Reversible and Irreversible Effects of Chemical Fixation on the NMR Properties of Single Cells Armin Purea 1 and Andrew G. Webb 2 * The effects of chemical ﬁxation are known to alter MR param- eters, such as relaxation times and the apparent diffusion co- efﬁcient (ADC) of water. It is often assumed that such changes are reversible after samples have been reimmersed in a buffer solution for a sufﬁcient period of time. In this study we charac- terize the changes associated with ﬁxation of single Xenopus laevis oocytes and their subsequent reimmersion in buffer. Sub- stantial reductions in both T 1 and T 2 values were measured for all compartments of the cell after ﬁxation, with the cytoplasm showing larger changes than the nucleus. After reimmersion in buffer, there were small but statistically signiﬁcant differences in MR parameters between fresh and reimmersed cells. Exper- iments with a gadolinium (Gd) contrast agent showed evidence of irreversible changes in the permeability of cellular membranes to small molecules. Magn Reson Med 56:927–931, 2006. © 2006 Wiley-Liss, Inc. Key words: magnetic resonance histology; chemical ﬁxation; relaxation times; membrane permeability; diffusion coefﬁcient The term “magnetic resonance (MR) histology” (1) typi- cally refers to the acquisition of high-resolution images, often with isotropic resolution in three dimensions, of ex vivo samples that have been chemically ﬁxed for tissue preservation. Although MR histology has a spatial resolu- tion on the order of tens of microns, which is much infe- rior to light and other forms of microscopy, it offers the advantages of true 3D data acquisition, a lack of morpho- logical changes associated with tissue dehydration and sectioning, and reduced data-processing time compared to histological sectioning. The noninvasive nature of MR his- tology is particularly important when the samples are unique and irreplaceable (e.g., samples that have been collected over a long period of time (2)). As in many types of microscopy studies, tissue speci- mens for MR histology are typically ﬁxed in buffered so- lutions of formaldehyde, glutaraldehyde, or a mixture of both. This chemical ﬁxation destroys autolytic enzymes, preserves tissue from decomposition due to bacteria or molds, and adds rigidity to very fragile tissue. Formalde- hyde dissolves in water to form methylene hydrate (HO- CH 2 -OH), which in its monomeric form is the active ﬁxa- tion agent. Formaldehyde’s mechanism of action is based on the reaction of the aldehyde group with primary amines in proteins. The initial reaction of formaldehyde with pro- tein is complete within 24 hr, but the formation of cross- links, or methylene bridges, between neighboring proteins (if the primary amines are close enough together) can take several weeks. Since formaldehyde penetrates tissue rap- idly, the rate-limiting step for ﬁxation is the cross-linking process. Substances such as lipids, nucleic acids, and car- bohydrates are not chemically modiﬁed unless ﬁxation is prolonged for several weeks, but can become trapped in a matrix of insoluble and cross-linked proteins. Another common ﬁxative, glutaraldehyde, has two aldehyde groups separated by three methylene bridges, which greatly increases the cross-linking compared to formalde- hyde. Glutaraldehyde reacts with proteins more rapidly than formaldehyde, but its physical penetration through tissue occurs more slowly. For example, a rat brain left in buffered glutaraldehyde solution for 12 hr shows pene- tration of only 2–3 mm. In the case of glutaraldehyde, there may also be unreacted aldehyde groups left over that can- not be removed from the tissue by washing. This is a major concern for MR studies, as discussed below, since these protons can potentially exchange with water even if all of the free ﬁxative has been washed out of the tissue. The unreacted aldehyde groups can potentially be “blocked” by treatment with glycine or similar small molecules with a primary amine group. The kinetics of formaldehyde and glutaraldehyde ﬁxation have traditionally been studied using radioactive isotopes (3,4), although recently MRI has also been used (5). Given the importance of ex vivo MR studies, many re- searchers have investigated how chemical ﬁxation affects MR parameters, such as relaxation times and diffusion coefﬁcients. In general, T 1 and T 2 relaxation times have been found to decrease upon chemical ﬁxation, with T 2 affected more than T 1 to a signiﬁcantly greater degree. The major mechanism for this reduction is chemical exchange between water and the protons in the aldehyde group of the ﬁxative. For example, Bossart et al. (6) showed that adding ﬁxatives to water has a relatively small effect on the water T 1 , but signiﬁcantly decreases the T 2 (a 2% formal- dehyde solution has a T 2 of 40 ms at 600 MHz, which drops to 8 ms for 8% ﬁxative; the corresponding T 1 values are 3.3 and 3.1 s, respectively). Many groups have performed experiments on human and animal tissue in an attempt to understand the altered contrast mechanisms in ﬁxed tissue compared to in vivo imaging, and to optimize the imaging parameters for ﬁxed tissue samples (6 –10). Since tissue heterogeneity makes it difﬁcult to deter- mine the exact mechanism of chemically induced changes, a number of simpliﬁed models have been developed. Tis- sue models based on erythrocyte ghosts (11) derived from human blood are a particularly elegant means of investi- gating changes in relaxation and diffusion properties, es- 1 Department of Experimental Physics 5, University of Wu¨ rzburg, Wu¨ rzburg, Germany. 2 Department of Bioengineering, Penn State University, University Park, Penn- sylvania, USA. Grant sponsor: Wolfgang Paul-Preis, Alexander von Humboldt Stiftung. *Correspondence to: A.G. Webb, Department of Bioengineering, Room 315, Hallowell Building, Penn State University, University Park, PA 16802. E-mail: agw@engr.psu.edu Received 14 November 2005; revised 14 June 2006; accepted 19 June 2006. DOI 10.1002/mrm.21018 Published online 29 August 2006 in Wiley InterScience (www.interscience. wiley.com). Magnetic Resonance in Medicine 56:927–931 (2006) © 2006 Wiley-Liss, Inc. 927