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 my lab to take images that rival the sharpness and resolution of pictures taken with significantly more expensive equipment; we basically have the cheapest Nikon microscopes. The tricks described here are ideas taken from other Arachnologists and provided by my students. If you find improvement 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 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 use on average 50 stack images to produce one combined picture. The stack images are taken by hand, keeping track of how the depth of field overlaps form top to bottom. More expensive equipment takes the stack images automatically with the same results.

We have used Helicon Focus for several years to combine stack images and highly recommend it, the combined images are need no editing or in some cases very little. This is important when thousands or images are required.

The digital images are taken with a Nikon DSU3 camera connected to a standard Dell Inspiron S660 desktop computer. 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, ours is the cheapest model of Nikon, but any digital camera should work.

Preparations setup
The Petri dish with sand. This has been known for many generations of Arachnologist and its practical value is appreciated when hundreds of specimens need to be sorted, identify or photograph. Bury 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. Coddington (1983) developed it and its thoroughly described in this book chapter; however, a brief description is provided here. Paste several glass slide covers on one side of a glass microscope slide, the height of this slides covers stack must be slightly taller than the structure. Cut a narrow glass stripe, from a long slide cover, and paste it with 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.

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 cleared 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 by a led lamp.

On a dissecting microscope paste a tiny drop of Vaseline in the dry surface of The Tiny Dish; if the surface is wet then the Vaseline will never stick. Create the required shape to receive the specimen and paste it to observe the required view. Cover the dish with a glass slide because the alcohol evaporates very fast. Transfer The Tiny Dish to a microscope for slides glass preparations and remove the glass cover to take the images. The Vaseline is removed from the specimen by shaking it for 15 seconds in a vial with chloroform or ether.

Images Composition Rules
This list will provide the rules to make the image composition constant among inventories and some ideas to improve the quality of the pictures, more tricks are presented below under that section.

  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 images composition or confuse data.
  2. Take all the habitus images horizontally and with the specimen looking to the left. Huge specimens can be oriented diagonally. Prosoma anterior view is always vertical. Composition of other images is free, but seeks to maximize information content.
  3. Sixteen Standard Views have proof 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 anterior views). Four images for epigyna (two with the epigynum attached and two dissected and cleared) and four images for the male left pedipalp. We have 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 is required, please check the section of microscopy tricks.

Image Name Codes
The back bone of the inventory pages uses the "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 crate 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 start with a MSDOS text file with the Folder Organization and Image Names. The Image Names in this text file have their addresses and can be splited by character number in its units such as: Family, Genus, Species, sex, body part, view, etc. These information is stored by the relative position of the code components in the file name, therefore errors such as shifted frames are detected using Excel dimanic tables. The revised names and Additional Specimen Data File are uploaded in the Main Excel spreadsheet. The HTML is created in a contiguous 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 serach engine. The Family and Species lists are creted in the same way. If you are interested in making similar pages for your inventory please conctact us and we will be happy to help; however, be aware that the algorithms may collapse or may not work depending on the organization of your data and files.