Future Directions Dvb
Flow cytometry is a rapidly expanding field worldwide where an enormous increase in its capability can be expected over the coming years. As indicated in the beginning of this chapter, there has been renewed interest in flow cytom-etry from the point of view of research where a major impetus is derived from its applications to genomics and proteomics. Recent advances in solid-state lasers, microfluidics, microarray technology, micro-optics, and miniaturized detectors provide challenging technological opportunities for developing small and compact flow cytometers with enhanced capabilities to simultane
ously monitor many more parameters than currently possible. It is refueling the expectation that perhaps a flow cytometer-on-a-chip is not such a distant dream. New applications of flow cytometry are already emerging and will continue to expand the dimensions of flow cytometry. An excellent review on emerging technology and future development in flow cytometry is by Stewart et al. (2002). Some of these future directions are listed here.
Research. A newly emerging field is single-molecule flow cytometry, which takes advantage of a variety of techniques that have been developed during recent years to detect individual fluorochrome molecules in solutions (Keller et al., 1996; Goodwin et al., 1996; Nie and Zare, 1997). Single molecule flow cytometry offers tremendous prospects for molecular biology. The technique can be used for DNA fragment sizing (Castro et al., 1993; Goodwin et al., 1993; Huang et al., 1996) and DNA sequencing (Ambrose et al., 1993; Goodwin et al., 1997). In this method, the concentration and flow is adjusted so that each molecule (or fluorescently labeled DNA fragment) flows through the illumination zone of a flow cytometer individually, one at a time, at the same rate, and experiences the same light intensity. The DNA sequencing information can readily be obtained by using fluorescence in situ hybridization (FISH) involving a pool of fluorescently labled oligonucleotide probes. This technique of hybridization using single-stranded oligonucleotides synthesized with a known base sequence has been discussed in Chapter 9, which describes biosensors for identifying the specific DNA and mRNA sequences in individual cells. A major limitation of the single-molecule flow cytometry is the background count, which can be significantly larger than that produced by single-molecule fluorescence. Various methods are being pursued to overcome this limitation. A promising new method utilizes single-molecule fluorescence detection by two-photon excitation (Mertz et al., 1995; VanOrden et al., 1999). Efficient two-photon excitation, provided by using ultra-short (femtosecond) pulses of high intensity but with low average power, facilitates single-molecule detection, because the excitation wavelength (in near IR) and emission wavelength (in the visible) are well separated. Recent reports of fluorochromes with a considerably enhanced two-photon (Bhawalkar et al., 1996) and three-photon (He et al., 2002) absorption to produce even population inversion and resulting stimulated emission provides further promise to single molecule flow cytom-etry. This also offers considerable opportunities for chemists to synthesize highly efficient multiphoton excitable fluorochromes for fluorescence in situ hybridization to be used in molecular flow cytometry.
Another intriguing prospect is to couple laser capture microdissection (LCM) with flow cytometry. In this approach, precise cell-type-specific microdissection using LCM yields pure specimen for nucleic acid analysis (DiFrancesco et al., 2000).
Another area of research activities is functional assays that utilize various activation markers to stimulate a cellular process and monitor the progress on a flow cytometer in real time. Using a pulse laser source and a vertical time-resolved scan over a length of the flow, one can monitor the dynamics. For detailed information, one can couple the time resolution with spectroscopic resolution, where the fluorescence signal obtained as a function of time (transit time in the flow) can be dispersed in a spectrograph and collected using an array detector.
A strategy, originally introduced by Liu et al. (1989), that is receiving more attention recently is that of multiplexing antibodies. It combines multiple antibodies having the same color fluorochrome to resolve multiple subsets of cells in a single tube. This strategy will benefit from the production of high affinity (strongly binding) antibodies and development of site-directed fluorochrome conjugation.
Another area of future development involves nanotechnology to produce 10- to 30-nm size highly efficient up-converting nanophores. These nanoparti-cles, when appropriately functionalized, can be used to target specific cells, can permeate through the membrane because of small size, and can be used to target a specific organelle where they can be detected by up-converted emission using excitation by a 970-nm laser. An important class of up-converting nanoparticles are those containing rare-earth ions (Chen et al., 1999; Kapoor et al., 2000). A major issue to address in this case is the long lifetime (in hundreds of microseconds) of the emitting rare-earth ions.
Technology. A major future direction of development in the technology of flow cytometry is in the area of miniaturization and use of robotics. Important development in the area of micro-lasers, detectors, micro-electro-mechanical systems (MEMS), dense wavelength division multiplexing (DWDM), and micro-optics is taking place, driven by their application to optical information processing. A monolithic integration of these components is already envisioned to produce a photonic chip. Significant progress has also been made in the area of microfluidics. These two developments, coupled together, can provide a fertile ground to produce a flow cytometer-on-a-chip, which is more versatile and offers expanded scope. Use of robotics to implement an automated system for specimen processing with increased number of probes will greatly enhance the capabilities of a flow cytometer in data acquisition, significantly reduce the specimen processing time, and permit operations with smaller volumes.
Another area of technical development is their use to detect and probe microbial activities. Although laboratory demonstration of detection of bacteria has already been successful, currently available commercial flow cytometers fall short of achieving this goal. Current advances in biomedical optics and lasers will provide designs to focus the beam to dimensions compatible with bacteria. It may also be able to immunophenotype specific organelles such as mitochondria, endoplasmic reticulum vesicles, golgi, lyso-somes, and so on.
An approach being introduced to provide high-throughput analyte analysis involves suspension array technology (SAT). The SAT method utilizes microsphere (beads) capable of quantifying up to 100 analytes in a single well, and one 96 well microtiter plate can be processed in less than one hour. The beads are functionalized with antibodies or genomic probes that can cova-lently couple to them to capture any analyte. Multiplexing in a single well, can be used to further enhance resolution. This assay format has recently been used for genomic evaluation (Iannone et al., 2001; Cai et al., 2000).
Applications. New applications of flow cytometry are constantly emerging. The future will see a considerable use of flow cytometers as a reliable research and clinical instrument for diagnosis and for monitoring the progress of a treatment.
Other new applications will be in the area of water and food quality control. Development of flow cytometers to detect microbial species will open up this vast application for continuous monitoring of water quality to detect any contaminants. Agriculture industry can also utilize flow cytometry to detect infection to plants and to develop new resistant species.
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