High intensity solid-state UV source for time-gated luminescence microscopy.
BACKGROUND
The unique discriminative ability of immunofluorescent probes can be severely compromised when probe emission competes against naturally occurring, intrinsically fluorescent substances (autofluorophores). Luminescence microscopes that operate in the time-domain can selectively resolve probes with long fluorescence lifetimes (tau > 100 micros) against short-lived fluorescence to deliver greatly improved signal-to-noise ratio (SNR). A novel time-gated luminescence microscope design is reported that employs an ultraviolet (UV) light emitting diode (LED) to excite fluorescence from a europium chelate immunoconjugate with a long fluorescence lifetime.


METHODS
A commercial Zeiss epifluorescence microscope was adapted for TGL operation by fitting with a time-gated image-intensified CCD camera and a high-power (100 mW) UV LED. Capture of the luminescence was delayed for a precise interval following excitation so that autofluorescence was suppressed. Giardia cysts were labeled in situ with antibody conjugated to a europium chelate (BHHST) with a fluorescence lifetime >500 micros.


RESULTS
BHHST-labeled Giardia cysts emit at 617 nm when excited in the UV and were difficult to locate within the matrix of fluorescent algae using conventional fluorescence microscopy, and the SNR of probe to autofluorescent background was 0.51:1. However in time-gated luminescence mode with a gate-delay of 5 mus, the SNR was improved to 12.8:1, a 25-fold improvement.


CONCLUSIONS
In comparison to xenon flashlamps, UV LEDs are inexpensive, easily powered, and extinguish quickly. Furthermore, the spiked emission of the LED enabled removal of spectral filters from the microscope to significantly improve efficiency of fluorescence excitation and capture.
Connally
Russell
R
Centre for Laser Applications, Division of Information and Communications Science, Department of Physics, Macquarie University, Sydney, Australia. rconnall@ics.mq.edu.au
Jin
Dayong
D
Piper
James
J
D000818:Animals; D002614:Chelating Agents / Q000737:chemistry; D005063:Europium / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005872:Giardia / Q000166:cytology / Q000276:immunology; D018796:Immunoconjugates / Q000737:chemistry; D008027:Light; D049449:Luminescence; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D013388:Succinimides / Q000737:chemistry; D013730:Terphenyl Compounds / Q000737:chemistry; D013997:Time Factors; D014466:Ultraviolet Rays; D005466:Fluorocarbons
D002614:Chelating Agents; D005063:Europium; D005456:Fluorescent Dyes; D018796:Immunoconjugates; D013388:Succinimides; D013730:Terphenyl Compounds; C518247:4,4-bis-(1,1,1,2,2,3,3-heptafluoro-4,6-hexanedion-6-yl) sulfonyl-aminopropyl-ester-N-succinimide-ester-o-terphenyl; D005466:Fluorocarbons