© 2005 The Royal Microscopical Society Journal of Microscopy, Vol. 218, Pt 1 April 2005, pp. 79–83 Received 30 August 2004; accepted 7 January 2005 Blackwell Publishing, Ltd. SHORT TECHNICAL NOTE A fast method to study the secretory activity of neuroendocrine cells at the ultrastructural level F. VAN HERP, T. COENEN, H. P. M. GEURTS*, G. J. A. JANSSEN* & G. J. M. MARTENS Department of Molecular Animal Physiology, Nijmegen Center for Molecular Life Sciences (NCMLS) and * Department of General Instrumentation, Faculty of Sciences, Radboud University Nijmegen, The Netherlands Key words. Cryo-FE-SEM, cryo-preparation, cryotransfer system, field emission scanning electron microscope (FE-SEM), neuroendocrine cells, POMC-producing intermediate pituitary melanotrope cells, secretory activity, Xenopus laevis. Received 30 August 2004; accepted 7 January 2005 Summary Cryo field emission scanning electron microscopy (cryo-FE- SEM) is a versatile technique that allows the investigation of the three-dimensional organization of cells at the ultrastruc- tural level over a wide range of magnifications. Unfortunately, cryopreparation of the specimens for this technique remains cumbersome, in particular because ice crystal formation must be prevented during freezing. Here we report that a light prefixation with glutaraldehyde and incubation in glycerol as cryoprotectant or a high-pressure freezing approach are both excellent procedures for cryopreparation of animal cells to be used in combination with cryo-FE-SEM. Using the proopiomelanocortin-producing intermediate pituitary mel- anotrope cells of Xenopus laevis as a physiologically inducible neuroendocrine system, we compared the ultrastructural characteristics of inactive and hyperactive neuroendocrine cells. The overall quality of the ultrastructural images was comparable for the two cryopreparation procedures, although some fine structures were better conserved using high-pressure freezing. Melanotrope cells in a secretory inactive state contained numerous storage granules and a poorly developed endoplasmic reticulum (ER), while large amounts of rough ER were present in hyperactive cells. Thus, the cryo-FE-SEM approach described here allows a fast ultrastructural study on the secretory activity of neuroendocrine cells. Introduction The scanning electron microscope (SEM) has been widely used in biomedical sciences as it provides vivid, seemingly three-dimensional images. Nevertheless, transmission elec- tron microscopy (TEM) has been primarily used for research on the fine structure of cells, because the resolution of conven- tional SEM is markedly inferior to that of TEM. By contrast, preparation of the biological specimens for TEM is technically complicated, time consuming (up to weeks) and the specimens must pass through several steps in the procedure (e.g. fixation, embedding and ultramicrotomy). With the development of the field emission SEM (FE-SEM) a considerable improvement in resolution could be achieved (Pawley, 1983) and cellular structures can be investigated with a resolution of a few nano- metres in a near life-like state. Furthermore, the development of first cryo-SEM and subsequently cryo-FE-SEM has allowed a simplified protocol for specimen preparation (for a review see Echlin, 1992). In cryo-FE-SEM, a cryotransfer chamber is directly coupled to the microscope and enables fracturing, pre- cise etching and coating of the sample within a few minutes and without the need to move the sample. This rather novel technique is a good alternative approach to the freeze- fracture/freeze-etch replication method for TEM. For freezing of native material, a light prefixation with glutaraldehyde and incubation in glycerol as cryoprotectant (Haggis et al., 1983) or high-pressure freezing (Walther, 2003) should be considered as the method of choice for cryo-preparation of the biological samples, as they prevent ice crystal formation during freezing and eliminate artefacts that would lead to misinterpretation of the ultrastructural results. The combination of high-pressure Correspondence to: Dr F. Van Herp. Tel.: +31 24 3610566; fax: +31 24 3615317; e-mail: F.vanHerp@science.ru.nl