Felix Koberling

Multi-target spectrally resolved fluorescence lifetime imaging microscopy

We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).

Self-diffusion and cooperative diffusion in semidilute polymer solutions as measured by fluorescence correlation spectroscopy

We present a comprehensive investigation of polymer diffusion in the semidilute regime by fluorescence correlation spectroscopy (FCS) and dynamic light scattering (DLS). Using single-labeled polystyrene chains, FCS leads to the self-diffusion coefficient while DLS gives the cooperative diffusion coefficient for exactly the same molecular weights and concentrations. Using FCS we observe a new fast mode in the semidilute entangled concentration regime beyond the slower mode which is due to self-diffusion. Comparison of FCS data with data obtained by DLS on the same polymers shows that the second mode observed in FCS is identical to the cooperative diffusion coefficient measured with DLS. An in-depth analysis and a comparison with current theoretical models demonstrates that the new cooperative mode observed in FCS is due to the effective long-range interaction of the chains through the transient entanglement network.

Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy

Quantitative distance measurements are difficult to obtain in spite of the strong distance dependency of the energy transfer efficiency. One problem for the interpretation of the Forster resonant energy transfer (FRET) efficiency is the so-called zero-efficiency peak caused by FRET pairs with missing or nonfluorescent acceptors. Other problems occurring are direct excitation of the acceptor, spectral crosstalk, and the determination of the quantum efficiency of the dyes as well as the detector sensitivity. Our approach to overcome these limitations is based on the pulsed-interleaved excitation (PIE) of both the acceptor and the donor molecule. PIE is used to excite the acceptor dye independently of the FRET process and to prove its existence via fluorescence. This technique enables us to differentiate a FRET molecule, even with a very low FRET efficiency, from a molecule with an absent or non-fluorescent acceptor. Crosstalk, direct acceptor excitation, and molecular brightness of acceptor and donor molecules are determined by analyzing the data with fluorescence correlation spectroscopy (FCS). FRET efficiencies of the same data set are also determined by analyzing the lifetimes of the donor fluorophores. The advantages of the PIE-FRET approach are demonstrated on a polyproline assay labeled with Alexa-555 and Alexa-647 as donor and acceptor, respectively.

Tip induced fluorescence quenching for nanometer optical and topographical resolution

Progress in nanosciences and life sciences is closely related to developments of high resolution imaging techniques. We introduce a technique which produces correlated topography and fluorescence lifetime images with nanometer resolution. Spot sizes below 5 nm are achieved by quenching of the fluorescence with silicon probes of an atomic force microscope which is combined and synchronized with a confocal fluorescence lifetime microscope. Moreover, we demonstrate the ability to locate and resolve the position of two fluorescent molecules separated by 20.7 nm on a DNA origami triangle with 120 nm side length by correlating topography and fluorescence data. With this method, we anticipate applications in nano- and life sciences, such as the determination of the structure of macromolecular assemblies on surfaces, molecular interactions, as well as the structure and function of nanomaterials.

Simultaneous single molecule atomic force and fluorescence lifetime imaging

The combination of atomic force microscopy (AFM) with single-molecule-sensitive confocal fluorescence microscopy enables a fascinating investigation into the structure, dynamics and interactions of single biomolecules or their assemblies. AFM reveals the structure of macromolecular complexes with nanometer resolution, while fluorescence can facilitate the identification of their constituent parts. In addition, nanophotonic effects, such as fluorescence quenching or enhancement due to the AFM tip, can be used to increase the optical resolution beyond the diffraction limit, thus enabling the identification of different fluorescence labels within a macromolecular complex. We present a novel setup consisting of two commercial, state-of-the-art microscopes. A sample scanning atomic force microscope is mounted onto an objective scanning confocal fluorescence lifetime microscope. The ability to move the sample and objective independently allows for precise alignment of AFM probe and laser focus with an accuracy down to a few nanometers. Time correlated single photon counting (TCSPC) gives us the opportunity to measure single-molecule fluorescence lifetimes. We will be able to study molecular complexes in the vicinity of an AFM probe on a level that has yet to be achieved. With this setup we simultaneously obtained single molecule sensitivity in the AFM topography and fluorescence lifetime imaging of YOYO-1 stained lambda-DNA samples and we showed silicon tip induced single molecule quenching on organic fluorophores.

Two-channel fluorescence lifetime microscope with two colour laser excitation, single-molecule sensitivity, and submicrometer resolution

We present results from a two channel confocal microscope set-up allowing one to efficiently record two-colour as well as polarization resolved time-correlated single molecule fluorescence data. In addition to their spectral characteristics, single molecules can be distinguished by their fluorescence lifetime and polarization. This provides independent distinctive information and results in enhanced detection sensitivity. The set-up we present uses two picosecond diode lasers (440nm and 635 nm) for fluorescence excitation and a piezo scanner for sample movement. A learning scan algorithm permits very fast piezo scanner movement and offers a superior positioning accuracy on single molecules. The time-correlated photon counting system uses Time-Tagged Time-Resolved (TTTR) data aquisition, in which each photon is recorded individually. This method allows for the reconstruction not only fluorescence decay constants of each pixel for the purpose of Fluorescence Lifetime Imaging (FLIM) but also to analyze the fluorescence fluctuation correlation function on a single spot of interest. Cross-correlation between two channels can be used to eliminate detector artifacts. Finally, fluorescence antibunching can also be analyzed. We show results obtained with immobilized and diffusing red and blue excited fluorescently labelled latex microspheres, as well as from single fluorophore molecules.

Recent advances in time-correlated single-photon counting

We report about the time-resolved confocal fluorescence microscope MicroTime 200, which is completely based on TTTR format data acquisition and enables to perform very advanced FCS, FRET and FLIM analysis such as Fluorescence Lifetime Correlation Spectroscopy (FLCS) or Two Focus FCS (2fFCS). FLCS is a fundamental improvement of standard FCS overcoming many of its inherent limitations. The basic idea of FLCS is a weighting of the detected photons based on the additional picosecond timing information (TCSPC start-stop time) when using pulsed laser excitation. 2fFCS goes even further, combining Pulsed Interleaved Excitation (PIE) with a time-gated FCS analysis. The basic implementation of 2fFCS uses two synchronized but interleaved pulsed lasers of the same wavelength but of different polarisation to generate two close by excitation foci in a pre-determined distance acting as a submicron ruler. In this case it it no longer necessary to have prior knowledge about the size and shape of the confocal volume. Maintaining the information about the photon´s origin, the dual focus data allows a precise calculation of absolute diffusion coefficients.

ns-Time Resolution for Multispecies STED-FLIM and Artifact Free STED-FCS

Stimulated Emission Depletion (STED) Microscopy has evolved into a well established method offering optical superresolution below 50 nm. Running both excitation and depletion lasers in picosecond pulsed modes allows for highest optical resolution as well as fully exploiting the photon arrival time information using time-resolved single photon counting (TCSPC). Non-superresolved contributions can be easily dismissed through time-gated detection (gated STED) or a more detailed fluorescence decay analysis (FLIM-STED), both leading to an even further improved imaging resolution. Furthermore, these methods allow for accurate separation of different fluorescent species, especially if subtle differences in the excitation and emission spectra as well as the fluorescence decay are taken into account in parallel. STED can also be used to shrink the observation volume while studying the dynamics of diffusing species in Fluorescence Correlation Spectroscopy (FCS) to overcome averaging issues along long transit paths. A further unique advantage of STED-FCS is that the observation spot diameter can be tuned in a gradual manner enabling, for example, determining the type of hindered diffusion in lipid membrane studies. Our completely pulsed illumination scheme allows realizing an improved STED-FCS data acquisition using pulsed interleaved excitation (PIE). PIE-STED-FCS allows for a straightforward online check whether the STED laser has an influence on the investigated diffusion dynamics.

Fast algorithms for the analysis of spectral FLIM data

The combination of simultaneous spectral detection together with Fluorescence Lifetime Imaging (sFLIM) allows collecting the complete information inherent to the fluorescence signal. Their fingerprint of lifetime and spectral properties identify the fluorescent labels unambiguously. Multiple labels can be investigated in parallel and separated from inherent auto-fluorescence of the sample. In addition, spectral FLIM FRET has the prospect to allow simultaneous detection of multiple FRET signals with quantitative analysis of FRET-efficiency and degree of binding. Spectral FLIM measurements generate huge amount of data. Suitable analysis procedures must be found to condense the inherent information to answer the scientific questions in a straightforward way. Different analysis techniques have been evaluated for a diversity of applications as multiplex labeling, quantitative determination of environmental parameters and distance measurements via FLIM FRET. In order to reach highest sensitivity in single photon detection, different detector types are investigated and developed. SPAD arrays equipped with micro-lenses promise superior detection efficiency while the integration of a spectrograph with a PMT array is easier to realize and allows for a higher number of detection channels. High detection speed can be realized through parallel TCSPC channels. In order to overcome the limits of the USB 2.0 interface, new interface solutions have been realized for the multichannel TCSPC unit HydraHarp 400.

Front Matter: Volume 8228

This PDF file contains the front matter associated with SPIE Proceedings Volume 8228, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.