Biomedical Engineering Reference
In-Depth Information
onto the lifetime detector via a biconvex lens, before which a filter that blocks
infrared light and transmits visible light (BG-36, www.chromatech.com) as well
as an emission filter is placed. The lifetime detector was a BH PMH-100-0 module
with a full width at half maximum (FWHM) response time of
150 ps, which was
recently upgraded to the BH hybrid HPM-100-40 module with a FWHM response
time of
120 ps. The hybrid lifetime detector uses a GaAsP PMT and offers higher
quantum efficiencies, lower dark counts, and no after-pulsing (see the applica-
tion note “HPM-100-40 Hybrid Detector” at www.becker-hickl.de/literature.htm),
which are the important characteristics for choosing a lifetime detector.
The TCSPC device is controlled using the BH SPCM software v8.91 and
contains PC plug-in boards (commercially available) that function as the time-
to-amplitude converter, time-to-digital converter, discriminator, and multichannel
analyzer [
177
] (also see the BH handbook available at
http://www.becker-hickl.
de/literature.htm#handb
)
. The TCSPC device synchronizes the lifetime detector to
the TPE pulse and the scanning clock and records both the arrival time relative to
the TPE pulse and the spatial (x, y,
z
) information for each detected photon. The
reference beam for a TPE pulse is acquired using a glass coverslip to reflect 4 % of
the MP laser to be converted by a photodiode to the reference signal that is fed to
the TCSPC device. Given a time period (a few seconds or minutes) of accumulating
emitted photons for thousands or millions of TPE pulses, a “photon counts” his-
togram (often termed as a decay profile) is built for each pixel of an image. The time
binning of the histogram depends on the time resolution of the TCSPC module: the
SPC-730 module used before provides a resolution of
156 ps or 64 time windows
in
10 ns; the SPC-150 module currently used offers a resolution of
39 ps or 256
time windows in
10 ns. The lifetime image is obtained by processing the decay
data using the BH SPCImage software v2.9.2.2989 (described below).
Since the lifetime detector is extremely sensitive, it is important to place the
lifetime detector in a sealed black box (see Fig.
3.6
) to minimize ambient light
detection. It is also suggested to cover the specimen chamber with a black cap
to avoid the reflection from the condenser of an inverted microscope. A TCSPC
FLIM system should be located away from any instruments which operate based on
magnetic fields, since the magnetic field will damage the lifetime detector. Magnetic
field interference can be avoided by putting a wire mesh around the whole system or
the detector. The FLIM system should be ideally placed on an antivibration table in
a room with a temperature of
20
ı
C. The room temperature should be consistent,
and its variance should not exceed
˙
2
ı
C.
3.4.3
Measuring the Instrument Response Function (IRF)
of the TPE-TCSPC FLIM System
In time-domain FLIM, a measured decay is the convolution form of the intrinsic
fluorescence decay and the IRF. The distortions can be caused by the finite rise
time, the width, and the decay of the excitation pulse, as well as the detector and
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