Dual channel detection of ultra low concentration of bacteria in real time by scanning fluorescence correlation spectroscopy

We describe a novel method to detect very low concentrations of bacteria in water. Our device consists of a portable horizontal geometry small confocal microscope with large pinhole and a holder for cylindrical cuvettes containing the sample. Two motors provide fast rotational and slow vertical motion of the cuvette so the device looks like a simplified flow cytometer without flow. To achieve high sensitivity, the design has two detection channels. Bacteria are stained by two different nucleic acid dyes and excited with two different lasers. Data are analyzed with a correlation filter based on particle passage pattern recognition. The passage of a particle through the illumination volume is compared with a Gaussian pattern in both channels. The width of the Gaussian correlates with the time of passage of the particle so one particle is counted when the algorithm finds a match with a Gaussian in both channels. The concentration of particles in the sample is deduced from the total number of coincident hits and the total volume scanned. This portable setup provides higher sensitivity, low-cost advantage, and it can have a wide use ranging from clinical applications to pollution monitors and water and air quality control.