 114, 137, 148, 153, 157, 311, 317, 383, 485 prevention of, 162, 164, 200, 212, 310, 319, 364, 367 removal of, 371 Convective processes cooling, 34 forced, 34 heat transfer, 33 natural, 34 laminar flow, 34 turbulent, 34 Cooling biochemical effects of, 49 controlled environment vitrification, 303-304, 307-309, 494 critical rates of, 41, 71, 75, 77, 82, 84, 91, 93, 270,492 comparison of, 77, 90-91, 93, 94 significance of, 91-92 curves for, 41 dehydration effects of, 49 effectiveness of, 99, 268, 313 effects on reaction rates, 50, 494 evaluation of, 94-96, 492 fluxes, 269 limiting processes of, 492 for scanning electron microscopy, 367 Countercurrent cooling, 78 Covalent bonds, Crevasses in sections, 127, 128 Critical excitation potential, 414 Cryoballistic cooling, 89 Cryocastle, 215 Cryocrystallography, 346-347, 495 Cryoelectron microscopy: see Low temperature transmission electron microscopy Cryoenzymology, 226 CRYOFIX computer programme, 92 Cryogens liquid, 34, 70-75 effectiveness of, 71 halocarbons, 73 helium, 70-71 nitrogen, 71-73 organic gases, 73-75 INDEX Cryogens (Cant.) liquid (Cant.) properties of, 71 solid, 75-77 effectiveness of, 76 properties of, 75 Cryoglues: see Low temperature glues Cryomicroscopy: see Low temperature light microscopy Cryomicrotomes, 110-117 basis requirements, 112 cryostat microtomes, 111-112, 113 cryoultramicrotomes, 113 Cryoplaning, 425, 496 Cryopliers, 86 Cryoprobes, 367 Cryopreparation units, 142-155, 361-362 Cryoprotection factor: see Radiation cryoprotection factor Cryoprotectants, 109, 242 artificial and nonpenetrating, 65-67 artificial and penetrating, 64-65 use with fracturing, 186-188 use with freeze-fracture replication, 168 use with freeze substitution, 232 use with light microscopy, 289 mode of action, 63 natural agents of, 64 with transmission electron microscopy, 299-300 Cryopreservation, 48, 95 Cryopumps, 207-209, 388 Cryosections collection of, 113, 117, 122-124, 139 different types, 102-103 flattening, 123-124 freeze dried, 102, 103, 104, 122-123, 126, 137, 204, 211, 444 frozen hydrated, 102, 103, 104, 122, 126, 129, 131, 135,298-299, 387, 445 for light microscopy, 125 quality assessment of, 122, 130 for scanning electron microscopy, 387 serial, 120 storage, 125, 137 transfer of, 125 ultra-thin, 298 Cryosectioning artifacts, 126-137, 140 cutting speeds for, 121, 132, 134 definition of, 101 equipment for, 110-117 forces developed during, 105, 108 heat generated during, 103 problems with, 126-137, 139 531 Cyrosectioning (Cant.) processes of, 102, 103, 106, 110, 114, 120-122, 130,492 sample preparation for, 118-120 theoretical models of, 107-109 thickness of, 102, 121-122, 125, 13l, 139 trough liquids for, 121 Cryosolvents, 226 Cryostats bath, 310 continuous flow, 310 microtome, III Cryotransfer devices, 311, 317-318 for high-voltage transmission electron microscopes, 317 for scanning electron microscopes, 358-362, 367 for transmission electron microscopes, 314, 317318 Crystallization, 14, 52 latent heat of, 33, 41, 53 processes of, 40 rates of, 51 Cubic ice, 20, 28-29, 165, 338 conversion to hexagonal ice, 25 crystalline structure of, 29 formation of, 28 Cutting, definition of, 101 Cytoskeleton, 15 Cytomatrix, 60 Cytoplasm, 60, 289, 393, 346 Damage by irradiation, 32, 245; see also Radiation damage Decoration artifacts, 171-172 Dehydration effects at subzero temperatures, 49 Denaturation, 232, 242 Desiccants, chemical, 200-201, 206, 210, 235, 238 Deuterium oxide, 201, 203-204, 209, 211 Devitrification, 30 during imaging, 336 during sectioning, 106 temperature, 32, 45, 46, 52 Differential scanning calorimetry, 45, 54 Differential thermal analysis, 45, 54 Diffusible substances, 103, 206 Digital image processing, 185,387,397,475-477 Digital scanning x-ray microanalysis, 396, 497 Directional solidification, 89-90, 281-282, 286, 402 Droplet cooling, 81-83 Drying agents, 200, 206, 210-211, 235, 237, 238 Enzyme reaction, effects of low temperatures on, 14 Elastic elongation, 102 532 Elastomers, 102 Electrolytes, 221, 260, 286, 452 Electron beam evaporation, 171 beam exposure, 244, 296, 455, 476-479 beam penetration, 380-382, 418, 431-442, 437, 439, 482 diffraction of ice, 22, 31, 32, 296, 329, 330 of cryosections, 22, 338 of organic specimens, 339 diffusion volume, 434 dose, 295, 332-345, 432, 455, 476, 480 energy loss spectroscopy, 261, 326-327, 446, 456, 468-469, 495 interaction with specimens in the scanning electron microscope, 379-392 in the transmission electron microscope, 322329 in the x-ray microanalyser, 430-431, 438-439, 442-446 irradiation of ice, 28 mean free path, 473, 483-484 probe dimensions, 433 range equations, 435-436, 439 scattering, 322, 325-326, 332-333, 338, 380381, 390, 430-431 cross section, 431, 432, 456 elastic events, 381, 415, 431-432, 432 inelastic events, 381, 415, 431-432, 444, 468, 482-484 stopping power, 334 Electron probe x-ray microanalysis: see Low temperature x-ray microanalysis Electrostatic charge, 115, 139, 287 Embedding agents, 67-68, 231, 493; see also Resins Emission spectra, 282 Emulsion droplet cooling, 82-83 Encapsulation, 67 Equilibrium cooling, 43 Etching by radiant heating, 146, 152, 155, 369-371 by conductive heating, 146, 155, 369-371 deep, 163, 166-169, 404 general feature of, 153 process of, 163-169 rates, 146, 165-166, 371 shallow, 165 Eutectic concentration, 43, 44, 45 mixtures, 43, 220, 235 temperatures, 43, 44, 45, 48 separation, 44 INDEX Eutectic (Cont.) solidification, 287 Evaporative coating methods, 147, 152, 167, 170173, 373-375, 448-451 damage caused by, 173-174, 298 Excitation spectra, 282 Extracellular spaces, 16, 153, 429, 467 ice, 49, 398-399 Field emission electron microscopes, 187, 355, 378, 445, 495 Fixation: see Chemical fixation Flashing, 290 Folds in sections, 127 Forced convection cooling, 79 Fourier analysis of images, 323-324, 331, 432 Fractures chonchoidal, 141, 160 complementary, 369-370 general features, 127 impact, 157-159 shear force, 141, 157 surface features of, 162 tensile stress, 141, 157, 166 Fracturing, 101, 141-191, 368, 425 applications of, 189-190 chemical fixation for cracking, 188 permeabilization, 189 polishing, 188; see also Cryoplaning thaw digestion, 188 thaw fixation, 186 equipment for, 142-155 complementary, 369 comprehensive devices, 149-155 simple devices, 142-143 integrated devices, 143-149 process of, 141, 159-160 under liquid gas, 163 under dry gas, 162 Free radicals, 333-334, 344 Free water, 9, 10 Freezing, 69 artifacts, 95-96 Freeze concentration factor, 47, 48, 405 Freeze damage: see Freeze injury Freeze drying, 193-222 analytical applications, 215-216 artifacts of, 219-221 consequences of, 194-196 equipment for, 201-207, 493 high vacuum, 202-206 low vacuum, 205-207 533 INDEX Freeze drying (Cant.) equipment for (Cant.) molecular distillation, 207-211 ultra-low temperature, 209-211 fixation of specimen for, 212-213 from nonaqueous solvents, 216-218 general features of, 193-194 monitoring, 201-202 morphological applications of, 194, 214-215 practical procedures for, 199-200 primary drying, 195, 203-204, 220 process of, 197-199 prolongation factor in, 197, 199,203,205-206 protocols for, 204, 206 rate of, 196, 198-199 resin embedding, 213-214 secondary drying, 195, 203, 220 sections: see Cryosections specimen handling, 211-213 theory of, 196-199 ultra-low temperature, 196 Freeze etching: see Freeze fracture replication Freeze fracture replication, 142, 155-191 applications of, 189-190 autoradiography, 179-184 monolayer, 179-182 double layer, 182 equipment for complementary, 158-159 fracture flip, 181, 184 general process of, 156 immunolabeling, 183-185 labeling and cytochemistry, 180-185 quantitation, 185 sample preparations, 157, 168 Feezing injury, 257, 265,402-405 Freeze substitution, 223-240 applications of, 259-260 chemical fixatives for, 229-231 desiccants for, 235, 237 effectiveness of, 258-259 equipment for, 251-252, 493 experimental schedules for, 235-237, 240 general outline, 227, 234 options for, 228 organic fluids for, 229, 234-237 measurements of, 238-239 rate of, 237-240 sample preparation for, 228, 253-255 temperature of, 237 Freeze thawing, 142 Freezing point depression, 55 Frozen hydrated material effects of ionizing radiation on, 334-335, 342345 Frozen hydrated material (Cant.) for scanning electron microscopy, 154, 370-371 for transmission electron microscopy, 298-299 Furrows in sections, 127 Gas-bubble formation in ice, 286-287, 292 Glass transition partially crystallized, 51, 55 point, 30, 491 temperature, 45, 46 Graininess in sections, 127 Health hazards, x, 218, 241 Heating fluxes, 269, 274-275 Heat transfer, 92, 276 Heterogeneous nucleation, 37, 39, 51 agents of, 38 as function of temperature, 38 in red blood cells, 38 Hexagonal ice, 20, 21-28, 165 boat configuration of, 24 chair configuration of, 24 crystal growth, 39 crystal symmetry of, 23 crystal structrue of, 24 electrical properties of, 25 formation of, 25 thermal properties of, 25 transformations, 28 High-pressure cooling, 21, 86-89 effectiveness of, 86, 88 equipment for, 87 freeze substitution following, 236 problems with, 89 High temperature superconductors, 356 Holey films, 301-303, 306 Homogeneous nucleation, 37, 39 as function of temperature, 36 temperature, 30, 36, 46 Hydration chambers for microscopy, 296 Hydration shells, 60, 225, 242, 248 Hydrogen bonds in water, 4, 5, in ice, 20, 27 Hydrophobic interactions, 8, 38 Hypercooled, 35; see alsa Undercooled Ice bend contours, 330 crystal damage, 66, 126-127, 134 crystal ghosts, 94-95, 126, 195, 259, 397,438 crystal growth, 40, 53, 61, 91, 257 crystal size, 37, 41, 109 dentritic crystals, 42 534 Ice (Cont.) as embedding agent, 296 films for transmission electron microscopy, 304-305 intracellular, 42, 285, 291-292, 398, 494 and liquid interfaces, 282 matrix, 109 mechanical properties of, 27 microcrystalline, 55, 86 nucleating agents of, 62 physical properties of, 19, 21 polymorphs of, 19-21 radiation effects on, 341-342 segregation of, 285 solidification of, 285 sublimation, 146, 196-199 rate of, 147, 196-197 tetrahedral configuration of, 19 thermal propeities of, 33 Image analysis, 186, 283, 285 contrast, 123, 249, 325, 428, 446 interpretation in light microscopy, 290-292 in scanning electron microscopy, 389-392 in transmission electron microscopy, 300 in x-ray microanalysis, 428, 442-446 processing, 389 recording, 283-284 Immobile water, 10 Immersion cooling, 77-79 Immunocytochemical studies, 180-185, 221, 226, 229, 236, 243-244, 246, 248, 250, 261-262 Impact cooling, 83-86 equipment for, 84-85 effectiveness of, 85, 98 use with microwaves, 492 Interfacial water, Inert dehydration, 225, 242 Intracellular darkening, 290-291 Ionic bonds interactions of, in water,S Isothermal freeze fixation, 227, 257-258 Jet cooling, 79-82, 492 Knives angles, 115, 116, 130, 139 for cryosectioning, 115-116, 125, 128, 139, 492 for cryofracturing, 142, 146 for freeze fracture replication, 159 making, 116,492 marks, 132-133, 162 temperature of, 113, 159 INDEX Laser probe analysis, 261 Latent heat of crystallization, 91 Leidenfrost, 71, 73 Light microscopy, 110-111, 123, 138, 224, 244, 260; see also Low-temperature light microscopy Low-temperature dehydration, 232-233 Low-temperature digital imaging and elemental mapping, 474-479 for bulk samples, 478-479 for sections, 475-477 Low-temperature embedding, 240-264 applications of, 259-260 equipment for, 253 experimental schedules for, 242, 249 of freeze dried material, 255-256 general outline, 227, 240-241 options for, 228 problems with, 244, 247 sample preparation, 228, 253-255, 493 tissue handling devices, 254-255 Low-temperature glues, 68, 97, 118-119, 123, 125, 366-367, 423 Low-temperature light microscopy, 265-293 applications of, 285-290 cold stages for, 49, 270-281 limitations of, 284, 292 optics of, 267-268, 271, 284-286 optimal features of, 271 Low-temperature microanalysis, 413-490 analytical protocols, 426 applications, 488-490 beam damage, 479-487 contamination, 487 loss of specific elements, 486 mass loss of organic material, 480-486 coating methods procedures for, 449-451 properties of, 448 confirmation of hydrated state, 471-474, 497-498 correction procedures, 422, 453, 454, 496 detection limits, 461-464 dry weight elemental concentrations, 468-469, 470 extraneous x-rays, 422 general description of, 413 high resolution, 479, 497 image visualization, 425, 428, 442-447 backscattered electron images, 443-444 energy loss image, 446 secondary electron images, 443 transmitted electron image, 442-443, 444-446 instrumentation, 419-423 cold stages, 421-423 detector geometry, 423, 437 INDEX Low-temperature microanalysis (Cont.) instrumentation (Cont.) energy dispersive spectrometers, 420-421 instrumental drift, 475 wavelength dispersive spectrometers, 420 matrix corrections, 458-459, 460, 463 measuring water content, 465-471, 497 bulk samples, 469-471 sections, 465-469 optimal condition for, 488 physical principals, 414-419, 430-442 characteristic x-rays, 415-417 continuum radiation or background, 417, 422 intensity of emission, 417-418 production of x-rays absorption effect (A), 419, 456, 457, 458, 459, 462 atomic number effect (2), 418, 459, 462 fluorescence effect (F), 419, 459 surface topography, 419, 478 qualitative analysis, 424 quantitative analysis, 424, 429, 451-464 continuum-normalization method for sections, 453-457 elemental ratio method for sections, 452 ionization function [(pz)] for bulk samples, 460-462 peak to continuum ratio method for bulk samples, 460 peak to local background method for bulk samples, 458-460 peripheral standards method for sections, 452453 ZAF corrected peak intensities method for bulk samples, 457-458 sample preparation, 423-430, 447-451, 493 spatial resolution, 426-428, 431-442 in bulk samples, 434-436 depth, 436, 438, 439, 440-441 lateral, 436, 438, 440-441 in sections, 431-434, 451 specimen holders, 422-423 size of interactive volume, 418, 431-442, 447 electron range equations for, 435-439 ionization function (pz) curves, 439-442 standards for quantitation, 452, 462-465 for bulk samples, 459-463 independent matrix-matching, 462 independent non-matrix matching, 462-463 peripheral, 452, 462, 466-467 for sections, 463 types of samples bulk specimens, 417, 425-426, 430, 434, 441, 448, 485, 496 535 Low-temperature microanalysis (Cont.) types of samples (Cont.) freeze dried, 429-430, 437, 438, 452 frozen hydrated, 428-430, 423, 437, 438, 440, 452,457 microdroplets, 426, 496 partially dried specimens, 430, 466 sections, 417, 422, 427-428, 430, 431, 440, 452, 485 wet weight elemental concentrations, 465, 469-470 whole cells and particles, 426 ZAF corrections, 456, 458, 460 Low-temperature microspectrofluorometry, 282-283 Low-temperature resins, 241 embedding schedules, 244-245 London resin, 249-251, 259, 261, 493 Lowicryl, 221, 233, 241, 243-248, 255, 256, 261, 493 ultra-low temperature embedding, 493 low-temperature scanning electron microscopy, 349-411 advantages of, 400, 408-409 anticontaminators for, 357-358 applications, 399-407 artifacts, 397-400 frozen-hydrated vs freeze-dried images, 391, 397-398 ice crystals, 398-399 beam damage, 392-397 contrast mechanisms atomic number, 381 topographic, 381-382, 389 disadvantages of, 410 Low-temperature scanning electron microscopy, 349-411 advantages of, 350-351, 400, 408-409 anticontaminators for, 357-358 applications, 399-407 artifacts, 397-400 frozen-hydrated vs freeze-dried images, 391, 397-398 ice crystals, 398-399 sample shrinkage, 399 beam damage, 392-397; see also Radiation damage beam heating, 394-396 cold stages for, 351-358 direct cooled, 352-354 flexible coupling cooled, 352-354 general principles of, 351-352 Joule-Thompson refrigerator, 352, 356-357 liquid helium, 355-356 contrast mechanisms atomic number, 381 536 INDEX Low-temperature scanning electron microscopy (Cant.) contrast mechanisms (Cant.) topographic, 381-382, 389 as convenient imaging system, 400 disadvantages of, 410 cold stages for, 351-357 examination of samples, 379-392 contrast changes, 389-391 image quality, 389-391 high resolution imaging, 495 signal generation, 379-389 signal processing, 389-392 frozen-hydrated samples, 371 image formation, 349-350, 379-389 as an innovative imaging system, 401-407 specimen attachment methods, 364-367 mechanical devices, 364-365 adhesives, 366-367 specimen coating methods, 372-379 evaporative, 373-375 high resolution, 375, 378, 386, 496 sputtering, 375-379 specimen holders, 362-364 specimen preparation devices, 361-362 specimen preparation procedures, 362-379, 495496 etching, 368-371, 391 fracturing, 368 micromanipulation, 368 specimen selection, 362-363 specimen transfer devices, 358-362, 367 airlock systems, 359-361 non-air-Iock systems, 358-359 versatility of, 350-351 Low-temperature sinks, 269 Low-temperature solvent drying, 216-218 Low-temperature storage, 56, 96-97 Low-temperature transmission electron microscopy, 295-348 applications, 297-298, 345-348 high voltage TEM, 345-346 high resolution, 347, 495 scanning tunneling, 346 beam damage, 330-345 comparative doses, 341 in hydrated specimens, 342-345 in ice, 341-342 observable effects, 336-337 measurable effects, 337-341 mechanisms of, 343-344 processes of, 332-334 thermal effects, 335-336 See also Radiation damage Low-temperature transmission electron microscopy (Cant.) cold stages for, 309-317 image appearance, 325-329 bright field, 325-326, 328 dark field, 325-326 electron energy loss, 326-327 scanning transmission, 325 image interpretation, 326-329 image observation, 322-330 amplitude contrast, 322-323, 327, 331 contrast transfer function, 323, 330 diffraction contrast, 327-328 phase contrast, 323-324 image processing, 329-330 image recording, 329-330 electron diffraction patterns, 329 low dose imaging, 329 image stability, 310, 315, 316, 326, 337, 495 specimen holders, 314 specimen preparation, 297-309 frozen sections, 298-300, 323, 326 thin liquid suspensions, 301 support films for, 301, 494 thin aqueous layers, 303-306 Macromolecular collapse, 177, 193, 194, 198,430 Mass loss, 244, 333, 334, 338, 340-342, 344, 396, 423, 467, 474, 480-486 Mass thickness, 298, 432, 456, 465 Mass spectrometer, 201-202 Melting during fracturing, 162 processes of, 54 temperature, 52 during sectioning, 105, 134-137 transient, 105, 108, 127 Metal mirror cooling: see Impact cooling Metastable water, 12 Microanalysis, 153; see also Low temperature x-ray microanalysis Microcomputer controls, 280 Microcrystalline ice: see Ice Micromanipulation, 368 Microplaning, 425; see also Cryoplaning Microtome, 157 Molecular distillation, 207-211, 263; see also Freeze drying Nanofilms, 494 Nitrogen slush, 72 Noncrystalline ice: see Amorphous ice; Vitreous ice Nonequilibrium cooling, 43 Nucleate boiling, 69 537 INDEX Nucleation, 35-39, 52 agents of, 39, 62-63 artificial, 62 natural, 62, 491 physiological effects of, 63 probability of, 36, 50 processes of, 36, 40, 61, 66, 286 rate of, 36 Optical darkening, 290 Oxygen atoms in hexagonal ice, 23, 24 in cubic ice, 29 Peltier cooling, lll, 206, 278-279 Penning sputtering, 174 Perturbed water, 10 Phase boundary, 288 Phase diagrams, 20, 42, 43, 44, 46 of multi-component systems, 46-49 of simple systems, 44-46 Phase equilibria, 42 Phase separation, 54 phases, 43 components, 43 Phase transitions, 46, 55, 306, 347 Phi-ro-z curves, 439-442, 461-462 Plastic deformation, 101, 102, 106, 132, 134, 141, 162, 177 embedding, 101, lIO materials, 102, 138, 162 sections, 130 Plunge cooling, 77-79 equipment for, 80-81 specimen holders for, 78 procedures for, 78-79 Polar interactions, Polar dehydration, 233, 237, 243 Polar embedding agents, 243 Polymers, 102, 138, 162, 189, 190, 343, 347, 400 Polymerization of resins by chemical catalysts, 224, 244-245, 250 by ultraviolet light, 224, 244-245, 255 exothermic reactions of, 247-249 Polymorphs of ice, 19-22 Polypeptides as nucleating agents, 39 Polysaccharides, as nucleating agents, 39 Progessive lowering of temperature, 227,233-234,493 Prolongation factor in freeze drying, 197, 199, 203, 205 Quadrupole mass spectrometer, 361 Quench cooling, 69; see also Rapid cooling Radiation cryoprotection factor, 334, 338, 342-344, 396, 486-488 Radiation damage, 123, 296, 298, 301, 330-345, 392-397, 429, 479-487 comparative doses, 341 consequences of, 344-345 effects on specimens, 344-345 processes of, 332-335 ionizing radiation effects, 336-345, 396 thermal effects, 335-336, 394-396 units of dose, 334-335 Radiolysis of ice, 341-344 Rapid cooling procedures, 69-90 Recrystallization, 52-54, 127, 220, 290 damage by, 239 irruptive, 54 migratory, 53 processes of, 52-54 spontaneous, 52, 54 temperature, 45, 240 Refrigerators, 251-253 Rehydration of frozen sections, 126, 134, 136 Replicas cleaning, 177 complementary, 158-161-167, 169 composite, 169 formation of, 170-174 fracture flip, 169 removal, 177 simple, 159, 169 simulcast, 169 Replication process, 169-174 Resins, 223, 226, 256; see also Embedding material Ripples in sections, 133-134 Rotary shadowing, 168, 174-176 Salting out, 47, 50 Scanning electron microscopy, 102, 104, 128, 147, 150-151, 178, 186, 189, 212, 427, 443; see also Low-temperature scanning electron microscopy beam-specimen interactions, 380-392 electron optics, 349-350 high resolution, 495, 496 low voltage, 187, 190, 260, 379, 495 Scanning transmission electron microscopy, 249, 387,427,444-446,469,497 Scanning tunneling microscopy, 178-179, 346 Scheiner's halo, 28 Secondary electrons, 379, 382-383 Sections: see Cryosections Semiconductors, 355, 385 Shadow coating, 170 538 Shrinkage, 194, 220, 242, 259, 328-329, 399, 453, 455, 466, 474 Slam cooling: see Impact cooling Solidification, 277, 288 velocity of, 289 Solid-liquid phase diagram of water, 20 Solvent drying, 216-218 Solvent environment, 225 Solute concentration, 286, 289, 303 Solute polarization, 286-287 Specimen bulk, 362-379 cooling, 59-101 geometry, 77 holders for, 77-78, 96, 157, 159, 169, 283, 362-364 liquid suspensions, 301; see also Thin films pretreatment before cooling chemical, 60-68 nonchemical, 68-69 size and shape, 68 strategies for, 54, 57, 68-69 supports, 122, 362-363, 423 transfer at low temperatures, 97, 143 types, 77 Spray cooling, 81-83 Sputter coating, 144, 146, 174, 375-379 Sputter shadowing, 174 Stains, 232, 246 Storage at low temperatures, 56, 96-97 containers, 96 dewers,96 Sublimination, 277; see also Etching and Freeze drying Substitution fluids, 196-198; see also Freeze substitution Surface heat transfer, 33 Surface modified water, Surface topography effects, 426, 478 Supercooled water, 12 Supercritical nitrogen, 72-74 Superconducting electron microscopes, 296 lenses, 296, 309, 316 Supersaturation, 43 Support films, 122, 301-303, 340, 344, 494 carbon, 301-302 holey, 302, 306 hydrophilic, 302, 494 hydrophobic, 302, 494 Surface tension, 301 Temperature control, of light microscope cold stages, 267, 279-281 INDEX Temperature gradients, 281 Temperature measurement, 92-94, 492 electrical, 94 fluorescent, 94 Thawing, 55 Thermal capacitance, 270 Thermal conductivity, 75, 76, 84 Thermal inertia, 75-76 Thermistors, 267 Thermocouples, 81, 92-94, 267, 273 calibration, 320 thin wire, 92, 279, 320 thin film, 93, 279, 283, 322 Thin film specimens aqueous suspensions, 296, 298, 303-309 liquid suspensions, 301, 304-305, 309 vitrified layers, 297, 301, 305, 324 Time resolved, stop-flow methods, 306-309, 494 procedures, 307-309 studying chemical reactions, 306-307 Transfer devices: see Cryotransfer Transmission electron microscopy, 102, 138, 178, 189, 202, 249, 259-260, 385-387; see also Low-Temperature transmission electron microscopy image formation, 322-324 Triple point of water, I Trough liquids, 139 Twitching, 291-292 Unfrozen water, 11, 328, 491 Unperturbed water, 11, 15, 109 Undercooled biological material, 13 conditions for, 13 plant material, 14 processes of, 55 temperature region, 30 samples, 33 solutions, 42 water, 12, 13, 21, 27, 35, 37, 39 Ultramicrotomy processes of, 110 equipment for, 110 See also Cryosectioning Ultrahigh vacuum, 157, 162, 207-209 Vacuoles, 16 van der Waals forces in water, Vitreous ice, 29, 42, 209, 337-338; see also Amorphous ice Vitrification, 31, 32, 51-52, 82, 86, 298 Free ebooks ==> www.Ebook777.com 539 INDEX Vitrified samples solids, 67, 195 cuting of, 106, 130 drying of, 195, 207 radiation resistance of, 343 sections, 327 suspensions, 297 Vitrification solutions, 67 Water in cells and tissues, 14-16 density of, dynamic processes in, 6, 11, 12 exchange rates of, 10 flickering structure of, glass-like state of, 13 heat capacity of, 27 heat of vaporization of, hydrogen bonds in, isothermal compressibility of, latent heat of, metastable, 35 molecules, 3, 4, as clusters, 492 as tetrahedrons, 3, 6, 7, 19, 23, 30 rate of movement of, 51 partial pressure of, 200, 202, 203, 317 physical properties of, polygons, Water (Cont.) pure liquid, 3, 12, 55 self-diffusion of, 2, 13 shear viscosity of, solvent properties of, specific heat of, specific volume of, 47 structure of, 3-7 thermal conductivity of, thermodynamic properties, 12 three dimensional network of, triple point of, I unfreezable, 328 viscosity of, 2, 13 Wrinkles in sections, 127 X-ray diffraction patterns for ice, 28, 31 techniques, 226, 229 X-ray mapping, 476, 479 X-ray microanalysis, 103, 126, 202, 216, 213, 261, 334, 340; see also Low-temperature x-ray microanalysis Young's modulus of ice, 27 ZAF corrections, 456, 458-459 Z contrast, 326 Zero-loss imaging, 326, 495 www.Ebook777.com ... procedures, low- temperature microscopy and analysis can take place in a safe, productive (and happy) environment The arguments for adopting cryotechniques and low- temperature microscopy and analysis. .. ==> www.Ebook777.com Low- Temperature Microscopy and Analysis www.Ebook777.com Low- Temperature Microscopy and Analysis Patrick Echlin University of Cambridge Cambridge, England Springer Science+Business... Studies 7.7.3 Low- Temperature Embedding with Lowicryl 7.7.4 Improvements and Modifications to Lowicryl Embedding 7.7.5 Other Low- Temperature Embedding Resins Freeze Substitution and Low- Temperature