Biomedical Engineering Reference
In-Depth Information
timing apparatus. Therefore, an iterative deconvolution routine with an input IRF
is often employed to estimate the lifetime information from measured decay data
based on a weighted least-squares numerical approach. Determining the IRF of a
TCSPC FLIM system is essential to achieve accurate lifetime information. Although
the IRF can be numerically estimated from acquired decay data, it is preferable
to directly measure the IRF experimentally. The IRF should be representative
of the experimental conditions and thus is ideally measured under the same
conditions used for biological specimens, such as using the same objective lens and
optical configurations and applying the same TCSPC and detector settings. More
importantly, the IRF of a TCSPC FLIM system should be checked periodically and
ideally measured before each experiment, since the IRF can change by reflections
in the optical path or by poor mode locking of the MP laser.
The IRF is commonly measured by recording either the scattered excitation
light or the decay of a fast reference dye such as Rose Bengal in phosphate
buffer pH 7.4 (lifetime
D
70 ps) [
178
]. For visible light excitation, a strongly
scattering specimen such as nondairy coffee creamer is conventionally used for IRF
measurements [
151
]. For infrared light excitation, the IRF can be measured using a
sample that yields second-harmonic generation (SHG) signals (see the application
note “Recording the Instrument Response Function of a Multiphoton FLIM System”
at www.becker-hickl.de/literature.htm). SHG is an ultrafast nonlinear process that
delivers a signal at one-half of the excitation wavelength. Two IRFs measured from
our TPE-TCSPC FLIM system by recording the SHG signals of the urea crystal
sample are shown in Fig.
3.7
: (1) use the 880-nm excitation wavelength and record
the SHG signals at 440 nm using a 460/50-nm emission filter, and (2) use the 940-
nm excitation wavelength and record the SHG signals at 470 nm using a 480/40-nm
emission filter. The IRF is not influenced by using different TPE wavelengths
because the pulse width of the MP laser (<150 fs) is much shorter than the time
response of the lifetime detector (
150 ps), which is the main determinant of the
FWHM of the IRF.
3.4.4
Calibration of the TPE-TCSPC FLIM System Using
Fluorescence Lifetime Standards
Prior to any scientific investigation, a FLIM system must be calibrated with standard
fluorophores of known lifetimes. There are many standard fluorophores that can
be used for the calibration of a FLIM system (see Appendix II in [
13
]and
also an online source at
http://www.iss.com/resources/reference/data tables
)
. It is
preferable to choose standards whose excitation, emission, and fluorescence lifetime
properties are close to those of the fluorophores in the intended samples, so that
the same imaging setup used for biological samples can be applied for calibration.
Very importantly, the fluorescence lifetime standard has to be carefully prepared
according to the reference, such as solvent, pH, and temperature, etc., because these
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