New Technique for Examining Invertebrate MembranesUsing High Resolution Scanning Electron MicroscopyV.R. TOWNSEND, JR.,* S. R. HAZELTON, B. E. FELGENHAUER, AND J.H. SPRINGDepartment of Biology, University of Louisiana at Lafayette, Lafayette, LA 70504

INTRODUCTIONMuch has been learned from recent advances in re-

search methods (TEM of thick serial sections and com-puter reconstruction, freeze-fracture, freeze-etching)adding to our understanding of intracellular structure.These methods are limited, however, in that they cansurvey only small areas of the cell at one time. Byemploying High Resolution Scanning Electron Micros-copy (HRSEM) to observe cells that have been fixed,frozen, cleaved, thawed, and subjected to cytosol ex-traction, we were able to study broad areas of the cellsin three-dimensional depiction. Although a similartechnique has been applied to mammalian tissue(Tanaka and Naguro, 1981; Lea et al., 1992), this is thefirst demonstration of its usefulness in studying themembrane dynamics of invertebrates.

MATERIAL AND METHODSMalpighian tubules (Mts) were taken from adult fe-

male Acheta domesticus and prepared using a modifiedversion of the protocol described by Lea et al., (1992).Specimens were fixed in (4°C) 2.5% glutaraldehyde in a0.1 M sodium cacodylate buffer for approximately 18hours. Following primary fixation, Mts were washed insodium cacodylate buffer, postfixed in 1% OsO4 for 1.5hours, rinsed with distilled water, placed in 25%DMSO for 30 minutes, and 50% DMSO for 60 minutes.DMSO was employed as a cryoprotectant to prevent icecrystal formation during rapid freezing (Haggis, 1992).Specimens were immersed in a liquid nitrogen slushand fractured using precooled forceps and a razorblade. Specimens were thawed in 50% DMSO, washedin buffer, placed in 1% OsO4 for 1 hr, and then rinsedwith buffer. Specimens were placed in 0.1% OsO4 for3–6 days (for extraction of the cytosol). Following ex-traction, specimens were washed in buffer, placed in1% tannic acid for 1 hr, washed in buffer, placed in 1%OsO4 for 1 hr, washed in buffer, and dehydrated in agraded ethanol series. Specimens were dried using thechemical drying agent, hexamethyldisilizane (in Na-tion, 1983), mounted on aluminum stubs with doublestick tape and sputter-coated with gold for 90 seconds.Specimens were observed with a JEOL 6300-F fieldemission scanning electron microscope at an accelerat-ing voltage of 20 kV.

RESULTS AND DISCUSSIONBest results were obtained with the 5 and 6 day

extractions. In 3 and 4 day, we could only discernnucleus and calcium spherites, but not mitochondria orendomembrane. Earlier investigations using TEM re-vealed that the basolateral membrane of the Mts con-

sisted of series of infolds (Spring and Felgenhauer,1996) and endomembrane located in the perinuclearregion (Hazelton, unpublished observations). In themid-tubule, spherites were present along with manymitochondria. However, due to the two-dimensionalnature of TEM, the relationship between the betweenthe membrane components and the continuity of thesystem was not clear (Fig. 1A). By using freeze-cleav-ing followed by cytosolic extraction, we were able to geta more precise representation of the complexity of themembrane system (Fig 1 B–F). This procedure hasadvantages over previously employed methods (freeze-cleaving, freeze-etching and three-dimensional stereo-scopic TEM reconstruction) in that it is does not re-quire infiltration by resins, post-embedding stainingwith heavy metals, or require stereo pair micrographs.The clarity of the representation of membranes andorganelles is evident when compared with informationobtained through cryo SEM (Bradley, 1989) or TEMreconstruction (Bergeron and Thiery, 1981). The majorstrength of this method is the ease in preparation ofspecimens and the clear, precise representation of themembranes due to the removal of the cytosol.

ACKNOWLEDGEMENTSThe authors thank Dr. T. Pesacreta of the University

of Louisiana at Lafayette Microscopy Center for hishelp.

REFERENCESBergeron M, Thiery G. 1981. Three-dimensional characteristics of the

endoplasmic reticulum of rat renal tubule cells, an electron micros-copy study in thick-sections. Biol Cell 42:43–48.

Bradley TJ. 1989. Membrane dynamics in insect Malpighian tubules.Am J Physiol 257:R967–R972.

Haggis G H. 1992. Sample preparation for electron microscopy ofinternal cell structure. Microsc Res Tech 22:151–159.

Lea PJ, Hollenberg MJ, Temkin RJ, Khan PA. 1992. Chemicalextraction of the cytosol using osmium tetroxide for high resolu-tion scanning electron microscopy. Microsc Res Tech 22:185–193.

Nation JL. 1983. A new method for using hexamethyldisilizane forpreparation of soft insect tissue for scanning electron microscopy.Stain Technol 58:347–351.

Spring JH, Felgenhauer BE. 1996. Excretion in the house cricket,Acheta domesticus. Effects of diuretics on the structure of the mid-tubule. J Morphol 230:43–53.

Tanaka K, Naguro T. 1981. High resolution scanning electron micros-copy of cell organelles by a new specimen preparation method.Biomed. Research (Suppl.). 2:63–70.

Contract grant sponsor: NSF; Contract grant number: IBN 9807948.*Correspondence to: Victor R. Townsend, Jr., Department of Biology, Box

42451, University of Louisiana at Lafayette, Lafayette, LA 70504-2451. E-mail:vrt5719@usl.edu

Received 19 October 1999; accepted in revised form 17 December 19999

MICROSCOPY RESEARCH AND TECHNIQUE 49:209–211 (2000)

© 2000 WILEY-LISS, INC.

Figure 1

Fig. 1. Comparison of ultrastructural features of the cells of theMalpighian tubules of A. domesticus as observed by Transmission Elec-tron Microscopy (TEM) and High Resolution Scanning Electron Micros-copy (HRSEM). A. TEM micrograph of a cross-section of a cell, showingbasolateral folds, mitochondria and spherites. 25003. B-F are resultsobtained by using the HRSEM protocol. B. Cross section of the mid-tubule revealing the monolayer epithelium with an extensive brushborder. 15003. C. Apical surface of a cell showing the abundance ofmicrovilli extending from the apical surface, nucleus, complexity of en-domembrane with mitochondria and spherites dispersed within mem-brane folds. 80003. D. Higher magnification of the brush border.15,0003. E. Basolateral infolds with associated mitochondria and spher-ites. 70003. F. Calcium phosphate spherites with concentric rings thatappear to be separated due to the extraction of the protein matrix at theborder of the lamella. 11,0003. Abbreviations: bl5basolateral infoldings,m5mitochondria, n 5 nucleus, s5spherites.

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