We have used an experimental arrangement comprising two photomultipliers and time-correlated single photon counting
(TCSPC) detection to measure time and polarization-resolved fluorescence decays and images simultaneously.
Polarization-resolved measurements can provide information which may be difficult to extract from lifetime
measurements alone. The combination of fluorescence lifetime and time-resolved anisotropy in an imaging modality
with two detectors minimizes the errors arising from bleaching of a sample between consecutive measurements.
Anisotropy measurements can provide evidence of fluorescence resonance energy transfer between chemically identical
fluorophores (homo-FRET). This phenomenon is not detectable in spectral or lifetime changes, yet a lowering of the
anisotropy and a faster anisotropy decay can provide evidence for close proximity (≤ 10 nm) of adjacent fluorophores
including dimerization and oligomerization of molecules. We have used FLIM and fluorescence anisotropy to measure
variations in fluorescence lifetimes and anisotropy of GFP-tagged proteins in cells in immunological synapse samples
and also acquire images of BODIPY-stained carcinoma cells.
We demonstrateWide-Field Time-Correlated Single Photon Counting (WiFi TCSPC) imaging based on an image
intensifier and a high-speed camera running at 30,000 frames per second. The timing of photon events is thus
performed in parallel, simultaneously on every pixel. The system is applied to lanthanide lifetime measurements
and time-resolved imaging of the lanthanide complex Europium Polyoxometalate (Eu POMs). We measure a
lifetime of 2.98 ms for Eu POMs in solid state, which is in excellent agreement with the literature value.
We present a novel time-resolved photon counting imaging technique and its use in multi-dimensional luminescence
spectroscopy. By using an ultrafast camera coupled to an image intensifier on a microscope, we demonstrate
the potential of wide-field time-correlated single photon counting, with a count rate of up to 5 Mhz. This system
has the advantage of allowing the detection of single photons in parallel in every pixel. We measured the
luminescence decay of Europium Polyoxometalate (POM), and observed contrast on lifetime images of Eu-POM
on silver nanocrystals.
Fluorescence imaging techniques are powerful tools in the biological and biomedical sciences, because they are
minimally invasive and can be applied to live cells and tissues. The fluorescence emission can be characterized not only
by its intensity and position, by also by its fluorescence lifetime, polarization and wavelength. Fluorescence Lifetime
Imaging (FLIM) in particular has emerged as a key technique to image the environment and interaction of specific
proteins in living cells. Using a time-correlated single photon counting (TCSPC)-based FLIM set-up, we find that the
fluorescence lifetime of GFP-tagged proteins in cells is a function of the refractive index of the medium the cells are
suspended in. In addition, combining Fluorescence Recovery After Photobleaching (FRAP) of fluorescently labeled
proteins of different sizes in sol gels with time-resolved fluorescence anisotropy measurements, we demonstrate that we
can measure their lateral and rotational diffusion. This allows us to infer the size and connectivity of the pores in the sol
gel matrix. Moreover, wide-field photon counting imaging, originally developed for astronomical applications, is a
powerful imaging method because of its high sensitivity and excellent signal-to-noise ratio. It has a distinct advantage
over CCD-based imaging due to the ability to time the arrival of individual photons. The potential of time-resolved wide-field
photon counting imaging with a fast CMOS camera applied to luminescence microscopy is demonstrated.