In this work, we exploited time-resolved fluorescence polarization anisotropy (TRFA) to characterize un-doped and doped carbon dots (CDs). The rotational correlation time related to the size of the particle through classical Stokes-Einstein -Debye equation. The TRFA technique applied in this study achieves picoseconds time resolution, which approximately corresponds to the particle size determination at sub-nanometer precision. The calculated diameter of the CDs from the measured depolarization time constants in aqueous solution is well matches with the actual size of the CDs within the precision. This study proves that the TRFA method is highly complementary with other size determination techniques.
Imaging viscosity and its spatiotemporal patterns can provide valuable insight into the underlying physical conditions of biochemical reactions and biological processes in cells and tissues. One way to measure viscosity and diffusion is the use of fluorescence recovery after photobleaching (FRAP). We combine FRAP with FLIM and time-resolved fluorescence anisotropy imaging (tr-FAIM), by acquiring time- and polarization-resolved fluorescence images in every frame of a FRAP series. This allows us to simultaneously monitor translational and rotational diffusion. This approach can be applied to measuring diffusion in homogeneous and heterogeneous environments, and in principle also allows the study of homo-FRET. Another way to measure viscosity and diffusion is through specific flexible dyes, e.g. fluorescent molecular rotors, whose fluorescence quantum yield and fluorescence lifetime depend on the viscosity of the environment, in combination with fluorescence lifetime imaging (FLIM). We show that a bodipybased fluorescent molecular rotor targeting mitochondria reports on their viscosity, which changes under physiological stimuli. Both methods can optically measure viscosity and diffusion on the micrometer scale.
We report the simultaneous combination of three powerful techniques in uorescence microscopy: Fluorescence Lifetime Imaging (FLIM), Fluorescence Anisotropy Imaging (FAIM) and Fluorescence Recovery After Photobleaching (FRAP), also called F3 microscopy. An exhaustive calibration of the setup was carried out with several rhodamine 6G (R6G) solutions in water-glycerol and from the combination of the FAIM and FRAP data, the hydrodynamic radius of the dye was directly calculated. The F3 data was analyzed with a home-built MATLAB script, and the setup is currently explored further with Green Fluorescent Protein (GFP). Some molecular dynamic (MD) simulations are currently being run in order to help with the interpretation of the experimental anisotropy data.
Fluorescence-based processes are strongly modified by the electromagnetic environment in which the emitters are placed. Hence, the design of nanostructured materials with appropriate electromagnetic properties opens up a new route in the control of, for instance, the spontaneous rate of emission or the energy transfer rate in donor-acceptor pairs. In particular, hyperbolic plasmonic metamaterials have emerged as a very flexible and powerful platform for these applications as they provide a high local density of electromagnetic states due to their peculiar mode structure which is governed by both the structural nonlocal response and the dispersion properties. Here, we will discuss an experimental and theoretical study of the influence of a hyperbolic metamaterial comprised of an array of gold nanorods on the radiative properties of quantum emitters and the energy-transfer processes between them.
Time-correlated single photon counting (TCSPC) is a widely used, sensitive, precise, robust and mature technique to measure photon arrival times in applications such as fluorescence spectroscopy and microscopy, light detection and ranging (lidar) and optical tomography. Wide-field TCSPC detection techniques, where the position and the arrival time of the photons are recorded simultaneously, have seen several advances in the last few years, from the microsecond to the picosecond time scale. Here, we summarise some of our recent work in this field with emphasis on microsecond resolution phosphorescence lifetime imaging (PLIM) and nanosecond fluorescence lifetime imaging (FLIM) microscopy.
We report the use of Time-Correlated Single Photon Counting (TCSPC) in a polarization-resolved Fluorescence Lifetime Imaging (FLIM) setup for the investigation of cell membrane structural and dynamic properties. This technique allows us to study the orientation and mobility of fluorescent membrane dyes, namely di-4-ANEPPDHQ and DiO, in model bilayers of different lipid compositions. Dipole alignment and extent of rotational motion can be linked to membrane order and fluidity. Comparison of the time-resolved anisotropy decays of the two fluorescent dyes suggests that rotational motion of membrane constituents is restricted in liquid-ordered phases, and appears to be limited to the region of aliphatic tails in liquid-disordered phases. In living cells, understanding the membrane structure provides crucial information on its functional properties, such as exo- and endocytosis, cell mobility and signal transduction.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.