Abstract
Many naturally occurring substances are intrinsically
fluorescent (autofluorescent) when exited at an appropriate
wavelength and emission can occur throughout the visible
spectrum. Autofluorescence is typically a short-lived phenomena
with a lifetime (t) measured in nanoseconds and
this property is exploited in Time-Resolved Fluorescence
(TRF) microscopy to enhance detection of labelled pathogens
against autofluorescence background. The TRF methods
are based on the use of immunofluorescent labels with
long fluorescence lifetimes (~600 μs) to ensure that
labelled target is viable long after short-lived autofluorescence
has faded. Pulsed excitation is used to excite fluorescence
from the sample and this is followed by a gate-delay
phase to permit decay of short-lived fluorescence. When
flashlamps are used as the excitation source, the gate-delay
period must be extended (>50 μs) to ensure that light from
the decaying plasma has decayed to zero. The extended
gate-delay results in a significant loss of fluorescence intensity
from the synthetic label and this is avoided with solidstate
excitation sources. A high-power (>100 mW) Light
Emitting Diodes (LEDS) (λ 365 nm) was substituted for the
flashlamp and found to give excellent background suppression
and strong label fluorescence compared to flashlamp
excitation.
Original language | English |
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Pages (from-to) | 724-725 |
Number of pages | 2 |
Journal | Cytometry Part A |
Volume | 75A |
Issue number | 8 |
DOIs | |
Publication status | Published - Aug 2009 |
Event | 14th Leipziger Workshop/7th International Workshop on Slide Based Cytometry - Leipzig, Germany Duration: 2 Apr 2009 → 4 Apr 2009 |