# New User is attempting color analysis of algae in water

#1

Hello all!

I am a high school teacher supporting a student who is working on a research project that involves growing algae under different conditions. We are trying to use the photographs she has take to visually determine the density of the algae in the water of her various units, based on color. We are trying to teach ourselves imagej to do this with the most accuracy… any help or guidance would be VERY appreciated.

If there is anyone on this forum who would be able to tell us how to make this happen or tell us where we need to go, it could be a life saver!

Eri

#2

Good day Eri,

please post a typical raw image in the original TIF- or PNG-format and explain in detail what you like to achieve.

I assume that you’ve already studied the ImageJ User Guide:
https://imagej.nih.gov/ij/docs/guide/index.html

Regards

Herbie

#3

If you are unfamiliar with the theory of densitometry, that’s is the term you want to look for. The Beer-Lamber (also Lambert-Beer) law links absorbance (=densitometry at a certain wavelength) to a concentration.

The global setup will be a light source behind an aquarium (or a cuvette, a beker with straight, parallel, walls) of which you measure the density/absorbance. The intensity of the light with an empty container will be your reference intensity I0. Without changing the intensity of the illumination, or the settings of your camera, you have your sample in a similar container (ie. same length of path of light from source to detector) and measure the reduction in amount of light, this will be your In. A divide operation gives you the percentage transmission of the sample (0-100%), which can be converted using the above mentioned law into an absorbance value A.

Finally, you need to obtain a molar extinction coefficient for your samples; a calibration standard, which probably you need to make one yourself using a dilution series of a known concentration range. ImageJ then can help you to fit a Rodbard function for obtaining quantified numbers from unknown samples (i.e. your experiment).

Measuring at different (monochromatic) wavelengths (using proper colour filters) on the one hand gives you more precise results and on the other hand might even allow you to distinguish between different populations of algae.

Importance of monochromatic light is mentioned in this article and this one goes over the theory with some formulas. Wikipedia also might be of further help. I have no conflict of interes with either publication.

(@EriRox: edited to indicate importance of constant illumination, and of camera setting which should be of constant location, aperture and exposure time)