Historical Development

The development of modern cryobiology lies tightly intertwined with the advent of cryomicroscopy. Robert Hooke invented the first compound microscope and used it to observe the cellular structure of plants. He also took his microscope outside to look at snowflakes and watched ice formation in living plant tissue. It wasn't until the early part of the nineteenth century, when German botanists began wondering once again about the nature of life and death at low temperatures. They first took their microscopes outside in the winter to observe what happened when ice formed in living tissues and later designed the first cryomicroscopes.

Fig. 14.3.1 Robert Hooke's Microscope

In 1830, Goeppert first observed plant cells during freezing by going outdoors with his microscope and observing tissues as they cooled to reach the outside temeprature. He refuted the view of earlier botanists that freezing death is caused by ice forming and expanding inside cells, thereby tearing and rupturing them. Goeppert observed ice forming between cells, not inside them, causing the cells to shrink as a result of dehydration. This was the first of many cryomicroscopical observations that would influence our ideas about cryobiology.

Sachs, in 1860, put his microscope in an open window during periods when the outside temperature fell to -5°C to observe plant cells during freezing. This seemingly minor innovation greatly improved the comfort of the microscopist, allowing longer observational schedules to be achieved. It also sped up the process of preparing new samples for mounting. Sachs confirmed Goeppert's observation that ice only forms between cells and went on to propose that cells die on thawing, not freezing.

Müller-Thurgau took the next logical step of placing his microscope inside a box with a surrounding ice-brine bath to keep the temperature of the microscope below freezing. He noticed that tissues needed to be supercooled before ice formation would occur inside the tissue, and that there was no injury when held at these sub-freezing temperatures in the absence of ice. Müller-Thurgau showed that the rate of thawing was of little or no consequence to the survival of most plant cells, refuting Sach's proposal that slow thawing might improve recovery. Müller-Thurgau was able to show that the temperatures at which lethal injury occured correlated with the conversion of the majority of tissue water to ice. He argued that cell injury was due to dehydration.

At the close of the nineteenth century, Hans Molisch built the first dedicated cryomicroscope. It consisted of an insulated wooden box with an ice-brine bath surrounding the inner cavity. The microcope was contained within this cavity, with controls for focussing and panning extending to the exterior of the box. A thermometer was placed beside the sample and a relatively constant temperature of about -10°C could be maintained for several hours with a room temperature of +5°C (room- temperature cryomicroscopy was still a dream of the future!). The temperature was controlled by varying the salinity of the ice-brine bath.
Fig. 14.3.2 Hans Molisch' Cryomicroscope

Using this cryomicroscope, Molisch confirmed the findings of Müller- Thurgau and found that partially dehydrating leaves before freezing could lessen the severity of freeze-thaw injury. Molisch proposed that the freeze-concentration of solutes in the cytoplasm could be toxic or could lead to reactions that created toxic compounds.

Schander and Schaffnit built the first cryostage (an independent microscope stage that could be bolted on to a normal microscope) in 1918. Cooling was accomplished by passing compressed CO2 through ether and circulating it through channels in the stage. This apparatus was able to achieve rapid, though uncontrolled, cooling down to about -30°C.

In 1931, Chamot and Mason improved upon this concept and designed a sophisticated stage for observing chemical phenomena. They circulated a cold liquid through channels in a solid metal block. In the center of the block, a cylinder was removed and a glass slide placed over the top. The sample was mounted on this glass slide and a thermometer bulb was inserted into the cylindrical opening to monitor temperature. They suggested mounting a microthermocouple on the glass slide to give more rapid and accurate temperature readings.

Following the discovery of the protective action of glycerol, Harmer developed a simple cryostage to investigate this phenomenon. He put copper strips on either side of a perspex slide and immersed the copper in either liquid air or warm water to control the temperature. A thermocouple was placed beside the sample to monitor the temperature. Polge, Smith, and Smiles later modified this design by changing the copper strips to a brass block with a cylindrical opening, embedding the thermocouple in the slide underneath the sample, and wrapping the brass block with heating coils.

In the late 50's, Luyet and Rapatz returned to a low-tech approach by sealing a cell suspension between two cover slips using vaseline. A pre- cooled alcohol bath was maintained on the microscope stage and the cover slip apparatus was simply placed into the alcohol. Using this equipment, they noted that deformation of the cells between ice channels did not cause cell lysis upon warming.

Ken Diller entered the picture in the early 70's armed with the emerging technology of microelectronics. He developed a computer-controlled cryostage that allowed sophisticated cooling and warming protocols to be used. Diller used cold nitrogen gas as a coolant and electric current for warming the sample. The sample was mounted on a glass slide that had a metal oxide sputtered onto one side, allowing the slide itself to serve as the resistance heater. The computer measured the temperature using a thermocouple and then put out a current to the heater to keep the temperature controlled.

Fig. 14.3.3 Ken Diller's Convection Cryostage

Convection Cryostage

The convection stage is cooled by injecting a refrigerant gas into a chamber below the sample. At constant flow rates, the cooling to the glass slide that the sample is mounted on is constant. Generally, nitrogen gas is taken from a sealed dewar (drawn through a coil that is immersed in the liquid phase to cool it). Pressure for the system is usually derived from a heater in the dewar. To enhance convective heat transfer, the inlet and outlet ports are usually situated to facilitate turbulence within the chamber. The glass slide that forms the top of the chamber has a transparent metal oxide (tin oxide is common) coating that serves as a resistive heater when current is passed through it. In the center of this slide, a foil thermocouple is bonded beneath a thin coverslip using transparent epoxy. The cell suspension is delivered to the top of this slide (about 2 ul) and a cover slip is placed over it.

A computer is used both to program the desired thermal protocol and to control the temperature at the thermocouple (by controlling the current going through the resistive heater) to follow that protocol. The temperature measurement can be calibrated by observing the thermocouple output when an ice front in distilled water is held steadily over the thermocouple. Ice formation must be artificially seeded or else the sample will supercool to a signficant degree before spontaneous nucleation occurs. Usually, a wire cooled in liquid nitrogen is held just above (or brought in contact with) the edge of the cover slip. This cools the region of the sample below the wire signficantly but is far from the thermocouple (and hence, the region being observed) so it does not cause artifacts.

One of the main advantages of the convection design is that it allows ice nucleation to be performed at whatever temperature is desired. Such experiments can take a lot of the complexity associated with analysis of events that occur during freezing.

Conduction Cryostage

The conduction cryostage sets up a horizontal temperature gradient by cooling a block of metal over which a slide is mounted. The slide passes over a cylindrical hole in the block, allowing the sample to be seen on the microscope, as well as providing a region that is only cooled by heat conduction through the glass slide. The temperature at the center of the slide is controlled by changing the temperature of the metal block (a refrigerant passes through the block). As with the convection stage, a thermocouple is placed in the center of the field of view to monitor the temperature. Unlike the convection stage, though, there is no need for seeding of ice since it will always form over the cold region above the block. As the temperature of the sample is lowered, ice moves in from the surrounding regions toward the center of the slide.

The main advantage of the conduction design is that it allows for a much steadier temperature at the site of observation. There is also no need for the ice nucleation step, which can be time consuming and difficult.

Directional Solidification Cryostage

The conduction cryostage suggested a further possibility for cryomicroscopy in which the growth rate of ice could be controlled and the interface tracked under the microscope. The directional solidification cryostage is the realization of this idea. The glass slide bridges two metal bases. One is maintained at a higher temperature than the other, but the temperature of both are controlled to develop a linear temperature gradient across the slide. The glass slide is moved at a known velocity that matches the rate of crystal growth corresponding to the temperature gradient.

Although the directional solidification stage was developed to view events at the ice interface, it also provides a mechanism for viewing larger tissue slices that are cooled at constant rates.

Common Problems encountered doing Cryomicroscopy

- focus changes with temperature change (contraction of materials)
- loss of electrical contact
- fogging of lenses
- difficulty seeding ice
- ice growth lifts cover slip - cells move, focus changes
- removal of cover slip
- overheating/slide breakage
- refrigerant flow adjustments

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Document last updated Mar. 22, 1999.
Copyright © 1999, Ken Muldrew.