Biomedical Engineering Reference
In-Depth Information
photoactivatable (dark to bright) fluorescent probes and intermittently turn on
individual fluorescent molecules which are imaged and then photobleached. This
process allows the molecules within the same diffraction-limited volume to be
temporally separated and the location of each activated molecule under a sub-
diffraction-limited resolution to be determined. The final superresolution image is
obtained from merging all of the single-molecule positions localized by repeated
cycles of photoactivation followed by imaging and photobleaching.
3.4
FLIM and Implementation of a TPE-TCSPC FLIM
System
Fluorescence lifetime is the average time a molecule spends in the excited state
before returning to the ground state, typically with the emission of a photon, and it
carries information about events in the probe's local microenvironment that affect
the photophysical processes (see Chapter
1
in [
142
]). For most of the fluorescence
microscopy methods, the fluorescent signal is quantified by integrating the emitted
photons over a period of time which is usually much longer than the fluorescence
lifetime - referred to as steady-state (or intensity-based) imaging. In contrast,
FLIM measures the fluorescent signal at a very high temporal resolution (down to
picoseconds (ps)) and is thus able to resolve the fluorescence lifetime information.
Tab le
3.2
summarizes several advantages and limitations of FLIM compared to
steady-state (intensity-based) imaging microscopy.
3.4.1
Overview of FLIM Techniques and Applications
The first nanosecond fluorescence lifetime measurements in optical microscopy
were made in 1959 [
143
]. Since then, numerous FLIM methodologies have evolved
for various biological and clinical applications [
142
]. Especially in the last 10 years,
commercial FLIM systems have become available from companies, including
Becker & Hickl (
www.becker-hickl.de
)
, PicoQuant (
www.picoquant.com
)
, ISS
(
www.iss.com
)
, Intelligent Imaging Innovations (
www.intelligent-imaging.com
)
,
and Lambert Instruments (
www.lambert-instruments.com
)
.
FLIM techniques are generally subdivided into the time-domain (TD) and the
frequency-domain (FD) methods [
142
] (also see Chapters
4
and
5
in [
55
]). The basic
physics that underlies the two methods is essentially identical, since both TD and FD
are finite Fourier transforms of each other. The TD method uses a pulsed light source
synchronized to high-speed detectors and electronics to measure the fluorescence
decay profile at different time windows after each excitation pulse. The fluorescence
lifetime is estimated by analyzing the recorded decay profile. Many TD FLIM
techniques have been developed. For example, the TCSPC FLIM technique has
been commonly implemented on single-photon confocal or multiphoton scanning
microscopes using PMTs with high time resolutions (25-300 ps) [
144
-
148
]. Design
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