Molluscan Research: Techniques for collecting, handling, preparing ...

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Most digital SLR cameras can be connected to a computer
and the monitor image provides adequate resolution to verify
focus after the image has been taken.
Intermediately priced compact digital cameras with
supposed macro capability generally produce inferior results.
As an example, the Nikon Coolpix 8000 has a
macrofunction, flash and adjustable f-stop. However, the
maximum f-stop is f/8 (as opposed to f/22 on all dedicated
macrolenses), making the depth of field very shallow; at
closest focus, the flash does not illuminate the image area,
because the lens barrel produces a shadow; and the LCD
screens on the camera as well as in the view finder do not
permit accurate focus adjustments.
Improper file manipulation (e.g., working on .jpeg
rather than .tif/psd files, or in CMYK rather than RGB/Lab
colour space, or in 8 bit rather then 16 bit per channel, if
available) will produce inferior results. Please consult
appropriate works on digital imaging for further information
(e.g., Davies and Fennessy 2001; Sedgewick and Sedgewick
2002). The little known Lab colour space offers particular
advantages for un-sharp masking of colour images. In the L-
channel, the sharpening will only have effects on the
brightness value of the pixels, while not affecting their
colour value stored in the a and b channels (see Margulis
2005). In RGB, the brightness and colour values are a joint
value in each of the R, G and B channels and sharpening can
lead to colour artifacts.
Furthermore, particularly in flash-photography, the
exposure meter assumes 18% reflection, hence, exposure
compensation is often required. For instance, when
photographing a white shell against bright background, the
automatic exposure will assume that 18% of light is reflected
and produce a dull-grey image. Thus the photographer has to
instruct the camera to overexpose the image to obtain the
true white of the shell (see Geiger 2006b for step-by-step
instructions). The black box of matrix metering may increase
the percentage of acceptable images, but will inevitably lead
to failures. A thorough understanding of exposure and
exposure compensation is imperative. Many of these
adjustments can also be accomplished afterwards with digital
image manipulation, but the final result will be affected by
the quality of the source files.
The Bayer pattern of most digital cameras (CCD,
CMOS sensors) is a significant issue for digital colour
photography, as 2/3 of the colour information in each image
is interpolated. Three layer photo-sensors such as the Foveon
X3, currently only available in Sigma cameras, and co-site
sampling technique as implemented in the Zeiss microscope
camera Axiocam HRc, have overcome this limitation. The
opinions on the Foveon X3 chip are divided, as it has a lower
resolution compared to current CCDs and CMOS sensors,
while on the other hand, the larger pixels have a better
signal-to-noise ratio and capture all three colours at each site.
The signal-to-noise question also applies to the issue of 2/3
vs full-size digital sensors; at the same number of pixels, a
larger chip has larger pixels and a better signal-to-noise ratio.
Three chip cameras also avoid the Bayer pattern problem,
but light intensity reaching each sensor is only one third of
GEIGER ET AL. (2007) MOLLUSCAN RESEARCH, VOL. 27
the original intensity because of the beam-splitter. The lower
light levels will cause somewhat elevated signal-to-noise
ratios. The latter can be improved by cooling of the imaging
chip. The two main cooling methods are fan and Peltier
stage. Because Peltier devices have no moving parts, they
cannot produce any vibrations, in contrast to a fan.
Stereo-microscope
For magnifications above 5x, a stereo-microscope with
photo attachment is advisable. Models with trinocular heads
or a dedicated photo-tube are preferable over ocular-mounted
systems. All photo-ports of modern stereo-microscopes use
only one of the light paths and, as the two light paths are at
an angle for stereoscopic viewing, lateral image shifts occur
when changing focus regardless of whether the
stereomicroscope is of Greenough or Telescope design.
Some instruments can counteract this image shift with
special attachments (e.g., Zeiss Discovery V8, V12 with
objective slider), which moves the objective so that the lightpath
is in line with the photo-tube.
There are some older models designed for photography
(e.g., Wild M400 series, Zeiss Tessovar system). These
microscopes look like a stereomicroscope, although they
only have a single light path, hence true stereoscopic viewing
of specimens is impossible. As they are primarily intended
for imaging, this design feature should rather be viewed as an
asset than a deficiency.
Lenses for stereo-microscopes come in many different
quality ranges. Plan-apochromatic lenses produce flat images
and are fully colour corrected, but are also expensive. Plan
lenses are corrected to produce a flat imaging plane, but may
show pronounced colour fringes (= lateral colour, e.g., Leica
Plan 1x with yellow/blue fringes). In some cases, the image
plane is distinctly curved, resulting also in apparent
distortion of the object. One can test the image flatness and
distortion by photographing graph paper with 1 mm ruling;
ideally the image is sharp from the center to each corner and
the lines are exactly parallel to the edge of the image.
Lighting
Some of us prefer continuous light (incandescent,
fluorescent, LED) with long exposure times. The lighting is
more predictable, because the effect of any changes can be
observed in real time. Issues with colour temperature of the
light can either be addressed with colour filters or with a
custom white-balance in digital systems. Some of us prefer
flash photography because the ultra short exposure time
eliminates any possibility of vibrations (shutter and mirror in
SLR cameras, fan of fiber optics illuminators, person moving
in room) which may deteriorate the image sharpness and the
colour temperature is a well-defined. With some experience,
the results are equally predictable and, with digital capture,
rapid assessment of the results is possible. Use a high-power
flash unit, as the flash duration is proportional to the
discharge proportion: at 10% discharge, the flash duration is
around 1/10,000 s, whereas a full discharge will take
approximately 1/200 s. Some portable flashes have built-in
focusing lights and studio strobe systems generally have both