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Proceedings Paper

Frequency domain fluorescence lifetime microwell-plate platform for respirometry measurements
Author(s): M. R. Chatni; G. Yale; A. Van Ryckeghem; D. M. Porterfield
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Paper Abstract

Traditionally micro-well plate based platforms used in biology utilize fluorescence intensity based methods to measure processes of biological relevance. However, fluorescence intensity measurements suffer from calibration drift due to a variety of factors. Photobleaching and self-quenching of the fluorescent dyes cause the intensity signal to drop over the lifetime of sensor immobilized inside the well. Variation in turbidity of the sample during the course of the measurement affects the measured fluorescence intensity. In comparison, fluorescence lifetime measurements are not significantly affected by these factors because fluorescence lifetime is a physico-chemical property of the fluorescent dye. Reliable and inexpensive frequency domain fluorescence lifetime instrumentation platforms are possible because the greater tolerance for optical alignment, and because they can be performed using inexpensive light sources such as LEDs. In this paper we report the development of a frequency domain fluorescence lifetime well-plate platform utilizing an oxygen sensitive transition-metal ligand complex fluorophore with a lifetime in the microsecond range. The fluorescence lifetime dye is incorporated in a polymer matrix and immobilized on the base of micro-well of a 60 well micro-well plate. Respiration measurements are performed in both aqueous and non-aqueous environment. Respirometry measurements were recorded from single Daphnia magna egg in hard water. Daphnia is an aquatic organism, important in environmental toxicology as a standard bioassay and early warning indicator for water quality monitoring. Also respirometry measurements were recorded from Tribolium castaneum eggs, which are common pests in the processed flour industry. These eggs were subjected to mitochondrial electron transport chain inhibitor such as potassium cyanide (KCN) and its effects on egg respiration were measured in real-time.

Paper Details

Date Published: 23 April 2010
PDF: 9 pages
Proc. SPIE 7674, Smart Biomedical and Physiological Sensor Technologies VII, 767407 (23 April 2010); doi: 10.1117/12.852472
Show Author Affiliations
M. R. Chatni, Purdue Univ. (United States)
G. Yale, Purdue Univ. (United States)
A. Van Ryckeghem, Fumigation Services and Supply (United States)
D. M. Porterfield, Purdue Univ. (United States)

Published in SPIE Proceedings Vol. 7674:
Smart Biomedical and Physiological Sensor Technologies VII
Brian M. Cullum; D. Marshall Porterfield; Karl S. Booksh, Editor(s)

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