Blu-ray cytometer

Abstract

The flow cytometers are one of the most powerful devices used to measure the properties of individual particles passing through a sensing region. In this thesis, a low cost cytometer using a Blu-ray pickup is analysed and further developed. This includes the characterisation of the existing system and its performance as well as the evaluation of count rate for different flow rates and focuses. Based on that, the best parameters for measurement conditions have been determined. The system was set up for forward scatter as well as for fluorescence, both were to be measured at the same time and it was found that both could be recorded and counted. In addition, it was found that fluorescing particles can give a fluorescent count as well as scatter count and it was noticed that different sizes of non-fluorescent particles can produce scatter count. The count rate for a mixture of fluorescent and non-fluorescent particles was measured. This required the installation of two different amplifiers (for fluorescent and non-fluorescent particles) and building a new LabView program containing two channels.

In a later part of the thesis, the system was improved to progress towards a setup of avalanche photodiode as detector for forward scattering as well as fluorescence. This gives a very good counting accuracy of 90% at speed of 10μl/s. However, this counting accuracy drops to 80% at speed of 1μl/s when mixing fluorescent and non-fluorescent particles. The counting accuracy is the ratio (in percent) of the measured count to the actual one. As a new detection approach, a beam stop was installed in front of the photo detector so scattered laser light from particles was detected instead of a reduction of laser intensity. This part of the project proves Mie theory which states that the the forward scattered light is in the forward direction. To double check this result, a beam pass was installed in front of the photo detector and the calculated percentage of counting accuracy was very close to the one without a beam stop. It was also checked that the system was capable of detection fluorescent particle after the installation of a high bass filter which passes fluorescent wavelength signals (green) and blocks the blue ray signals. Finally, it was found that this system is able to detect small size particles, such as 1μm particles and to distinguish between 1μm and 10μm particles.