Fluorescence Lifetime Imaging (FLIM) with a focus on frequency domain FLIM with pco.flim
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Fluorescence Lifetime Imaging (FLIM) with a focus on frequency domain FLIM with pco.flim

Fluorescence lifetime imaging or FLIM in the time and frequency domain. The differences in fluorescence
lifetimes in luminophores can be used as an imaging
technique called FLIM. This can be achieved both in the
time and the frequency domain. Established examples of
time domain equipment are point scanning TCSPC systems. These point scanners count
the emission photons in time allowing you to reconstruct
a luminescence decay curve. This makes TCSPC suited for mixed,
fast and weak emission signals. On the downside the recording
speed of the process is slow, due to its point scanning nature. One frame of a 1000 times a 1000 pixels can take up to several minutes. And because it depends on
single photon counting it has a limited dynamic range. To prevent under or over exposing it has to be tuned for
either dim or bright signals. Another kind of time-domain FLIM
uses a digital image sensor. In this setup the sample is excited by a period train of rectangular light pulses. Each excitation pulse results in a slightly delayed
weaker emission signal. Starting with a sloped rise of the
emission intensity giving their rise time and a decaying emission after the excitation
light has been turned off giving the fall time. Repeatedly integrating over
the same narrow section of the decay curves leads to the first image. Then, the integration interval is slightly shifted to integrate over another
section of the decay curves giving the second image. This section-wise
integration is repeated until the whole
decay curve is sampled. The limitation of this approach are
the shortest possible exposure times of image sensors. The shortest known exposure time
at the release date of this video is about a 120 nano seconds. By the development of a novel fast
modulatable CMOS image sensor, the QM-FLIM-2, the so-called frequency domain
method became more attractive. In this frequency domain approach the
intensity of the excitation light is continuously modulated
using sine or square waves. The modulation source is the camera which provides modulation frequencies ranging from five kilohertz
up to 40 megahertz as well as the signal for dark gating. Using a sinusoidal excitation waveform the emission waveform is also a
sinusoid with the same frequency. It is delayed in time and
shows a decreased amplitude, a decreased constant component, and a decreased modulation depth. By comparison of the excitation
and emission sinusoids the time or phase shift can be determined, the phase angle Phi. Also the excitation and emission amplitudes A-excitation and A-emission become clear. And the constant components
B-excitation and B-emission. These characteristic parameters
can be determined by sampling and reconstructing the
emission signal by a kind of half-wave integration measured at at least four
different phase angles. The first half period integration is done
at a phase angle of zero degree giving the image i-1. Subsequently the next half period
corresponds to a phase angle of 180 degree resulting in image 1-3. If then the onset of the camera
integration is shifted by 90 degree, and the detection is repeated, the first integrated image i-2 will
correspond to a phase angle of 90 degree While the fourth image i-4 represents
a phase angle of 270 degree. By repeating these integrations
during a given exposure time a sufficient amount of light can be
captured to obtain a better signal. Finally, a sine curve is fitted into the measured
values in the images i-1 to i-4 for each pixel. This sine curve provides
the phase angle Phi the amplitude A-emission and the
constant component B-emission which are used to determine the
corresponding fluorescence lifetime values. These lifetime images extend the information obtained
by common intensity images. The reconstruction of the emission
signal by means of half wave integration requires a fast
modulatable image sensor. This is where the modulatable high-frequency
sensor QMFLIM-2 comes into play. It was developed by CSEM
and PCO from 2007 to 2012. In this unique image sensor each of the 1008 times 1008 pixels has two charge collection areas, tap A and tap B. An externally applied two-level modulation signal controls whether the generated charge
carriers pass through tap A or tap B. By modulating the excitation light and
the image sensor at the same frequency, specific phase shifts can
be measured in each pixel. At phase zero tap A is active. All charge carriers are
collected in charge bucket A. This is image i-1. At phase 180 tap B is active and all charge carriers go to charge bucket B. Resulting in image i-3. This kind of charge swing enables the simultaneous record of i-1 and i-3 in the same camera exposure
creating a double image. The image sensor modulation is shifted
relative to the excitation light, then a measurement is performed
at a phase angle of 90 degree. The charge carriers collected
in tap A create image i-2. Tap B samples the information
at a phase angle of 270 degree, creating image i-4. At a maximum modulation
frequency of 40 megahertz minimum half period integration intervals
of 12.5 nanoseconds can be achieved. So this is what makes
the pco.flim camera a versatile frequency
domain FLIM system. It can be used for the
measurements of a huge range of luminescence lifetimes
from tenths of microseconds down to 100 picoseconds. The PCO frequency domain FLIM system
is best in its class because: it covers the largest
range of lifetimes from tenth of microseconds up
to hundreds of picoseconds. It’s easy to apply. It has the highest resolution and the highest frame rate of the
available FLIM systems at the moment. And it’s price performance
is hard to beat. pco.flim is the perfect FLIM system
for fluorescence lifetime imaging, FRET measurements, dynamic
life cell experiments, optical chemical measurements like optical
oxygen measurements and more. For more information
please visit www.pco.de

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