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with excess Mel at 5°o vernight. The reaction mixt was evapd to dryness and the residue was crystd from Me,C0-Et,0 to furnish colorless needles of VIb in almost quantitative yield: mp 195°; the quaternary 7V-Me group signaled at 3.30 (9 H, s) in the nmr (D,0) spectrum.2,3-Dihydrohelenalin (II). 2,3-Dihydrohelenalin could be readily synthesized in quantitative yield from any one of the following 3 methods.(a) A soln of VIb (30 mg) in , (1 ml) was heated on a steam bath for 30 min. The reaction mixt was acidified with 5% HC1 and extd with CHCl,, washed with , , dried (NajSOJ, and evapd. The residue was purified in CHCl, by passing through a column of silica gel (0.6 X 3.5 cm) to provide II as fine colorless silky needles after one recrystn from CH,Cl,-hexane: mp 154-155°; nmr 4.88 (1 H, t, /= 7.5 cps, H-8), 4.30 (1 H, d,/= 3 cps, H-6), 1.08 (3 H, d,/ = 6 cps, C10-CH,) and 0.78 (3 H, s, C,-CH,).(b) A soln of VIb (67 mg) in MeOH (20 ml) was treated with excess of freshly prepared Ag,0 and stirred for 30 min at room temp. The reaction mixt was filtered and evapd in vacuo to yield II as an oil. This was chromatogd in CHCl, on silica gel (0.3 X 3 cm) to give colorless needles (35 mg) after one recrystn from PhH-EtOH: mp 154-155°.(c) A soln of VIb (18 mg) in 5% aq NaHCO, soln (5 ml) was stirred at room temp for 1 hr. The reaction mixt was acidified with dil HC1 and extd with CHCl,. The CHCl, ext was washed with , , dried (NajSO,), and evapd to yield 10 mg of II as colorless needles after 1 recrystn from benzene-EtOH: mp 154-155°.Helenalin from Method b. Treatment of Vb (20 mg) in a similar manner as described for II (method b) afforded I as colorless needles. The identity of this compd with helenalin was established by tic, ir comparison, and mmp determination.11.13-DihydrohelenaUn (III) was prepd according to the method of Adams and Herz.4 2.3.11.13-Tetrahydrohelenalin (IV) was prepd by the method of Clark5 and melted at 171-173°.Helenalin Piperidine Adduct (Vg). A soln of helenalin (I) (100 mg) in freshly distd piperidine (1 ml) was allowed to stand at room temp overnight. The reaction mixt was dild with H,0 and extd with CHCl,. The CHCl, layer was washed with , , dried (Na^OJ, and evapd under reduced pressure to yield a residue (30 mg) which crystd upon addn of Et,0. Recrystn from CH,C1,-Et,0 gave colorless

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The scanning electron microscope appearances and shrinkage of blocks of canine endocardium prepared by freeze-drying directly, by freeze-drying after replacing tissue water with tertiary butanol (2-methyl propan-2-ol) and by critical point drying were compared. All three methods demonstrated endothelial cells which showed nuclear prominences, microvilli and intercellular boundaries. The microvilli varied in size and number from dog to dog but were generally less well defined in specimens freeze-dried from water. Shrinkage due to t-butanol dehydration was significantly less than that which occurred in ethanol in the critical point drying method. Overall the reduction in surface area was significantly less in specimens freeze-dried directly at -65 C (6.8%) than in those dried from t-butanol at -20 C (15.4%) and those prepared bly critical point drying (22.1%). However the amount of shrinkage observed in t-butanol treated tissue was not significantly different from that which was critical point dried. It was not possible to distinguish between comparable samples prepared by these two methods on the basis of their scanning electron microscopic appearances. Thus the relative simplicity and convenience of the t-butanol method, together with its saving of time, its use of standard freeze-drying equipment and the avoidance of ice-crystal artefact justify its consideration as an alternative method of preparing wet biological tissue for scanning electron microscopy.