fulltext16 - Time-Gated Luminescence Microscopy RUSSELL E....

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Time-Gated Luminescence Microscopy R USSELL E. C ONNALLY AND J AMES A. P IPER Centre for Laser Applications, Department of Physics, Division of Information and Communication Sciences, Macquarie University, Sydney, Australia Autofluorescent algal samples were spiked with europium beads for analysis on a novel all-solid- state, time-gated luminescence (TGL) microscope. Pulsed UV excitation (365 nm) was provided by a high-power UV-LED source fitted to an Olympus BX51 microscope. An “Impactron” electron multiplying charge-coupled-device (CCD) camera acquired images in delayed luminescence mode. Second, we evaluated sensitivity of the instrument by acquiring images of immunofluorescently labeled Giardia cysts with a single-exposure period of 3 ms. The camera was triggered 3 µ s after the LED had extinguished to yield a 14-fold increase in signal-to-noise ratio within a single 33 ms capture cycle. This novel instrument could be switched instantly from prompt epifluorescence mode to TGL mode for suppression of short-lived fluorescence. Key words: time-gated luminescence; autofluorescence; lanthanide; phosphorescence; Giardia ; EMCCD; UV LED Introduction Fluorescence-based techniques provide a powerful means for both the qualitative and quantitative detec- tion of biomolecules. 2 Fluorescence methods afford a sensitive means for the detection of single molecules, 5 however probes lose much of their discriminatory power in the presence of autofluorescence. Organic and inorganic autofluorophores are ubiquitous in na- ture and some materials fluoresce with great intensity, obscuring or diminishing the visibility of synthetic flu- orescent probes. 7 Spectral selection techniques (emis- sion and excitation filters) can reduce the problem but are not always applicable due to the abundance and spectral range of autofluorescence. 9 Time-gated luminescence (TGL) techniques oper- ate within the time-domain and are directed toward detection of events that occur at much longer time- scales (phosphorescence). For TGL operation, the detector is gated off while a brief pulse of light is used to excite emission from the sample. The detector is maintained in the off-state for a resolving period (gate-delay) while short-lived ( < 1 µ s) fluorescence fades beyond detection. The detector is then enabled to capture luminescence in the absence of autofluorescence, greatly increasing the signal-to- noise ratio (SNR) (F IG .1). Address for correspondence: Russell E. Connally, Centre for Laser Ap- plications, Department of Physics, Division of Information and Commu- nication Sciences, Macquarie University, Sydney, NSW 2109, Australia. [email protected] While it is possible to discriminate probe fluores- cence from autofluorescence using time-resolved flu- orescence (TRF) techniques, a simpler and much less costly TGL instrument can be employed if a suitable luminescent probe is available. Lanthanide (Eu 3 + or Tb 3 + ) chelate luminescent probes have exceptionally long phosphorescence lifetimes (
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This note was uploaded on 07/11/2010 for the course SPECTOGRAP 545 taught by Professor Gdf during the Spring '10 term at AIB College of Business.

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fulltext16 - Time-Gated Luminescence Microscopy RUSSELL E....

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