Open Access Dissertation
Date of Award
Electrical and Computer Engineering
Amar S. Basu
MULTIPLEXED PHOTOMETRY AND FLUORIMETRY USING MULTIPLE FREQUENCY CHANNELS
KHALED M. DADESH
Advisor: Dr. Amar Basu
Major: Electrical Engineering
Degree: Doctor of Philosophy
Multispectral photometry and fluorimetry are useful for quantifying and distinguishing samples during flow injection analysis, flow cytometry, and ratiometric absorbance measurement. However, multispectral detectors, including spectrometers, typically require arrayed or multiple light detectors, optical components, and path alignments, all of which increases the size and cost of the detection system. Several previous efforts have attempted the use of time division multiplexing or frequency division multiplexing (FDM) techniques to minimize both size and cost of multispectral photometry equipment by using only a single light detector. Although many of these designs achieved low cost, they generally operated at <50 KHz, which limited the detection speed of the overall system. An alternative frequency multiplexing design operated at 3MHz; however, it required electro optical modulators , which are too expensive and bulky for portable applications. In contrast to both approaches, the objective of this research is to use frequency division multiplexing to perform multispectral photometry and fluorimetry while achieving both low cost and high frequency operation (up to 100 MHz). The multiplexing is performed electronically using low cost optoelectronic sources, a single light detector, and a single high-throughput interrogation window. It enables us to perform multi-parameter biological analysis at lower costs and less complexity. Multiple monochromatic light sources, each with a unique wavelength, are electronically modulated at distinct frequencies, and their combined light emission is directed to the sample detection cell. The light transmitted by the sample (absorbance mode) or emitted by the sample (fluorescence mode) is directed to a single light detector. The received light is then converted to a voltage signal and demodulated into the frequency channels using phase-sensitive electronics. Each recovered channel therefore provides either absorbance or fluorescence at its respective optical wavelength. The system is designed to operate at high speed in order to be used in high throughput detectors such as flow cytometers. As a proof of concept, we apply the FDM technique in two detection systems: 1) a three-color absorbance photometry detector and 2) a two-color laser induced fluorescence (LIF) detector. In the first system, three LEDs are operated with 150 KHz, 200 KHz, and 250 KHz modulation frequencies, and the system achieves a 1 ms measurement time constant at an overall component cost <$10. We perform absorbance photometry of four different organic dyes in flow injected solutions and in discrete droplet microreactors with throughputs in the 10's of samples per second. In both cases, the system is able to simultaneously discriminate between them . In the LIF system, first two laser diodes operated at 1 KHz and 1.5 KHz, respectively, are used to excite fluorophores at the respective frequencies. This system is able to distinguish low speed (1 drop/sec) water-in-oil droplets containing fluorescein or rhodamine-6G generated in a microfluidic junction. Second two laser diodes operated at 25MHz and 40 MHz, respectively, are controlled using a developed high frequency FDM system to excite Fluorescein and Alexa 680 dyes at the respective frequencies. Because of the high frequency operation, this system is able to distinguish alternating high speed (300 drops/sec) droplets containing the two fluorescent dyes. In both case, the developed In previous experiments we use an inverted fluorescence microscope with a specific optical cube to excite dyes and collect fluorescence signals. These two FDM-LIF systems identify the different fluorophores based on their excitation frequency rather than their emission band, giving it a unique ability to distinguish fluorophores with overlapping emission spectra. However, overlapping excitation spectra is a problem in the FDM-LIF system, and any assay has to be prepared using fluorophores with minimal excitation overlap. Therefore, fluorophores with sharp excitation lines such as lanthanide ions are the best candidate material in use with FDM-LIF system. The system uses high frequency (100 MHz) modulation which enables multiplexed time constants on the order of 1 µs. Achieving this high bandwidth allow us to apply the system towards high throughput analysis such as cell cytometry, where it could substantially reduce cost and size of the system. Therefore, the FDM-LIF system is installed in an old BD bioscience cytometer, which is available in the cell cytometry laboratory in Karmanos Cancer Research Center (KCRC) located at Detroit Medical Center (DMC). A biological assay containing Alexa Fluor® 680 Goat Anti-Mouse IgG (H+L) and Alexa Fluor® 430 Goat Anti-Mouse IgG (H+L) with BDTM CompBead Anti-mouse Ig, κ beads is tested using the FDM-LIF system. The system is capable to count the two different antigens simultaneously, which gives the possibility of incorporating this system in cytometers. This technology promises to reduce cost and complexity of future cytometers.
Dadesh, Khaled M., "Multiplexed Photometry And Fluorimetry Using Multiple Frequency Channels" (2013). Wayne State University Dissertations. 757.