Laser biospeckle method for a fast and reliable microbioassay

Abstract

Recently, the direct toxicity assessment (DTA) approach or bioassay has attracted much attention, as it can comprehensively evaluate water toxicity without examining a specific/individual chemical component. Bioassays are used to investigate how organisms respond to pollutants compared to controls. In this technique, a microscopic observation is required to know the critical features such as alive/dead, and swimming ability of microorganisms. As the size of the microorganism becomes smaller, observation becomes more difficult due to the narrower focal depth of the imaging system. In our study, to overcome the difficulties, we propose, to our knowledge, a novel technique for the microbioassay utilizing a biospeckle in the diffraction field generated from its movement. We employed Paramecia caudatum sized around 200–300 µm. The sample cell containing Paramecia was illuminated with a laser light with a diameter 15 mm and wavelength 635 nm. The specular component of the transmitted light was filtered out using a spatial filter placed at the Fourier plane and the biospeckle patterns in the diffraction field were acquired using a CCD camera as a movie. It is obvious that the dynamic properties of the biospeckle precisely reflects the movement of the scattering particles or Paramecia. Paramecia were exposed to HCl solutions with pH7.1, control, to 3.7 where all were dead. The swimming abilities of the Paramecia were successfully evaluated based on the crosscorrelation function of the frames of biospeckle patterns. The advantage of the method is that, owing to the non-imaging system, an enormous number of Paramecia can be processed as a whole, and a statistically significant estimation of the swimming ability of Paramecia under the influence of environmental pollution can be derived immediately.