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.
We report a new approach to an unidirectional photonic wire based on fluorescent dyes as chromophores and DNA as a rigid scaffold. The physical functioning of the wire is realized by dipole-dipole intreraction, i.e. resonant energy transfer, between chromophores. The use of four dyes (Alexa 430, TAMRA, Cy3.5, and Cy5) with different excited state energies creates an energy cascade constituting the driving force of the energy current and providing the unidirectionality of the device. The unique molecular properties of DNA, its scaffold-like structure, combined with straightforward synthesis methods allowed the engineering of a 30 base pair double-stranded DNA with inter-dye distances of 10 base pairs (3.4 nm), respectively, a range where electronic interactions between the chromophores can be neglected but dipole-dipole induced fluorescence resonance energy transfer (FRET) is expected to be still highly efficient. Steady-state and time-resolved ensemble spectroscopic measurements show an overall energy transfer efficiency of approximately 0.60. That is, the unidirectional transport of photonic energy over a distance of approximately 10 nm and a spectral separation of approximately 250 nm. Furthermore, pulsed diode laser excitation at 440 nm in combination with spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) was applied to characterize the effectiveness of individual photonic wires dispersed on glass coverslips.
We present a new technique for high-resolution colocalization of fluorescent dyes. The technique is based on polarization modulated excitation and spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM) as well as on coincidence analysis of the detected photon counts following pulsed laser excitation. The method takes advantage of single fluorescent dyes that can be efficiently excited by a single pulsed diode laser emitting at 635 nm but differ in their emission maxima, and in their fluorescence lifetime. A combined analysis of the fractional intensities and fluorescence lifetimes recorded on two spectrally-separated detectors enables the classification of the portion of each dye per pixel in a point-spread-function (PSF) image with high accuracy, even though only a limited number (generally a few thousand) photons are detected per single dye. From these portions two separate PSF images are calculated and fitted to two-dimensional (2D) Gaussian functions to localize their centers with a precision of a few nanometers. To reveal the number of absorbing and emitting molecules polarization modulated excitation and coincidence analysis of the detected photon counts is used. We demonstrate that by the use of appropriately selected dyes, the presented technique permits (1) the counting of the number of molecules present in the observation volume, and (2) the determination of the distance between two single molecules down to approximately 30 nm with a precision of approximately 10 nm without any chromatic aberrations. The developed techniques are promising for applications in molecular biology, e.g. to determine the number of polymerase molecules active within a transcription factory and/or to measure their distances to nanscent transcripts.
The technology to rapidly manipulate and screen individual molecules lies at the frontier of measurement science, with impacts in bio- and nano-technology. Fundamental biological and chemical processes can now be probed with unprecedented detail, one molecule at a time. These single molecule probes are most often fluorescent dye molecules embedded in a material or attached to a target molecule, such as a protein or nucleic acid, whose behavior us under study. The fluorescence from a single dye molecule can be detected, its spectrum and lifetime measured and its absorption or emission dipole calculated. From this information, the rotational and translational dynamics of the fluorophore can be calculated, as can details of its photophysics. To the extent that these properties reflect the properties of the target molecule, we can use these fluorescent tags to probe the dynamics and structure of the target. In this work we discuss the dependence of the physical and photophysical dynamics of fluorescent molecules on their local environment, and we use confocal microscopy to study single molecules in thin films, on surfaces, and in various liquid and gaseous environments.