Sorting of biological cells in a microfluidic channel using holographic optical tweezers combined with raman spectroscopy

Investigation of single cells is crucial, since the measurement from population gives an average value that does not necessarily represent every individual in the population. Besides, sorting and sometimes counting the desired kind of cell is a measure of how the concentration of that cell. These types of micro-scale measurements are sometimes necessary, for example, to monitor the cell cycle, cell-signaling processes. Serving to these purposes, first a holographic optical tweezers (HOT) setup was constructed. This setup was built by improving the current optical tweezers setup in the Laser Spectroscopy Laboratory in Istanbul Technical University. A brand new Holoeye Spatial Light Modulator (SLM) was installed in the system after it was calibrated using an interferometric setup. The holograms were calculated using a Gratings and Lenses algorithm written in MATLAB and implemented by the homemade GUI that manages all the tasks needed for trapping and spectroscopy. The test particles to characterize the HOT setup were polystyrene (PS) particles. An array of four trapped PS particles was manipulated to the Raman spectrum measurement region by the software. The software communicates with the spectrometer and obtains multi-track spectra from the array that gives individual Raman spectra of each particle. These spectra identified by the software using the correlation coefficient of this measurement with the dataset that has already been prepared using PS particles and yeast cells. The test Raman measurements of biological cells using yeast cells and E.coli cells were made to investigate the signal level in low exposure times. The measurement showed that, our system would not respond enough signal to classify particles when the sample is E.coli and the exposure time is one second. However, the yeast cells responded better in short exposure time. Considering these, the sorting test experiments were made using yeast cells and PS particles. In a mixed solution of PS particles and yeast cells, an array of four particles whose members are randomly chosen was moved to the measurement region. According to the measurement and the following classification, the particles are targeted to the previously defined positions in the measurement cell. In these experiments, classifications were made with success. A study to determine the embryo quality by Raman spectroscopy was made in collaboration with Istanbul University Medical School. In this study, it was aimed to find an objective method to assess embryo viability. The preliminary results showed that the sensitivity and specificity of these measurements are %93 and %77, respectively. A Mann-Whitney U test was applied on the band areas and all band area ratios obtained by band component analysis. The most significant one was found to be the band area ratio of 903/942 cm-1. Comparing the measurements of amino acids samples, it was determined that glutamine, glycine, proline, and valine has the most intense bands in the region that includes these significant bands. Among these, glutamine and glycine are the amino acids that contribute to embryo development most, according to the literature.