The use of
confocal laser scanning microscopy (CLSM) for the study of inclusions in amber
is a recent development. Böker & Brocksch (2002) produced a series of images of
insects in Baltic amber using CLSM and identified the potential for 3D imaging
of minute detail of taxonomically important morphological structures such as the
mandibles and genital organs. Since then, however, very little has been
published on CLSM analysis of amber inclusions despite the apparent benefits.
using this technique, amongst other microscopic techniques, looked at some
Spanish amber from Álava (Ascaso et al. 2003, 2005). The study looked at a
protozoan with fungal hyphae trapped in amber and produced a 3D image based on a
series of optical sections recorded by the CLSM. It is perhaps surprising,
considering the detail and quality of the images that can be produced, that more
studies have not incorporated this technique. More recently, Speranza et al.
(in press) have used light microscopy, CLSM & widefield fluorescence microscopy
to image microscopic fungi embedded in amber, thus confirming the benefits of a
present in the vast majority of angiosperms and have been considered for some
time to be of importance in comparative systematic studies (Theobald et al.
1979). They have an important role as defensive structures, especially in
repelling phytophagous insects (Levin 1973). There is often more than one
trichome type on any one taxon, but certain types may be more common to one
taxon than another (Theobald et al. 1979). The taxonomic value of trichomes is
therefore limited, but the Baltic trichomes are not inconsistent with them being
from a type of oak.
In this study,
certain elements of the microflora are examined. The “stellate hairs”, or
trichomes, are common in Baltic amber and have been considered as a
characteristic of this type of amber (Weitschat & Wichard 2002). These trichomes
are found associated with, and attached to, the male oak flowers and are
therefore thought to belong to oak also (Weitschat & Wichard 2002) although
there may be more than one type of trichome present. During this study samples
from Chiapas, Mexico were also examined and were found to contain abundant
trichomes. No previous record of trichomes in Mexican amber has been found in
Wichard (2002) describe the ‘stellate hairs’ found in Baltic amber as structures
that develop on the flower and leaf buds of the oak that are shed in great
numbers every year. They also state that no studies have been able to clarify
the origin or their significance for amber.
interpretation of what the inclusions in Baltic amber represent in terms of
their ecology has been the subject of much debate as many of the insect
inclusions suggest a wide range of biotopes (Weitschat and Wichard 2002). Even
individual pieces of amber seem to contain species from what would currently be
both temperate and tropical regions (Weitschat 1997). This may suggest that
species represent within the amber could have had a wider range then than their
extant relatives (Weitschat and Wichard 2002).
In the present
study we present a technique which uses CLSM and 3D imaging to analyse the
structure of trichomes.
Amber from the
Baltic Region is well known for its trichome inclusions. One amber nugget was
sliced and polished to allow examination of the trichomes (GLAHM 114416/3).
A piece of
Mexican amber from the La Quinta Formation (Middle Miocene) from the mines in
the municipality of Simojovel of the Chiapas region in southern Mexico
containing numerous trichomes was sliced and polished (GLAHM 131494/3). The vast
majority of inclusions in the Mexican amber are small flying insects belonging
to the dipteran order (Solorzano Kraemer 2007). Solorzano Kraemer suggested that
the fauna showed a close similarity with the amber inclusions of the Middle
Miocene deposits of the Dominican Republic and Hispanola as a whole. It was
concluded that the ecology was typically that of a lowland tropical dry forest
with elements suggesting more open forest with a mangrove region. The plant that
is thought to have produced the resin that became the amber in Hispanola is the
angiosperm Hymenaea protera (Iturralde-Vinent and MacPhee 1996, Poinar et al.
amber in the collections of the Hunterian Museum, University of Glasgow (GLAHM)
were examined using a Nikon SZM-2T trinocular stereoscopic zoom microscope to
determine the abundance of trichome inclusions. The samples with the most
trichomes were then carefully sliced using a fine diamond saw at slow speed
close to the visible trichomes. This was done to maximise the number of
trichomes close to the cut surface. The surface was then finely polished. We
imaged trichomes within 200µm of the surface to accommodate the working distance
of high power objectives and reduce the optical aberrations caused by the
mismatched refractive index of amber (~1.5) and air (1). Penetration of the
amber by the laser beam was dependant on the colour and clarity of the amber
analysis was performed using a BioRad Radiance 2100 fitted to a Nikon Eclipse
TE300 inverted microscope. Optimum excitation and emission parameters were
sought to maximise the signal to background ratio. Since amber is known to
exhibit UV auto-fluorescence we confined our investigations to the green-red end
of the spectrum. Optimum imaging was achieved using the red-diode 637nm line
with a 660LP emission filter. Pinhole radius was set to 1.1mm. A single
trichome was chosen from the Baltic amber and was scanned using 2.6 zoom, 10X
objective and 1024 x 768 pixel resolution. A group of several trichomes from
Mexican amber were also scanned using 1.2zoom, 10X objective (NA 0.5) and 521 x
512 pixel resolution. Lambda scanning was used to produce images which were the
average of 3 scans (i.e. Lambda=3) The CLSM produced 2D image slices by varying
the plane of focus in the z-axis at 1µm increments. The images collected were
8-bit greyscale. Optical sections (2D) were pre-processed in MetaMorph prior to
being reconstructed in 3D using AMIRA. The software produced a surface-rendered
image of the trichomes. The images of the Mexican trichomes were then compared
to light microscope images taken of the same trichomes immersed in Johnson’s
Baby Oil. The oil immersion helps to reduce reflections caused by internal
fractures and because of the similar refractive indices of the amber and oil (Crighton
& Carrió 2007).
both Baltic and Mexican amber exhibited the optimum signal to background noise
ratio when excited with a wavelength of 632nm and collected 660nm. This produced
excellent image data which facilitated sharp thresholding and segmentation and
in turn enabled surface rendering in AMIRA (figure 1).
1. Stereo pairs of Baltic trichome (GLAHM 114416/3) showing (a) visible side,
and (b) opposite side not seen from top surface of polished amber (scale =
2. Outline of Baltic trichome (GLAHM 114416/3) with indication of number of
radii (scale = 0.2mm).
in Baltic amber also fluoresced at lower wavelengths, but not as pronounced. The
amber immediately surrounding the Baltic amber trichomes also fluoresced
particularly in the green wavelengths. The Baltic trichome chosen had a single
long radius and 16 shorter radii (figures 1, 2; animation 1).
chosen in this study from the Mexican amber form a group of linked stellate
trichomes (figures 3, and 4; animation 2) of eight to ten radii in one plane
(rotate – terminology used is according to Theobald et al. 1979) with a central
disk (slightly lepidote?) from which the radii eminate. Although some of the
trichomes studied had less than ten radii, the form of these trichomes is
similar to that found in the Euphorbiacean angiosperm Croton (Webster et al.
3. Trichome group in Mexican amber showing (a) visible side using CLSM, and (b)
the same group using a transmitted light microscope (scale = 0.2mm).
4. Trichome group in Mexican amber (GLAHM 131494/3) showing (a) opposite
side using CLSM, and (b) the same group in outline with three trichomes with
their radii coloured (scale = 0.2mm).
An attempt was
also made to look at insect inclusions using the same method following the
success of Böker & Brocksch (2002). The insects did not fluoresce in the far red
as the trichomes did, but in the ultraviolet part of the spectrum. As the insect
inclusions were generally larger than the trichomes, it was more difficult to
produce a 3D image as the inclusions extended deeper than the imaging
capabilities of our system and were larger than the field of view of the
objective. Using a lower power objective may help by increasing the field of
view, but there may still be a problem with the penetration. The amber also
fluoresced in the ultraviolet, making it difficult to distinguish the insect
from the amber. The white fungus commonly found in Baltic amber fluoresced
differently from the surrounding amber and was clearly visible (figure 5).
5. Beetle in Baltic amber (GLAHM 114409) shown using (a) natural transmitted
light and (b) ultraviolet fluorescence. The white fungus (white arrow) on the
cuticle of the beetle (a) fluoresces slightly more in ultraviolet than the amber
(b). Flow structures (black arrows) of the original resin can also be seen in
the amber using ultraviolet (scale = 1mm).