Methods, Equipment, Chemicals
All specimens where caught by net sweeping. The whole net
contents is killed in fumes of ethyl acetate and transferred to
airtight glass jars and stored in a deep freezer at -22°C for
later examination. Single specimens were put immediately to a deep
freezer. After unfreezing the specimens look like freshly caught.
I usually first make a photo of the whole body of the fresh
specimen, preferably in the pinned state. Proper orientation of
pinned specimens is done with the "entoball" from
firstname.lastname@example.org. To adjust the brightness of the
background I found it sufficient to use a black, grey and white
card, depending on the brightness of the specimen. Whole body
images ususally are focus stacks consisting of ~10 exposures,
photos of details consist of fewer exposures or are not stacked at
all. As I found it more convenient to remove the terminalia and
other parts in the unpinned state, I use folded pieces of card
(black, grey, white) for orientation. Small gnats and midges which
dry and distort too quickly, and material intended for barcoding
is sometimes shown in alcohol, where dusting patterns are
sometimes impossible to show.
Clearing of terminalia, legs, ... is done by leaving the parts
overnight in 10% KOH, then they are tranferred to pure water (~10
min), to glacial acetic acid (~10 min) and finally to glycerol.
When the streaks visible after moving the parts have almost
disappeared (~ 1h), they are transferred to a slide with a rounded
excavation into a drop of glycerol about 1/3 the diameter of the
excavation. I wait again for ~10 min and then start to dissect and
to orient the part under the binocular and make a photo under the
compound microscope. To be able to work without a coverslip, I use
a 20x and 40x Objective from an inverse microscope with a long
working distance (and lower resolution). Parts too large to be
placed under the compound microscope, I'm shooting with the
binocular in a watch glass with incident light.
Clearing the whole specimen, after having documented colours and
dusting, is sometimes useful. The wings should be kept in alcohol,
because they are damaged by KOH and before transferring the
specimen to acetic acid head, legs and terminalia should be
separated to avoid breaking by the quickly forming CO2 bubbles. To
remove gas bubbles the parts are placed in isopropyl alcohol and
then back to glycerol.
For the best possible resolution the dissected parts are embedded
in Malinol (artificial Canada Balsam). Before they are placed for
~1h in 2-propanol (isopropyl alcohol) washing out all hydrophilic
substances. Very small parts I transfer with a glass pipette
from the alcohol to the slide, add an appropriate drop of
mountant, and, after a few minutes, orient the part and add the
coverslip. Care must be taken that the alcohol is not evaporated
before mountant is added.
After a few days the viscosity of Malinol is high enough to
orient the part by moving the coverslip and pressing it down at
one side. This way one can show arbitrary orientations provided
enough mountant has been added. Using too much mountant degrades
the quality of the photos, because high resolution objectives are
calculated for thin samples/coverslips. (The refraction index of
Malinol is very similar to the index of the coverslip.)
Most of the image processing and creation of html stuff has been
automatized by some simple scripts.
The additional software used is Gimp, two components from the
Hugin suite (align_image_stack and enfuse), and exiftool.
For more details see Basic Introduction
and Working at Higher Resolution.