This section will describe how to take digital images of excellent quality with cheap equipment. The tricks described below enable us to take images similar in sharpness and resolution to pictures taken with significantly more expensive equipment; we basically have the cheapest Nikon microscopes and digital cameras. The techniques described here are ideas taken from other Arachnologists and provided by my students. If you find improvements please share them.
All images were taken with the following Nikon equipment. The first workstation has two digital cameras a DS-Fi1 connected to the dissecting microscope SMZ1270 and a DS-Fi2 connected to the glass slides microscope E200, both cameras are controlled with a desktop Dell Inspiron 660s. The second work station has a SMZ1000 and E200 microscopes, both sharing a DS-Fi3 connected to a HP Slimline 270-a0xx. Illumination was provided with custom made lamps made of two 1.5 watts LEDs of 2 cm in diameter. The LEDs are mounted at the end of flexible tubes on aluminum heads. The maximum size of the images in pixels is width 2560, height 1920, with a resolution of 300 pixels/inch. I recommend buying a digital camera special for microscopes, but any digital camera should work.
Small lenses produce sharper images, but with very narrow depth of field. The 10X and 20X objectives of a regular microscope for glass slides produces images considerably sharper than a dissecting microscope at the same magnification.
The depth of field problem is resolved by combining several images at different focal lengths. We combine on average 50 images to produce one combined picture. The stack of images is taken by hand, keeping track of how the depth of field overlaps from top to bottom. More expensive equipment takes the stack of images automatically with similar results.
We have used Helicon Focus for several years to combine stack images and highly recommend it, the combined images didn't require editing, or in some cases very little. This is important when thousands or images are required for web pages.
Images Composition Rules
This list will provide the rules to make the image composition constant among inventories.
The Petri dish with sand. This has been known for many generations of Arachnologists and its practical value is appreciated when hundreds of specimens need to be sorted and photographed. Cover the specimen partially in the sand until the desired position is maintained and remove alcohol until the surface is close to the specimen, a lot of alcohol will result in blurry images.
Microscope for preparations setup
Temporary slide (top right). This temporary slide is used only for cleared structures either with clove oil or methyl salicylate. Jonathan Coddington developed it and its thoroughly described in a book chapter difficult to find; therefore, a brief description is provided here. Paste several glass slide covers on one glass side for microscopes, the height of this covers stack must be slightly taller than the structure to observe. Cut a narrow glass stripe, from a long slide cover, and paste it with petroleum jelly (Vaseline) to the top of the stack of slide covers. Add a drop of clove oil or methyl salicylate to unite both pieces of glass and cover the specimen.
The cleared specimen must be submerged in alcohol or clove oil at all times to avoid bubbles. Move the specimen, with a transfer pipette, between the microscope slide and the tallest part of the narrow glass stripe. Manipulate under a dissecting microscope the structure until the desired view is maintained; only then transfer it to the microscope for slides. Tiny adjustments can de made by gently taping the long slide cover, but major changes must be done in the dissecting microscope. Check that the width of the glass stripe protects the objective form the clove oil.
The Tiny Dish (bottom right). This preparation setup is used for non-cleared structures or specimens less than 1 mm. Get a regular microscope glass slide and paste a plastic lid in the middle as in the "Preparations setup" image. The plastic lid has to be white or translucent. The diameter has to be wide enough to allow the objective lens to enter and tall enough to contain enough alcohol to cover the specimen. Our plastic lid has a internal diameter of 1.53 mm, external of 1.66 mm and a height of 0.9 mm. Illumination is provided with a led lamp.
Looking through a dissecting microscope paste a drop of petroleum jelly in the bottom of "The Tiny Dish". The surface must be dry, otherwise the jelly will never stick; only then: fill the dish with alcohol, remove jelly to get the required shape to receive the specimen and paste it. Cover the dish with a glass slide because the alcohol evaporates very fast. Transfer The Tiny Dish to a microscope for slides preparations and remove the cover to take the images. The petroleum jelly is completely removed from the specimen by shaking it for 15 seconds in a vial with chloroform or ether.
Alcohol base gels are not recommended because of: bubbles, these gels have a light refraction index that creates more optical distortions than the air-alcohol transition, cover completely the specimen and are impossible to thoroughly remove.