Laboratorio de Aracnología   Facultad de Ciencias UNAM


Microscopy methods

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.

Microscope for preparations setup

Microscope tricks
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.

The digital images are taken with a Nikon DSU3 camera connected to a standard Dell Inspiron S660 desktop computer and a E200 Microscope. 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.

Preparations setup

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.

Temporary slide (top right). This temporary slide is used only for cleared structures either with clove oil or methyl salicilate. 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 salicilate 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.

Images Composition Rules
This list will provide the rules to make the image composition constant among inventories.

  1. First remove all legs and pedipalp from the left side of the specimen. Dissect the cephalothorax appendices in the membrane between the coxa and trochanther and check for cuticle or muscle fragments that may spoil the image.
  2. Take all the habitus images horizontally and with the specimen looking to the left. Large specimens can be oriented diagonally or splited in two images. Prosoma anterior view is always vertical. Composition of other images is free, but seek to maximize information content.
  3. Sixteen Standard Views are enough to document the anatomy of a species with both sexes and provide data for reliable identifications. Eight images document the overall anatomy (habitus and prosoma). Four images for epigyna (two with the epigynum attached and two dissected and cleared) and four images for the male left pedipalp. We have recommend an upper bound of 20 images per species.
  4. Keep the same width and height in pixels for all images. If the structure looks too small in the image composition, then an instrument with more magnification or better optics is required, please check the section of microscopy tricks.

Image Name Codes
The back bone of the inventory pages is "File Name Code" and "Body Parts File" naming protocols created by the Planetary Biodiversity Inventory (PBI) of the spider Family Oonopidae research team. These Arachnologists created an elegant and simple code that with four letters can name 232 body parts and its orientation. We are very thankful to them.

These are the links to the "Image Name Code" and the "Body Part File". Examples of these Image Name Codes can be observed in any image file name.

Additional Specimen Data
These data links the voucher specimens codes with the additional information required to create the inventory pages. This worksheet can be organized in Excel and must include the following data:

  1. Family Name.
  2. Species or morphospecies name.
  3. The specimen voucher code.
  4. Specimen locality
  5. Taxonomic identification authors.
  6. Number of female specimens.
  7. Number of male specimens.
  8. Taxonomic notes.

Pages creation process
The process starts with a MSDOS text file of the Folder Organization and Image Names. The Image Names in this text file can be splited by character number in its units such as: Family, Genus, Species, sex, body part, view, etc. This information is stored by the relative position of the code components in the file name and the folders organization. Errors such as shifted frames are detected using Excel Dynamic Tables. The revised names and Additional Specimen Data File are copied to the Main Excel spreadsheet. The HTML is created in a second sheet that write the code with many INDEX, MATCH, IF, AND, OR statements. This code is saved as a text file and read by several Perl scripts that create, split or combine the pages for each search menu. The Family and Species lists are created in the same way.

If you are interested in making similar pages for your inventory please contact us and we will be happy to help; however, be aware that the algorithms may collapse or may not work if the organization of your data and files does not follow the rules described above.