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Proceedings Paper

Novel flashlamp-based time-resolved fluorescence microscope reduces autofluorescence for 30-fold contrast enhancement in environmental samples
Author(s): Russell Connally; Duncan Veal; James A. Piper
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Paper Abstract

The abundance of naturally fluorescing components (autofluorophors) encountered in environmentally sourced samples can greatly hinder the detection and identification of fluorescently labeled target using fluorescence microscopy. Time-resolved fluorescence microscopy (TRFM) is a technique that reduces the effects of autofluorescence through precisely controlled time delays. Lanthanide chelates have fluorescence lifetimes many orders of magnitude greater than typical autofluorophors, and persist in their luminescence long after autofluorescence has ceased. An intense short pulse of (UV) light is used to excite fluorescence in the sample and after a short delay period the longer persisting fluorescence from the chelate is captured with an image-intensified CCD camera. The choice of pulsed excitation source for TRFM has a large impact on the price and performance of the instrument. A flashlamp with a short pulse duration was selected for our instrument because of the high spectral energy in the UV region and short pulse length. However, flash output decays with an approximate lifetime of 18μs and the TRFM requires a long-lived chelate to ensure probe fluorescence is still visible after decay of the flash plasma. We synthesized a recently reported fluorescent chelate (BHHCT) and conjugated it to a monoclonal antibody directed against the water-borne parasite Giardia lamblia. Fluorescence lifetime of the construct was determined to be 339μs ± 14μs and provided a 45-fold enhancement of labeled Giardia over background using a gate delay of 100μs. Despite the sub-optimal decay characteristics of the light pulse, flashlamps have many advantages compared to optical chopper wheels and modulated lasers. Their low cost, lack of vibration, ease of interface and small footprint are important factors to consider in TRFM design.

Paper Details

Date Published: 9 July 2003
PDF: 10 pages
Proc. SPIE 4964, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing X, (9 July 2003); doi: 10.1117/12.477977
Show Author Affiliations
Russell Connally, Macquarie Univ. (Australia)
Duncan Veal, Macquarie Univ. (Australia)
James A. Piper, Macquarie Univ. (Australia)

Published in SPIE Proceedings Vol. 4964:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing X
Jose-Angel Conchello; Carol J. Cogswell; Tony Wilson, Editor(s)

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