Cryobiology and Electron Microscopy reading list

aurellem

Written by:

Robert McIntyre

Books

Bozzola – Electron Microscopy (1999)

ISBN
0-7637-0192-0

Very practical book; it goes into specific details about how to stain, embed, cut, and image samples for electron microscopy. This is basically how I do EM myself.

You could set up your own EM lab with workable protocols just from the specifics described in this book, some money, and Electron Microscopy Science's catalog.

Griffiths – Fine Structure Immunocytochemistry (1993)

ISBN
978-3-642-77097-5

This is the ultimate review book for electron microscopy, at least for everything up till the mid 80's. The scholarship of this book is just amazing – it has most comprehensive reference sections I've ever seen! I personally discovered the Azide trick by reading this book, and have been able to explore many other ideas I would have never found thanks to its excellent scholarship.

If you want to dive into the entire body of scientific literature surrounding electron microscopy, this is the book for you.

Hayat – Basic Techniques for TEM (1986)

ISBN
0-12-333925-1

Goes into great detail about the various ways to prepare biological specimens for electron microscopy. It's basically a collection of EM techniques from the literature up until 1986.

Read this if you want to optimize your techniques for a specific biological specimen.

Hayat – Fixation for Electron Microscopy (1981)

ISBN
0-12-333920-0

This is considered to be THE textbook for electron microscopy. It's a very useful read and can get up speed on the practice of electron microscopy. Hayat's book is a collection of ideas and techniques from the literature, but its reference section is not nearly as good as Griffiths' book. Sometimes, he will say to do things a certain way, but won't provide any references or reasons why.

You should read this if you want a good theoretical introduction to the field of electron microscopy.

Life in the Frozen State (2004)

ISBN
0-415-24700-4

This is THE textbook for Cryobiology, and gives a great summary of ice formation, freezing injury, extremophiles, and the state of the art in cell and tissue preservation (though doesn't talk about vitrification much). I really enjoy the pictures in the book, and the different perspectives on Cryobiology (like many textbooks, it's a compilation of different articles).

Papers

1949 Polge – Revival of Spermatoza after Vitrification and Dehydration at Low Temperatures (Nature)

DOI
10.1038/164666a0

Nature 164, 666-666 (15 October 1949)

Short report about reviving sperm after freezing them down to dry ice temperature. This is one of the earliest "cryobiology" papers. Sperm are a great first model organism, because you can tell whether they survived the process just by looking at them under a microscope and seeing whether they can still swim!

1962 Palay – Fixation of Neural Tissues for Electron Microscopy by Perfusion with Solutions of Osmium Tetroxide (J. Cell Bio)

DOI
10.1083/jcb.12.2.385

The Journal of cell biology, v.12: no.2, 1962, p.385-410

This is the first paper that described how to perfuse-fix brain tissue for electron microscopy. They are beyond hardcore, and actually perfused OSMIUM TETROXIDE (the stuff that blinds / kills you) into mouse brains. (This was before people figured out the glutaraldehyde primary fixation / Os secondary fixation trick.) Before this, people had preserved tissue only via diffusion.

With the source of illumination adjusted to the appropriate angle and brilliance, the gray matter of the cerebral cortex, the neuropil, and the nuclear regions of the brain stem appeared a dull, dark gray, and the white matter, both in the great tracts and commissures and in the slender bundles that interlace everywhere, all appeared a glistening black.

They report that it's very difficult to actually stain the entire brain with Osmium. For some reason, the Osmium just doesn't get everywhere.

Even in the most successful perfusions, a few small white spots, usually not visible to the unaided eye, but detectable upon 9 X magnification, persisted in the slices. The most common site of focal failure was the center of the corpus callosum. Other sites were unpredictable and usually asymmetrical.

For their purposes, they just select the parts of the brain that were adequately stained.

They talk about how the outer membranes of cells have "holes", and that by adding 0.5% w/v CaCl2 they managed to prevent these holes. This doesn't seem to be as important when you're using Glutaraldehyde as the primary fixative.

They use Sodium Nitrite as a vasodilator at 1% in an initial injection for one minute.

Sodium nitrite is the only vasodilating drug that was tested, because it gave satisfactory results on the first trial. Use of a vasodilator is essential for the success of vascular perfusion with osmium tetroxide solutions. On occasions when we have intentionally omitted or have forgotten the nitrite, the perfusion has always failed.

They recommend warming the perfusate:

Warming of the initial perfusion fluids is also an important step in the procedure. Preliminary and unsuccessful trials were made with precooled solutions and with solutions at room temperature. The explanation for these failures is not immediately obvious. Possibly chilled osmium tetroxide induces arteriolar constriction which prevents passage of the fixative into the capillary bed. In contrast, warm solutions may dilate the arterioles, and once they have become immobilized in an open position, further perfusion with a chilled solution becomes possible. Moreover, it should be remembered that warming the solutions 10-15°C above room temperature significantly increases the diffusion rate of the solutes.

They recommend EPON over methacrylate:

The myelin sheath in methacrylate embedded tissue invariably displayed explosions, distortions, and focal disarray of the lamellae. These disturbances were almost completely eliminated merely by embedding the tissue in EPON; those which were not could be attributed to faulty sectioning.

WTF guys?

The best quality surgical "amber" rubber tubing was used. Although it blackened immediately upon exposure to osmium tetroxide, it remained flexible for many months of use.

1963 Lovelock – The Mechanism of the Protective Action of Glycerol against Haemolysis by Freezing and Thawing (Biophysica)

DOI
10.1016/0006-3002(53)90005-5

Biochimica et Biophysica Acta, Volume 11, 1953, Pages 28–36

Lovelock puts forward the idea of "solution effects" injury to account for freezing damage. The idea is that as ice forms, it pulls water out of solution, and concentrates salts in the remaining water. Eventually the high salt concentration kills the cells. Adding glycerol to the solution helps prevent solution effects injury because it prevents the salt concentration from rising too high.

He does his experiment on human red blood cells, and measures hemolysis by examining free hemoglobin after centrifuging – a clever way to count how many cells burst!

CuSO4 causes RBCs (red blood cells) to become impermeable to glycerol:

The property possessed by copper ions of rendering red blood cells practically impermeable to glycerol at 0° provides a convenient means of determining the protective action of glycerol within the cell.

So he froze red blood cells with various concentrations of glycerol, and found that:

  1. Glycerol protects the RBCs only if it permeates the cells.
  2. More glycerol protects the cells better.
  3. Large salt concentrations in a certain temperature range seem to be killing the cells. Very cold temperatures (< -35°C) slows this damage down.
  4. They find a particular salt concentration (0.8 Molal NaCl) where cells begin to be destroyed, independent of glycerol concentration and temperature (above -35°C).

1963 Mazur – Kinetics of Water Loss from Cells at Subzero Temperatures and the Likelyhood of Intracelluar Freezing (J. General Physiology)

DOI
10.1085/jgp.47.2.347

JGP vol. 47 no. 2 347-369

Mazur is a prolific Cryobiologist and this is one of his first papers!

He creates a differential equation to model the loss of water from a cell as the amount of extracellular ice increases. It's based on balancing the vapor pressures of ice and water in the presence of 1.) Salts which accumulate as ice forms and depress freezing point of the remaining water and 2.) The presence of a cell with a membrane which is modeled as impermeable to salts with a temperature-dependent permeability to water.

He gets a formula with no closed form solution which must be solved numerically, and derives the classic shrinkage curves for various simulated cells with various cooling velocities. Solving these equations was actually a challenge back in 1963:

The equation was solved on an IBM 7090 digital computer by M. T. Harkrider of the Mathematics Division, Oak Ridge National Laboratory, using the Runge-Kutta method for second-order differential equations (Korn and Korn, 1961). I am most indebted to him for this essential part of the study.

For fun, I decided to model these same equations using octave and Sussman's scmutils: Water Loss Kinetics

fahy-mazur-residuals.png

Figure 1: Comparison of Mazur's equation and Fahy's simplified linear differential equation.

1965 Farrant – Mechanism of Cell Damage during Freezing and Thawing and its Prevention (Nature)

DOI
10.1038/2051284a0

Nature 205, 1284-1287 (27 March 1965)

Introduces "Farranting", which is where you slowly increase the concentration of something like DMSO while freezing a system. You allow ice to form and concentrate solutes, then you add more DMSO and water, then allow more ice to form by lowering the temperature, and you repeat this process over and over, chasing the freezing point by adding more DMSO. By doing this slowly enough, you are able to essentially freeze the whole system while keeping salt concentration at a lower level. Farrant argues that this means that what damages cells is not ice, but instead "solution effects" injury.

He also shows an alternate method where you add DMSO in increasing concentrations and keep depressing the freezing the freezing point while cooling without ice formation. Taking this idea to its logical extreme will eventually get you vitrification, though practically you have to worry about the permeability of the cells to your penetrating CPA at lower temperatures.

1965 Karnovsky – A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy (Cell Biology Abstract)

Karnovsky, M. J. (1965) J. Cell Biol. 27,137a-138a

This is obnoxious to find, because it's an ABSTRACT that was never actually published as a paper!

Karnovsky introduces an extremely high osmolality fixative composed of formaldehyde and glutaraldehyde. He finds that it doesn't cause much shrinkage when doing immersion fixation. Starts the myth that formaldehyde is for "fast" fixation and glutaraldehyde is for subsequent stabilization.

1968 Meryman – Modified Model for the Mechanism of Freezing Injury in Erythrocytes (Nature)

DOI
10.1038/218333a0

Nature 218, 333 - 336 (27 April 1968)

Meryman eventually became the president of the Society of Cryobiology. This is one of his first papers!

Meryman revisits the "solution effects" injury hypothesis and presents experiments on RBCs that indicate that it's not salt concentration that damages the cells, but an "osmotic pressure gradient" which kills them. He points out that RBCs shrink down to a certain minimum volume, but then they can't shrink anymore, and as ice continues to extract water from the extracellular solution, they are destroyed by the pressure gradient that forms. He isn't clear on how this happens, but he also points out that it's not clear how concentrated salt kills cells either.

So the main point of this paper is presenting the "minimum volume hypothesis" of freezing injury.

1970 Mazur – Cryobiology: the Freezing of Biological Systems (Science)

DOI
10.1126/science.168.3934.939

Science 22 May 1970: Vol. 168 no. 3934 pp. 939-949

This is an introduction to the then nascent field of Cryobiology. Mazur revisits his water loss curves, shows some images of ice damage, talks about extremophiles, and reviews the uses of cryobiology – organ transplants and cryosurgery.

1971 Meryman – Cryoprotective Agents (Cryobiology)

DOI
10.1016/0011-2240(71)90024-1

Cryobiology Volume 8, Issue 2, April 1971, Pages 173–183

Meryman reviews the types of cryoprotectants. He divides them into three categories:

  1. Penetrating Cryoprotectants
  2. Non-Penetrating Cryoprotectants
  3. Other

He suggests that the penetrating cryoprotectants work by reducing the freezing point of the solution, and preventing concentration of salts.

He doesn't know how the non-penetrating solutes work (and no one else at this time does either).

He also talks about other cryoprotectants that might work by "water binding", which is where a molecule such as a protein becomes hydrated and prevents its water of hydration from being osmotically active. He claims that this is how Mytilus (wikipedia) survives freezing to -10°C – it is able to keep around 20% of its intracellular water bound and osmotically inactive.

Meryman also talks about "loosing 65% of intracellular water" as being the main thing which kills cells during freezing. This is a more nuanced and correct view than the "solution effects" theory, because it accounts for the behavior of the Mytilus muscles which survive freezing. The ones that are freeze tolerant stay alive even as the osmolality of their extracellular solutions increases far beyond the salt concentration Lovelock claimed was lethal. They die when they loose 65% of their intracellular water, exactly the same as human RBCs and the non freeze resistant versions of Mytilus.

1972 Elford – Effects of Electrolyte Composition and pH on the Structure and Function of Smooth Muscle Cooled to -79°C in Unfrozen Media (Cryobiology)

DOI
10.1016/0011-2240(72)90015-6

Cryobiology Volume 9, Issue 2, April 1972, Pages 82–100

Elford takes smooth muscle and uses Farranting to cool it down to -79°C while increasing the DMSO concentration to 60% w/v. He found that he could get 92.1% functional recovery using a high potassium PIPES buffer set to pH 7.4. His electron micrographs on the muscle tissue show how muscles are damaged by the cooling process. This is a good example of a well done cryobiology paper with good electron microscopy and a nice functional assay of viability.

1973 Rapatz – Cryoprotective Effect of Methanol During Cooling of Frog Hearts (Cryobiology)

DOI
10.1016/0011-2240(73)90027-8

Cryobiology Volume 10, Issue 2, June 1973, Pages 181–184

Rapatz preserves frog hearts at -30°C by cooling them while perfusing with increasing concentrations of methanol in frog Ringer's solution. They do get the frog hearts back, but they found that they hit a wall – they can't go to -35°C or below or the methanol solution will crystallize, and they can't increase the concentration of methanol or it will kill the frog heart. It's a neat paper because is shows perfusion introduction of CPA into an organ and functional recovery.

1980 Fahy – Analysis of "Solution Effects" Injury (Biophys J.)

DOI
10.1016/S0006-3495(80)85019-3

Biophys J. 1980 Nov; 32(2): 837–850.

Describes equations which model the phase diagrams for two ternary systems, DMSO-NaCl-Water and Glycerol-NaCl-Water. These equations only apply for temperatures > 50°C and for slow freezing. They're intended to be useful to analyze solution effects injury during different freezing protocols.

1982 Meryman – Isotonicity in the Presence of Penetrating Cryoprotectants (Cryobiology)

DOI
10.1016/0011-2240(82)90185-7

Cryobiology Volume 19, Issue 5, October 1982, Pages 565–569

This is a very simple paper about how to balance the osmolality of solutions containing cryoprotectant. Turns out you need to keep a constant molar concentration instead of a molal concentration! When I make my solutions, I just make a concentrated buffer and then mix it with the appropriate cryoprotectant. If you fail to do this and instead make a cryoprotectant solution by mixing an isotonic salt solution with cryoprotectant, you will explode the cells you're working with!

1988 Takahashi – Mechanism of Cryoprotection by Extracellular Polymeric Solutes (Biophysics)

Introduces what I call the "Saran wrap" model of cryoprotection by extracellular cryoprotectants, which are molecules like PVP, which are unable to actually penetrate into cells. He says that basically, these extracellular cryoprotective agents form a shield around the cell which prevents water from escaping, which prevents fatal water loss from the cell.

1993 Rostgaard – Improvements in the technique of vascular perfusion-fixation employing a fluorocarbon-containing perfusate and a peristaltic pump controlled by pressure feedback

Journal of Microscopy Volume 172, Issue 2, pages 137–151, November 1993

DOI
10.1111/j.1365-2818.1993.tb03405.x

This paper is AWESOME! They make a major advance in fixation by using a fixative that can deliver massive amounts of oxygen to tissues, similarly to blood! They claim that they are able to preserve tissues in a non-anoxic state, and that traditional fixation causes anoxic damage, because fixation itself requires oxygen, so while you're fixing tissue, you're stealing oxygen and causing problems. Plus, most fixatives can only carry around 1/20th – 1/10th of the oxygen that blood can carry.

This is such an interesting approach that I had to try it myself. I'll have results on 2015-07-07!

1997 Saija – Changes in the permeability of the blood-brain barrier following sodium dodecyl sulphate administration in the rat

DOI
10.1007/PL00005725

Experimental Brain Research July 1997, Volume 115, Issue 3, pp 546-551

They take rats and inject small quantities of SDS (a chemical used in detergents). They found that it permeabilized the blood-brain barrier of the rat brain for around 30 minutes, and that the blood-brain barrier then sealed back up afterwards. They used Evans Blue (which normally does not cross the BBB) to see whether SDS worked as intended. They found that when they injected SDS, the entire brain turned blue! I use this research extensively in my own experiments to permeabilize the blood brain barriers of rabbit and pig brains to allow those brains to take up cryoprotectants. Yuiry Pichugin also analyzed surfactants specifically for CPA uptake a few years ago. All I could find by him is a provisional patent application which has now expired.

Author: Robert McIntyre

Created: 2017-05-18 Thu 15:17

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