The prevention of waterborne diseases requires rapid detection of pathogens in drinking water, with an ultimate goal of in-line monitoring in real time. Standard cultivation-based methods are too time-consuming and thus not suitable for this purpose. Many technologies were proposed to achieve this goal, such as ELISA, PCR, FISH, FTIR and flow cytometry. However, they still have limitations of non-specificity, complexity and high cost. Therefore, an optical system is proposed and developed towards the in-line monitoring of bacteria, which combines the advantages of FTIR and micro-flow cytometer for bacterial identification and precise quantification. The in-line use requires obtaining IR spectra of bacterial cells directly in water, which is achieved using a CaF2 liquid cell. The spectra of a series of bacterial samples are collected and analyzed using principal component analysis for their differentiation. A preliminary study on fabricating a CaF2 concentrator is conducted, in which a novel phenomenon on stress release of silicon nitride film on CaF2 substrate is discovered and studied. To determine the concentration of bacteria in drinking water, a micro-flow cytometer is built based on a micro-fabricated device that integrates on-chip beam-shaping optics and microfluidic channels. With this micro-flow cytometer and optimized data analysis for counting particles in real time, linearity with correlation coefficient of over 0.99 is achieved for the dependences of throughput on both volumetric flow rate and concentration of sample. With a one-dimensional hydrodynamic focusing, no degradation of the counting efficiency is demonstrated when the focused sample stream expands. The high accuracy of counting makes this micro-flow cytometer a promising candidate for low concentration applications. Counting of E. coli DH5α cell suspensions in phosphate buffered saline is performed using the micro-flow cytometer. Side-scattered light signals are used to count the E. coli cells. A detection efficiency of 92% is achieved when compared with the expected count from a haemocytometer. It is demonstrated that E. coli can be easily distinguished from beads of similar sizes (2-4µm) as their scattering intensities are different. / Thesis / Doctor of Philosophy (PhD)

This research mainly develops a new z-position measurement based on the chromatic aberration effect. An objective-type dark-field illumination scheme is built to produce diascopic chromatic aberration light, and aimed to enhance the signal-to-noise ratio. The xenon lamp is adapted to create white light with continuous spectrum, besides, lens with low Abbe number is needed to extend the degree of chromatic aberration, so lens made of PMMA is as a chromatic aberration component. In the proposed system, the depths of samples in micro-channel is illuminated by the dispersed light and scatter the optical signals, which are captured by a low numerical aperture (N.A.) objective lens. After the simple normalization, the intensity ratio of two selected wavelengths 450 nm (blue light) and 670 nm (red light) from the scattered spectrum becomes a reliable index for the depth information of the detecting objects. By means of establishing the relationship between depth and intensity ratio, every object flowing through diagnosed spot is able to be determined the depth level by cross-referencing the database. By using spectrometer as detector, delicate moving components for light filtering or electrical stage for light scanning can be excluded for high-speed z-position detection. Furthermore, in order to identify the depth level of sample with high flowing rate, avalanche photodiodes are adapted to achieve rapid detection. The experimental results show that the relationship between depth and intensity ratio is a parabola curve, but in this research, the region which tends to behavior linearly is adapted. The proposed system provides a linear detection range of ¡Ó15 £gm for particles with a diameter of 20 £gm. The lens with high Abbe number only obtains ¡Ó10 £gm with linear detection range though, the resolution for size is better than PMMA. The BK7 lens is capable to discriminate the depth change of 2 £gm micro-beads, note that there is no limitation of depth discrimination in this system, because of the measurement is achieved by cross-referencing the linear line. The use of UV-Vis-NIR spectrometer enable this system to analyze the depths of the samples in flow rate 0.5 mm/s. To gain the higher performance, the two avalanche photodiodes are utilized, and the short(CWL=450 nm, ¡Ó20 nm) and long(CWL=650 nm, ¡Ó20 nm) band pass filter are also equipped to represent enhancements of blue and red ray. The effective detection range extends to ¡Ó25 £gm and has high linearity(R square=0.99285) after the optimization of light stop. In high flowing rate detection, this system is able to identify the depth of sample when the flow velocity is 4.167 mm/s, the calculated throughput is 126 particles/s. It also successfully analyzes the depth of flowing human erythrocytes under the flow velocity is 2.778 mm/s, the velocity which the developed system is capable to analyze is about 5-8 folds to the conventional micro-PIV system. With this novel and simple approach, there will be the quantified information from z-direction of flowing body for bio-analysis, and also benefits estimating the performance of micro structure or device in the microfluidic chip, also the analysis of flow field. Except for dynamical detection, this system also be capable to apply in a open and static situation, such as cell or tissue proliferation assay.