SEN 02 Portable Algae Flow CytometerSEN 02.1 OverviewThe portable algae flow cytometer is a project that aims to expedite research in algae biology using microfluid-based and state-of-the-art detection technology. The project is a joint effort that will incorporate the expertise of two different groups, Dr. David Caron at USC and Dr. Yu-Chong Tai at Caltech. One main focus of the project is to develop a portable flow cytometer that is suitable for on-field monitoring of algae population and reduce test time. The overwhelming growth of microalgal population, the microalgal bloom, has negative effects on marine ecosystems. The temporally-distinct increase in biomass results in a net loss of oxygen through respiratory and degradation process. The hypoxic event leads to the mortality of larger organisms including fish, shellfish and seagrasses. Further, many bloom-forming species are capable of toxin production which is harmful to the marine lives even affects human health. However, factors driving selective proliferations of microalgae and, especially, harmful species are still poorly understood. Part of the reason for this lack of knowledge is the time- and labor-intensive nature of analysis of water samples for specific bloom-causing organisms. Many of today s ocean-observing systems provide only rough proxies for algal biomass (e.g. chlorophyll fluorescence, absorption, or backscattering) and couldn t distinguish different species. To solve this problem we build a portable algae flow cytometer system to provides a precise evaluation of the algae population. The flow cytometer measures individual algae cells for their size, chlorophyll fluorescence and other biological properties, which is important to distinguish different species, especially to resolve the harmful ones among algae communities. Also, the portable system can be used for constant vigilance in the pre-bloom stage to tie down processes contributing to the increased growth of algae.SEN 02.2 ApproachThe portable system built at Caltech uses the microflow (flow) cytometer technology. A disposable microfluidic chip is used as the flow cell of the cytometer to reduce the sample volume needed for each test. The dimension of the flow cell can be optimized for different sizes of the target algae cells. Laser-induced-fluorescence measurement and light extinction measurement are used to evaluate the properties of the algae population such as the cell size, Chlorophyll- fluorescence, viability, esterase activities, etc. Each algae cell is measured individually and several thousands of algae cells can be measured in a short period of time to achieve a precise evaluation of the algae properties. The portable algae flow cytometer provides the on-field testing capability of the algae population and its biological properties, which is very useful for applications such as the on-field monitoring of the harmful algae bloom. It can also be used together with Algae Culture Chip we developed to further expand its functions such as controlling the number of algae cells being loaded into the culture chip by upstream test and evaluating the biological properties of the cultured algae cells by downstream test.SEN 02.3 System Description and ExperimentsThe portable algae flow cytometerA protocol of the portable system is built in Caltech with off-the-shelf components for facility demonstration (Fig.1). A microfluidic chip is used as the flow cell of the cytometer. The flow cell, made by the standard soft lithography process, is disposable after each test. The dimension of the detection zone, as shown in Fig.1, can be optimized for the target size of the algae cells. A blue (488nm) solid laser module is used as the excitation source, and the signals (extinction intensity, fluorescence intensity) are measured by two photomultiplier tubes (PMT). A mini peristaltic pump is used to draw the sample for test.2010 Annual Report 234Center for Embedded Networked Sensing 2.11 Embeddable SensorsFig.1 The portable algae flow cytometer system (right) and the disposable flow cell (left).Fig.2 The optical configuration of the portable algae flow cytometer.Fig.2 shows the optical configuration of the system. Based on the filter configuration, two-color fluorescence signals or one-color fluorescence signal and the extinction signal can be measured. The extinction measurement provides an estimation of the algae cell size. The fluorescence measurement can be used to evaluate the algae properties such as the Chlorophyll- fluorescence, esterase activities (with fluorescein diacetate (FDA) staining), etc. The portable system is assembled in an aluminum case (12” x 9” x5”). It can be powered by the standard 110V AC or a 5V DC source. The testing data is read out through a USB port and can be visualized on a laptop computer.Fig.4 shows a portion of the typical signals measured on the system. The left figure is the red fluoresence (>590nm) from the Chlorophyll- content of the algae cells. Each peak represents an algae cell passing through the detection zone of the flow cytometer, and the height of the peak indicates the Chlorophyll  content in the cell. The right figure is the the green fluoresence from the FDA staining, which indicates the algae cell s esterase activities.Based on the measured signal, the number of algae cells, the size of each algae cell and their biological properties (Chlorophyll  fluorescence, esterase activities, viability, etc.) can be evaluated.Count of Algae PopulationFor counting algae population, the filter set of the flow cytometer can be easily optimized to measure the Chlorophyll- fluorescence signal and the extinction signal simultaneously. The number of algae is determined by the measured Chlorophyll- fluorescence signal as shown in Fig.4. Algae cells are identified by their unique Chlorophyll- contents among other particles and the number of algae cells are counted by the number of the peaks. With a known sample volume, the number of algae cell per sample volume can also be calculated.The size of the algae cell is closely related to the extinction signals, which means the amount of the excitation light shielded away by the algae cell. The size of each algae cell is individually measured and several thousand of cells are measured to obtain the average cell size and the size