Low temperature light microscopy and its application to study freezing in aqueous solutions and biological cell suspensions

Körber, Ch.; Englich, S.; Schwindke, P.; Scheiwe, M.W.; Rau, G.; Hubel, Allison; Cravalho, E.G.

doi:10.1111/j.1365-2818.1986.tb02721.x

Cited by 15 publications

(7 citation statements)

References 28 publications

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“…The solutions were first degassed, and the gas content was then adjusted prior to the experiment. Nucleation, growth and encapsulation were observed and evaluated dynamically by means of a cryomicroscope in combination with a video-recording system and digital image processing (Korber et al 1984(Korber et al , 1986. Two types of freezing stages were used, one of them yielding a constant and…”

Section: Experimental Results and Observationsmentioning

confidence: 99%

Phenomena at the advancing ice–liquid interface: solutes, particles and biological cells

Körber

1988

Quart. Rev. Biophys.

136

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Ice formation in aqueous solutions and suspensions involves a number of significant changes and processes in the residual liquid. The resulting effects were described concerning the redistribution of dissolved salts, the behaviour of gaseous solutes and bubble formation, the rejection and entrapment of second-phase particles. This set of conditions is also experienced by biological cells subjected to freezing. The influences of ice formation in that respect and their relevance for cryopreservation were considered as well. A model of transient heat conduction and solute diffusion with a planar ice front, propagating through a system of finite length was found to be in good agreement with measured salt concentration profiles. The spacing of the subsequently developing columnar solidification pattern was of the same order of magnitude as the pertubation wavelengths predicted from the stability criterion. Non-planar solidification of binary salt solutions was described by a pure heat transfer model under the assumption of local thermodynamic equilibrium. The rejection of gaseous solutes and the resulting gas concentration profile ahead of a planar ice front has been estimated by means of a test bubble method, yielding a distribution coefficient of 0.05 for oxygen. The nucleation of gas bubbles has been observed to occur at slightly less than 20-fold supersaturation. The subsequent radial growth of the bubbles obeys a square-root time dependence as expected from a diffusion controlled model until the still expanding bubbles become engulfed by the advancing ice-liquid interface. The maximum bubble radii decrease for increasing ice front velocities. The transition between repulsion and entrapment of spherical latex particles by an advancing planar ice-front has been characterized by a critical value of the velocity of the solidification interface. The critical velocity is inversely proportional to the particle radius as suggested by models assuming an undisturbed ice front. The increase of the critical velocity for increasing thermal gradients shows good agreement with a theoretically predicted square-root type of dependence. Critical velocities have also been measured for yeast and red blood cells. The effect of freezing on biological cells has been analyzed for human lymphocytes and erythrocytes. The reduction of cell volume observed during non-planar freezing agrees reasonably well with shrinkage curves calculated from a water transport model. The probability of intracellular ice formation has been characterized by threshold cooling rates above which the amount of water remaining within the cell is sufficient for crystallization.(ABSTRACT TRUNCATED AT 400 WORDS)

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Section: Experimental Results and Observationsmentioning

confidence: 99%

Phenomena at the advancing ice–liquid interface: solutes, particles and biological cells

Körber

1988

Quart. Rev. Biophys.

136

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“…As both experimental techniques, light cryomicroscopy (Scheiwe & Korber, 1982cKorber et al, 1986;Korber, 1988;Englich, 1988) and low-temperature DSC Jochem & Korber, 1987;Englich, 1988), have been described previously in detail, only a short outline is given here with mention of the most important steps and parameters.…”

Section: Materials a N D M E T H O D Smentioning

confidence: 99%

Intracellular ice formation: cryomicroscopical observation and calorimetric measurement

Körber

Englich

Rau

1991

Journal of Microscopy

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, differential scanning calorimetry, human lymphocytes, darkening and twitching types of intracellular ice formation, freezing injury, threshold cooling rate, crystallization temperature, influence of the cryo-additive dimethyl sulphoxide, nucleation mechanisms.The formation of ice crystals within biological cells is generally deleterious and results in a severe loss of cellular viability and function. With the aim of circumventing this lethal event, the mechanisms of nucleation and their dependence on governing parameters such as temperature, cooling rate and solute and/or additive concentration, and the correlation with the osmotically induced water transport across the cell membrane were investigated. Quantitative low-temperature light microscopy was used for this purpose as it offers the major advantage of studying the dynamics of the involved processes. T o substantiate further the visual observations of the morphological changes associated with intracellular ice formation, supplementary studies by differential scanning calorimetry (DSC) were performed under comparable conditions to measure the quantity of water actually transformed into the crystalline state due to the evolution of latent heat.Human lymphocytes were used as a biological model cell. In particular it could be shown that the twitching type of intracellular ice formation which is evident but difficult to observe under the cryomicroscope can be attributed to a liquid-solid phase change within the cells as determined by DSC. Good agreement was obtained between the results measured by both techniques with respect to the following dependencies of governing parameters: the fraction of cells exhibiting intracellular ice determined as a function of the cooling rate shows a sharp demarcation zone with an increase from 0 to loo",, at about the same threshold cooling rate. On the other hand, the temperatures at which intracellular ice forms were found to be only weakly dependent on the cooling rate. With respect to the effect of cryo-additive concentration at a fixed value of the cooling rate, the crystallization temperatures were seen to decrease with concentration. T h e D S C results may hence be regarded as a validation of the microscopic observations.

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“…Carte,8 as well as Bari and Hallett,9 discussed the formation air bubbles in ice, hoping that the study can help explain the formation of hailstone. Kõrber et al 10 found that air bubbles were formed in cooling cell suspensions. This is harmful in the preservation of cells and tissues, since the existence of air bubbles may cause different stress distributions in the ice and lead to the braking of ice, which may kill the preserved cells.…”

Section: Resultsmentioning

confidence: 99%

Microscopic Study of Crystal Growth in Cryopreservation Agent Solutions and Water

Liu

Hua

2002

Annals of the New York Academy of Sciences

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Ice formation inside or outside cells during cryopreservation is evidently the main factor of cryoinjury to cells. In the study described here a high voltage DC electric field and a cryomicroscopic stage were used to test DMSO and NaCl solutions under electric field strengths ranging from 83 kV/m to 320 kV/m. Dendritic ice crystals became asymmetric when the electric field was activated. This change in the ice crystal shape was more pronounced in the ionic NaCl solution. In addition, ice growth of distilled water without an electric field was tested under different cooling rates.

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Low temperature light microscopy and its application to study freezing in aqueous solutions and biological cell suspensions

Cited by 15 publications

References 28 publications

Phenomena at the advancing ice–liquid interface: solutes, particles and biological cells

Phenomena at the advancing ice–liquid interface: solutes, particles and biological cells

Intracellular ice formation: cryomicroscopical observation and calorimetric measurement

Microscopic Study of Crystal Growth in Cryopreservation Agent Solutions and Water

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