METHODS, IMAGING

Creating a small color and size reference card

A reference card in images can serve multiple purposes: on the one hand, a reference card is required to map size from pixels to real world units (e.g., from pixels to millimeters) and to calibrate color and brightness so that they become comparable across images and photographed scenes. On the other hand, including a reference card in every images will ensure that the images are comparable to each other - e.g., in the case of varying brightness or distance from camera and photographed specimen. In this post I will describe how I build small, waterproof reference cards that I used in many of my own studies, and that are increasingly used in other laboratories as well. The main idea is to take little parts of a commercial color reference card and a bit of millimeter paper, and laminate it to form a small, roughly stamp sized plastic card.

Thanks Erica Winslott for your help when making these :raised_hands:

1. Materials

What you need:

2. Cut up tiny squares

Cut out roughly 1 x 1 mm pieces from the color stripes. The exact size does not matter, but the relative size they will have in the picture later on. In other words, if in the final images the reference card is tiny because you place it next to a huge specimen, the squares should be larger so that enough pixels are featured in every color and gray scale. Note that I only use the color squares with full saturation.

3. Align the squares

Now place the squares in the opened laminating pouch. This is the tricky part: those pouches can be a bit stiff and may close again, so that the squares are launched all over the place. Patience and a calm hand is your friend here. Don’t forget to add a small stripe of mm-paper.

Alternatively, use tape to hold the little squares in place (this was Erica’s idea :smile:):

4. Laminate

After putting everything together, the pouch is ready to go into the lamination machine. Enter it carefully, so that the squares inside don’t move. Done! :sparkles: