Detection of Flowing Fluorescent Particles in a Microcapillary Using Fluorescence Correlation Spectroscopy

Abstract

Capillary flow experiments are described with fluorescent molecules, bacteria, and microspheres using fluorescence correlation spectroscopy as an analytical tool. The flow velocity in the microcapillary is determined by fitting autocorrelation traces with a model containing parameters related to diffusion and flow. The flow profile of pressure-driven flow inside a microcapillary is determined by using the fluorescence fluctuations of a small dye molecule. It was found that bacteria and microspheres are retarded in their flow by optical forces produced by the laser beam.

Design of a confocal microfluidic particle sorter using fluorescent photon burst detection

“…The confocal detection system consisted of an inverted fluorescence microscope (ConfoCor, Carl Zeiss, Jena, Germany, and EVOTEC Biosystems, Hamburg, Germany), especially designed to perform fluorescence correlation spectroscopy (FCS). 16,26 The 488 nm line from an argon laser was used to excite fluorescent molecules (Rhodamine Green, Rhodamine Green labeled dextrans) and fluorescent microspheres. Neutral density filters were used to decrease incident laser power when needed.…”

“…29 EOF can only be applied in channels smaller than ϳ100 m. The flow velocity of fluorescent molecules or particles through the channel can be determined with FCS as described previously. 7,16 Thus, a focused laser beam together with confocal detection optics was used to probe a tiny observation volume having a Gaussian three-dimensional (3D) intensity profile (ϳ0.5 by 3 m) inside the channel. Only those fluorescence photons emitted in the observation volume were detected and autocorrelated in real time yielding an autocorrelation curve.…”

“…30 In our case, the flow-FCS model was slightly adjusted to account for short-term phenomena like triplet-state kinetics of the molecules. 31 When there is active transport in the form of laminar flow (i.e., not turbulent), the autocorrelation function G͑ ͒ changes to 16,[32][33][34] …”

“…[11][12][13][14][15] Previous work in our laboratories reported on the detection of flowing fluorescent particles like bacteria and microspheres in a microcapillary mounted on a confocal fluorescence microscopy setup. 16 These results led to the design of a microfluidic biochip aimed at detection and sorting of relatively small particles in real time using the same confocal fluorescence microscope. The biochip is made from glass, containing a network of channels and reservoirs.…”

Global analysis of fluorescence fluctuation data

“…FCS measurements of molecules in solution include the observation of translational diffusion , rotational diffusion (Ehrenberg and Rigler 1974;Pecora 1975, 1976), intersystem crossing (Widengren et al 1995), chemical reactions (LaClair 1997, conformational dynamics of DNA hairpins (Bonnet et al 1998), photobleaching (Eggeling et al 2001), flavins and flavoproteins ; Van den Berg et al 2001), viscosity and photodynamics of red fluorescent proteins (Schenk et al 2004). FCS has also proved to be a suitable technique to detect nanoparticles and flowing particles (Akcakir et al 2000;Kunst et al 2002) and to study membrane-mimetic systems (Bastiaens et al 1994;Hink and Visser 1998;Hink et al 1999;Korlach et al 1999;Schwille et al 1999a;Fradin et al 2003). In addition, special studies have been addressed to investigate the flickering dynamics of variants of green fluorescent protein (GFP) (Haupts et al 1998;Schwille et al 2000;Heikal et al 2000), the hybridisation of DNA and RNA sequences (Walter et al 1996;Oehlenschla¨ger et al 1996), ligand-receptor interactions (Rauer et al 1996) and the binding of single-chain antibodies to gramnegative bacteria .…”