Characterization of the sensitivity of side scatter in a flow-stream waveguide flow cytometer

Mariella, Raymond P.; Huang, Zhengping; Langlois, Richard G.

doi:10.1002/(sici)1097-0320(19991001)37:2<160::aid-cyto10>3.0.co;2-3

Cited by 10 publications

(14 citation statements)

References 12 publications

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“…For another thing, the practical particle size limit for SSC detection has been reported as 0.15 µm although a significant amount of crocidolite fibers show smaller diameter. (23) In fact, crocidolite fibers have enough length for SSC detection and usually form bundles, so most of them should be detected by flow cytometric analysis, even though their influence on the degree of SSC increase may sometimes depend on their intracellular position.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of two distinct methods to quantify the uptake of crocidolite fibers by mesothelial cells

Yamashita

Nagai

Misawa

et al. 2013

J. Clin. Biochem. Nutr.

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Exposure to asbestos fibers increases the risk of mesothelioma in humans. One hypothetical carcinogenic mechanism is that asbestos fibers may directly induce mutations in mesothelial cells. Although the uptake of asbestos fibers by mesothelial cells is recognized, methods for the quantification of the uptake have not been well established. In the present study, we evaluated two distinct methods, using crocidolite fibers and MeT5A mesothelial cells. One method is histological evaluation using the cell-block technique, which allows for the direct cross-sectional observation of cells and fibers. We found the bright field observation with ×1000 magnification (oil-immersion) of the sample with Kernechtrot staining was most suitable for this purpose. The other method is flow cytometric analysis, which permits the evaluation of a much larger number of cells. We observed that the side scatter (SSC) increased with the intracellular fibers, and that the “mean SSC ratio (treated/control)” was useful for quantification. We could collect the cells with abundant internalized crocidolite fibers by sorting. Results of the two methodologies were correlated well in the experiments. The quantities of internalized fibers increased with incubation time and loaded dosage, but they were inversely associated with cellular density in culture.

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Section: Discussionmentioning

confidence: 99%

Evaluation of two distinct methods to quantify the uptake of crocidolite fibers by mesothelial cells

Yamashita

Nagai

Misawa

et al. 2013

J. Clin. Biochem. Nutr.

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“…The first reported example was implemented in a stream in air cytometer. In this instrument, the water stream in air naturally forms a liquid core waveguide due to air having a lower refractive index than water (15,17). Scattered light from cells was then conducted to a fiber optic placed in the fluid stream and measured by a PMT as in this study.…”

Section: Discussionmentioning

confidence: 99%

“…For generating additional fluorescence channels the potential exists to use a variety of dichroic mirrors and bandpass filters (Hammamatsu) to split out the various wavelengths of light and input them into their respective PMTs (17). However, this option is still subject to the rigors of proper alignment of each of the mirrors in the filtering units.…”

Section: Fiber Optic Splittermentioning

confidence: 99%

Flow cytometry without alignment of collection optics

Sitton

Srienc²

2009

Cytometry Pt A

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This study describes the performance of a new waveguide flow cell constructed from Teflon AF 1 (TFC) and the potential use of fiber optic splitters to replace collection objectives and dichroic mirrors. The TFC has the unique optical property that the refractive index of the polymer is lower than water and therefore, water filled TFC behaves and functions as a liquid core waveguide. Thus, as cells flow through the TFC and are illuminated by a laser orthogonal to the flow direction, scattered and fluorescent light is directed down the axis of the TFC to a fiber optic. The total signal in the fiber optic is then split into multiple fibers by fiber optic splitters to enable measurement of signal intensities at different wavelengths. Optical filters are placed at the terminus of each fiber before measurement of specific wavelengths by a PMT. The constructed system was used to measure DNA content of CHO and yeast cells. Polystyrene beads were used for alignment and to assess the performance of the system. Polystyrene beads were observed to produce light scattering signals with unique bimodal characteristics dependent on the direction of flow relative to the collecting fiber optic. (1) and have since been refined into highly developed, sophisticated instruments. Over this time, the basic principles have not been changed significantly. In traditional flow cytometry, the illuminating laser beam and the light collection optics are placed in the same plane perpendicular to the flow stream. In most current instrument configurations microscope objectives gather scattered or fluorescent light. In place of microscope objectives, optical waveguides butted against the flow cell wall have been proposed and shown to collect light similar to microscope objectives (2). The collected light is then passed through a series of dichroic mirrors and bandpass filters to measure light from fluorophores correlated with a specific aspect of cell physiology. For high accuracy and precision, this traditional arrangement requires a precise alignment between the illumination optics, the flow stream, the collection optics, and the dichroic mirrors and filters used. Properly aligning these elements is a sensitive task that requires considerable skill and experience.Current research to expand this technology has taken two directions: (i) development of higher-end cytometers with increasing signal strength, numbers of illuminating lasers, and fluorescence parameters analyzed (3-5) and (ii) development of instruments that are smaller, less expensive, and more accessible (Guava Technologies, Accuri) (6-8). This report describes an alternate configuration that has the potential to make cytometers less expensive, more accessible, and more robust.The described instrument configuration uses a Teflon AF capillary as the flow cell (TFC). The TFC has the unique optical property that the refractive index of the polymer (n 5 1.290) is lower than water (n 5 1.333) (9). Thus, a water filled TFC

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“…12 The APDS project was initiated in 1996 by LLNL as an internally funded project, but in 1997 transitioned to be part of the DOE's Chemical and Biological Nonproliferation Program as that program advanced. The APDS development efforts included a high-performance LLNL flow cytometer called the miniFlo 13,14 , successfully demonstrated at the DoD's 1996 Joint Field Trials III (see Figure 2). This early success contributed to the establishment of the DOE program and of the APDS project.…”

Section: Early Developmentmentioning

confidence: 99%