Investigations Using a Digital Camera To Observe and Quantify the Effects of Pollution on Aiptasia pallida, a Model Organism for Coral Presented by Jennifer Welborn June, 2012

Learning Goal This goal of this powerpoint is to introduce STEMDIGITAL participants to the application, theory and steps for finding the area of an image using a combination of ADI tools: Spatial Analysis, Enhanced Colors and Masked Color

Overview Background– Aiptasia as a model organism ADI procedures – theory, steps, sample images Pollution lab procedure Further Investigations

Aiptasia as a Model Organism for The Effect of Global Environmental Change on Corals Background Coral bleaching--serious consequence of global environmental change. Corals have a symbiotic relationship with algae * algae provide food * anemone provides shelter Coral bleaching– corals are stressed by environmental factors, release algae, turn white and die Environmental stress factors increased water temperatures (global warming) lower pH (ocean acidification) pollution effects

Background Continued Aiptasia pallida -- small anemones similar enough to coral to be used as a model organism in labs. Unlike corals, Aiptasia are hearty and will not die if stressed to the point of releasing their symbiotic algae. They are able to get food by stinging and eating small organisms living in the water. Aiptasia -- can be used for coral bleaching research without harming them. Aiptasia pallida The “bleached” Aiptasia on the right have released their algae due to environmental stress

How big is this anemone? This is a question of area (in a 2 dimensional image).. ADI masking tool is used to find area by making pixels within a certain range black. First, you need to figure out the colors and intensities which distinguish the critter from its background. The Aiptasia above is light-colored and is on a white background. Enhanced Colors option is used to isolate the critter, colorwise. A stressed Aiptasia pulls its tentacles in thereby having less area available for photosynthesis. You can compare the area taken up by the anemone before and after the addition of a potential stressor to see the effect. ADI and Area Measurements– A change in area can indicate stress or growth in living things

Contented anemone – tentacles out Stressed anemone— Tentacles retracted

Color Histograms  A color histogram is a graph of frequencies of the color intensities  Separate groupings of colors relate to different objects in the image  The white background has intensities of red, green an blue near100%.  The smaller color histograms (lower intensities) of RGB to the right correspond to the Aptasia  In the next slide, the ADI rectangle tool was used to take a sample of pixels inside the Aiptasia The

The three RGB peaks on the left (nearly 100% intensity) correspond to the white paper background. The RGB smaller bump, lower Intensity, on the right corresponds to the Aiptasia in the photo

Histograms of RGB Values in entire photo—large Peaks are the white background—smaller bumps Are the Aiptasia

Manually isolating the min/max values which appear to be associated with the Aiptasia, then masking those pixels gives an approximate image in black. Play around with min/max and mask colors to see the effect.

A more efficient and accurate way to mask pixels associated with the Aiptasia Is to use Enhance Colors option-this makes the critter stand out

Open a photo into enhanced colors

Red vs blue Normalized makes the critter show in red only

Zoom in to see the detail

Click on Mask Colors first Then, draw a Rectangle which encompasses a good sample of the range of red pixel intensities In the critter You will then get min/max values of the red pixels within the box Finally, click on Apply Mask Test 1 Click off green and blue to speed processing

You can toggle back and forth from the original image to the masked image to see how thoroughly the mask matches the real critter. Then, manually adjust the min. Max values (fine tune) until the mask is an accurate representation.

Once you are satisfied with your mask, go back to spatial Analysis and select polygon tool as the Measurement tool

Draw a polygon around only the Aiptasia. note the number of masked pixels inside the polygon

The next two slides show polygon enclosed unmasked and masked images of the same Aiptasia after being exposed to first crude oil, then dispersant

Number of masked pixels when exposed to oil: 12, 583

When exposed to dispersant and oil, the number of masked pixels is 7,573 = stressed out!

The Effect of Crude Oil and Dispersant on Aiptasia-- Experiment Research Questions What is the effect of crude oil and dispersant on Aiptasia pallida? How can a digital camera be used to quantify a change in area of the Aiptasia in response to an environmental stressor? Procedure 1.Groups 1-5 set up the lab according to the lab directions handout. (Testing oil alone and oil plus dispersant). 2.Photograph and analyze photos using enhanced color and masked pixels. Directions for ADI analysis are shown in this ppt and in the lab handout. 3.Groups 1-5 compare data with each other. 4.Groups 6-11 repeat steps 1-3 (testing dispersant only)

Further Investigations 1.Using an incubator, test the effect of increasing temperature on the color and behavior of Aiptasia over time. Rectangle tool, enhanced color and masked pixels 2. Using growlights, test the effect of light vs dark on the color and behavior of Aiptasia over time Rectangle tool, enhanced color and masked pixels 3. Test the immediate effect of slightly altering pH on the behavior of Aiptasia Rectangle tool, enhanced color and masked pixels