Organic Preservation of Biopolymers in Fossil Leaves - Biopolymers: A Molecular Paleontology Approach - page 45

Microscopic Analyses
Transmission (TEM) and scanning (SEM) electron microscopy were used to identify
the leaf components (cuticles, cellular structure, etc.) and to assess the quality of
leaf morphological preservation. Leaf pieces were taken from the same samples
as those used for chemical analysis. Fossil leaf pieces for electron microscopy were
treated with Hydrochloric acid and Hydrofluoric acid to remove minerals,
especially diatoms which obscured all surfaces in SEM of untreated material and
would also have caused major problems for sectioning work. For SEM leaf pieces
were halved and portions mounted with opposite surfaces upwards. From 2 to
5 mm 2 of surface area were examined for each subsample. Modern samples were
studied without use of fi xatives and some time (weeks) elapsed between collection
and study during which specimens were stored in foil in a refrigerator. For further
details of methods see Collinson et al. ( 1998 ) and references there cited. Interpre-
tations of structure and organisation were informed by comparison with results
previously obtained for other modern and fossil leaves (Collinson et al. 1998 ) .
Organic Geochemical Analyses
Figure 3.1 outlines the analytical strategy in the form of a fl owchart. The modern
and fossil leaf samples were extracted ultrasonically three times, 30 min each with
2:1- CH 2 Cl 2 (DCM): CH 3 OH (methanol) to yield the total lipid extract (TLE). The
insoluble residues were secured in glass vials, dried in a fl ow of N 2 and stored in the
freezer at −20 °C for analysis. The TLE of the modern plants was saponifi ed in
0.5 M 95 % methanoic NaOH for 1 h at 80 °C. The saponifi ed extract was methylated
with borontrifl uoride/methanol (14 % w/v) by heating at 70 °C for 1 h, and collected
in chloroform. The TLE of the modern plants was further separated into fatty acid
(FA), phospholipid fatty acid (PLFA) and neutral fractions using the Bond Elut
separation technique. Bonded aminopropyl solid phase cartridges were conditioned
fi rst with methanol followed by 2:1 v/v DCM: isopropanol. The neutral fraction was
eluted by passing 12 ml 2:1 v/v DCM:isopropanol through the column, followed by
12 ml 2 % acetic acid in diethylether (to elute the FA fraction). Finally, the column
was washed with 12 ml methanol to elute the PLFA fraction. The PLFA fraction was
saponifi ed in 0.5 M 95 % methanoic NaOH for 1 h at 80 °C. The insoluble residues
of the modern plants were saponified in 1 M 95 % methanoic NaOH at 80 °C
for 24 h (Mösle et al. 1997 ) to remove completely the hydrolysable constituents,
e.g. cutin. Microscopic analysis of modern Quercusrobur and Pinussylvestris
showed that the cuticle was successfully removed. Following this the saponifi ed
residues of selected leaves were subjected to acid treatment by heating for 1 h at
80 °C in 1:3, v/v, acetyl bromide-acetic acid to remove the polysaccharide component
of the leaf (Mösle et al. 1997 ).