Volker Buschmann

Confocal Fluorescence Lifetime Imaging Microscopy (FLIM) at the Single Molecule Level

We report on confocal fluorescence lifetime imaging microscopy (CFLIM) of single dye molecules adsorbed on glass surface. Applying a short-pulse diode laser emitting at 635 nm with a repetition rate of 64 MHz we studied the time-resolved identification of individual carbocyanine and oxazine dyes via their characteristic fluorescence lifetimes of 2.06±0.37 ns (Cy5) and 3.89±0.91 ns (JA242). Fluctuations in fluorescence intensity and lifetime of individual adsorbed molecules were investigated with millisecond time resolution. These jumps exhibit short off-states τoff of 0.5 ms which can be ascribed to the triplet state lifetime under dry conditions with an intersystem crossing yield YISC of ∼0.2 %. Besides triplet states, other quantum jumps into longer lived states (several milliseconds) with lower transition probability were observed. The correlation of rotational and spectral jumps of single and coupled fluorophores with changes in the observed fluorescence lifetime are discussed.

Use of a novel Förster resonance energy transfer method to identify locations of site-bound metal ions in the U2–U6 snRNA complex

U2 and U6 snRNAs pair to form a phylogenetically conserved complex at the catalytic core of the spliceosome. Interactions with divalent metal ions, particularly Mg(II), at specific sites are essential for its folding and catalytic activity. We used a novel Förster resonance energy transfer (FRET) method between site-bound luminescent lanthanide ions and a covalently attached fluorescent dye, combined with supporting stoichiometric and mutational studies, to determine locations of site-bound Tb(III) within the human U2–U6 complex. At pH 7.2, we detected three metal-ion-binding sites in: (1) the consensus ACACAGA sequence, which forms the internal loop between helices I and III; (2) the four-way junction, which contains the conserved AGC triad; and (3) the internal loop of the U6 intra-molecular stem loop (ISL). Binding at each of these sites is supported by previous phosphorothioate substitution studies and, in the case of the ISL site, by NMR. Binding of Tb(III) at the four-way junction and the ISL sites was found to be pH-dependent, with no ion binding observed below pH 6 and 7, respectively. This pH dependence of metal ion binding suggests that the local environment may play a role in the binding of metal ions, which may impact on splicing activity.

Fluorescence correlation spectroscopy for flow rate imaging and monitoring—optimization, limitations and artifacts

We recently demonstrated a new method for mapping fluid velocities in 3 dimensions and with exceptionally high spatial resolution for the characterization of flow in microfluidic devices. In the method, a colloidal suspension containing fluorescent tracer particles, dye doped polymer spheres, is pumped through a microchannel and confocal microscopy combined with fluorescence correlation spectroscopy is used to measure fluid velocities. In this report, we further characterize the technique and report on optimizations that allow a 5-fold increase in speed of single point velocity measurements. This increase in measurement speed will yield a 25 fold reduction in the time needed to collect a complete velocity image. The precision of measured velocities was characterized as a function of tracer particle concentration, measurement time, and fluid velocity. In addition, we confirm the linearity of the measurement method (velocity vs. applied pressure) over a range of velocities spanning four orders of magnitude. Furthermore, we demonstrate that an artifact in velocity measurements using fluorescence correlation spectroscopy (FCS) that was interpreted by others as being caused by optical trapping forces is actually an artifact caused by detector saturation and can be avoided by careful choice of experimental conditions.

Application of Fluorescence Correlation Spectroscopy for Velocity Imaging in Microfluidic Devices

In this paper we present and demonstrate a technique for mapping fluid flow rates in microfluidic systems with sub-micrometer resolution using confocal microscopy in conjunction with fluorescence correlation spectroscopy (FCS). Flow velocities ranging from ∼50 μm/s to ∼ 10 cm/s can be recorded using fluorescent polymer nanospheres as fluid motion tracers. Velocity profiles and images of the flow in poly(dimethylsiloxane)–glass microchannels are presented and analyzed. Using the method, velocity images along the horizontal (top view) and vertical planes within a microdevice can be obtained. This is, to our knowledge, the first report of FCS for producing velocity maps. The high-resolution velocity maps can be used to characterize and optimize microdevice performance and to validate simulation efforts.

Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells

In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

Tailor-made dyes for fluorescence correlation spectroscopy (FCS).

Abstract Two new fluorescent labels are presented that are optimized for excitation with He/Ne laser and red diode lasers. Application in FCS and labeling of proteins and oligomers are demonstrated. A strong rise of quantum yield and emission life time upon binding to biomolecules are characteristic features of the dyes.

Use of Time-Resolved Fluorescence To Improve Sensitivity and Dynamic Range of Gel-Based Proteomics

Limitations in the sensitivity and dynamic range of two-dimensional gel electrophoresis (2-DE) are currently hampering its utility in global proteomics and biomarker discovery applications. In the current study, we present proof-of-concept analyses showing that introducing time-resolved fluorescence in the image acquisition step of in-gel protein quantification provides a sensitive and accurate method for subtracting confounding background fluorescence at the photon level. In-gel protein detection using the minimal difference gel electrophoresis workflow showed improvements in lowest limit of quantification in terms of CyDye molecules per pixel of 330-fold in the blue-green region (Cy2) and 8000-fold in the red region (Cy5) over conventional state-of-the-art image acquisition instrumentation, here represented by the Typhoon 9400 instrument. These improvements make possible the detection of low-abundance proteins present at sub-attomolar levels, thereby representing a quantum leap for the use of gel-based proteomics in biomarker discovery. These improvements were achieved using significantly lower laser powers and overall excitation times, thereby drastically decreasing photobleaching during repeated scanning. The single-fluorochrome detection limits achieved by the cumulative time-resolved emission two-dimensional electrophoresis (CuTEDGE) technology facilitates in-depth proteomics characterization of very scarce samples, for example, primary human tissue materials collected in clinical studies. The unique information provided by high-sensitivity 2-DE, including positional shifts due to post-translational modifications, may increase the chance to detect biomarker signatures of relevance for identification of disease subphenotypes.

A Novel Analysis Technique for Transcutaneous Measurement of Glomerular Filtration Rate With Ultralow Dose Marker Concentrations

Objective: A novel high-precision approach [lifetime-decomposition measurement (LTDM)] for the assessment of the glomerular filtration rate (GFR) based on clearance measurements of exogenous filtration marker. Methods: The time-correlated single photon counting (TCSPC) acquisition in combination with a new decomposition method allows the separation of signal and background from transcutaneous measurements of GFR. Results: The performance of LTDM is compared versus the commercially available NIC-kidney patch-based system for transcutaneous GFR measurement. Measurements are performed in awake Sprague Dawley (SD) rats. Using the standard concentration required for the NIC-kidney system [7-mg/100-g body weight (b.w.) FITC-Sinistrin] as reference, the mean difference (bias) of the elimination curves GFR between LTDM and NIC-kidney was 4.8%. On the same animal and same day, the capability of LTDM to measure GFR with a FITC-Sinistrin dose reduced by a factor of 200 (35-μg/100-g b.w.) was tested as well. The mean differences (half lives with low dose using LTDM compared with those using first, the NIC-Kidney system and its standard concentration, and second, LTDM with the same concentration as for the NIC-Kidney system) were 3.4% and 4.5%, respectively. Conclusion: We demonstrate that with the LTDM strategy substantial reductions in marker concentrations are possible at the same level of accuracy. Significance: LTDM aims to resolve the issue of the currently necessary large doses of fluorescence tracer required for transcutaneous GFR measurement. Due to substantially less influences from autofluorescence and artifacts, the proposed method outperforms other existing techniques for accurate percutaneous organ function measurement.

Velocity imaging in microfluidic devices using fluorescence correlation spectroscopy

In this paper we present and demonstrate a technique for mapping fluid flow rates in microfluidic systems with sub-micron resolution using confocal microscopy in conjunction with fluorescence correlation spectroscopy. Velocity profiles and velocity images of the fluid within poly(dimethylsiloxane)-glass microchannels are presented and analyzed. Flow velocities ranging from a few μm/s to a few cm/s can be recorded using nanometer-scale fluorescent polymer spheres as fluid motion tracers. The method is applied to mapping the hydrodynamic flow velocity in complex geometries. This is, to our knowledge, the first report of FCS for producing 2-dimensional velocity maps.

The use of time-resolved fluorescence in gel-based proteomics for improved biomarker discovery

This paper describes a new platform for quantitative intact proteomics, entitled Cumulative Time-resolved Emission 2-Dimensional Gel Electrophoresis (CuTEDGE). The CuTEDGE technology utilizes differences in fluorescent lifetimes to subtract the confounding background fluorescence during in-gel detection and quantification of proteins, resulting in a drastic improvement in both sensitivity and dynamic range compared to existing technology. The platform is primarily designed for image acquisition in 2-dimensional gel electrophoresis (2-DE), but is also applicable to 1-dimensional gel electrophoresis (1-DE), and proteins electroblotted to membranes. In a set of proof-of-principle measurements, we have evaluated the performance of the novel technology using the MicroTime 100 instrument (PicoQuant GmbH) in conjunction with the CyDye minimal labeling fluorochromes (GE Healthcare, Uppsala, Sweden) to perform differential gel electrophoresis (DIGE) analyses. The results indicate that the CuTEDGE technology provides an improvement in the dynamic range and sensitivity of detection of 3 orders of magnitude as compared to current state-of-the-art image acquisition instrumentation available for 2-DE (Typhoon 9410, GE Healthcare). Given the potential dynamic range of 7-8 orders of magnitude and sensitivities in the attomol range, the described invention represents a technological leap in detection of low abundance cellular proteins, which is desperately needed in the field of biomarker discovery.