Autofluorescence of Bacteria

Autofluorescence is the spontaneous fluorescence of materials without added dyes. Many people are responsible for this work but principal credit goes to Cindy Maddera, who just received her Master's in my lab. Also contributing were Shelly Theiss, and Amy Huseby.

This work was sponsored, in part, by the Oklahoma State University Center for Sensors and Sensor Technology and the Oklahoma Center for the Advancement of Science and Technology.

Two-dimensional E. coli autofluorescence

We have explored the autofluorescence of bacteria and found some interesting properties. Those properties have been briefly encountered by others but we have added some new wrinkles - literally - as you will see later. We obtain our autofluorescence data by illuminating the bacteria with a specific color band and detecting the color of the light emitted by the bacteria. For example, we illuminate the bacteria with blue light and measure how much light is emitted from the bacteria. This data composes a two dimensional graph as illustrated below.

 

 

In the above graph the excitation wavelength varies from 300 nm to 500 nm while the emission wavelength of the instrument was held at 550 nm. Fluorescence measured the intensity of output of the light and varied from 0 to about 1.33 volts.This graph is an average of three separate trials; the vertical bars represent the variability among trials. Although this 2-D format is useful we have found that a three dimensional representation - hundreds of the 2-D experiments plotted together to construct a three-dimensional grid- is useful and much more interesting but hard to understand.

Three dimensional E. coli object

This object is from a fluorescence scan of Eschericha coli, a common bacterium and sometimes a very pathogenic one. In this diagram "Ex" stands for the excitation wavelength while "Em" stands for the emission wavelength. In this figure Ex varies from 200 NM to 500 NM Em varies from 400 NM (on the visible edge of ultraviolet) to 800 NM, in the infrared.Fluor stands for the value of the resulting fluorescence and has values from 0 to 0.6 volts. The image has been converted to pseudo-color so that differences in level are more apparent. Low values are represented as blue while higher values are represented as orange and red. (data from Cindy 3.23 E. coli)