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1 July 2006 Dual-color total internal reflection fluorescence cross-correlation spectroscopy
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We present the development and first application of a novel dual-color total internal reflection (TIR) fluorescence system for single-molecule coincidence analysis and fluorescence cross-correlation spectroscopy (FCCS). As a performance analysis, we measured a synthetic DNA-binding assay, demonstrating this dual-color TIR-FCCS approach to be a suitable method for measuring coincidence assays such as biochemical binding, fusion, or signal transduction at solid/liquid interfaces. Due to the very high numerical aperture of the epi-illumination configuration, our setup provides a very high fluorescence collection efficiency resulting in a two- to three-fold increase in molecular brightness compared to conventional confocal FCCS. Further improvements have been achieved through global analysis of the spectroscopic data.

Total internal reflection fluorescence microscopy (TIR-FM) is an important tool in life science. Taking advantage of a very thin (<100nm) optical excitation depth formed by an evanescent wave above a glass substrate, TIR fluorescence detection achieves an exceptional axial resolution and allows for the study of important cellular processes, in particular at or near the cellular membrane.1 Prominent biological applications of TIR-FM include real-time in vivo observations of dynamics of molecular motors,2, 3 or of various membrane trafficking events, such as vesicle fusion or signal transduction after receptor binding.4, 5 To extend the sensitivity and observation of such cellular assays, it is important to combine TIR-FM with fluorescence measurement and single-molecule fluctuation analysis. Fluorescence correlation spectroscopy enables to expose biological reactivity such as binding events on the basis of temporal fluctuations in the fluorescence emission of single molecules.6, 7 Dual-color fluorescence cross-correlation spectroscopy (FCCS) represents a further development of fluctuation spectroscopy, which achieves a significantly improved selectivity and applicability.8 FCCS institutes two differently colored labels and realizes the distinction between coinciding and separated occurrence of these labels. It thus introduces a useful tool to monitor various biological assays including molecular binding between two differently labeled binding partners. Typically, FCCS is based on a confocal epi-illumination microscope with multicolor laser excitation and single-photon detection. Nonetheless, for several applications, TIR illumination can provide further benefits, for instance when ligands interacting with receptors immobilized onto a surface are to be studied.9, 10, 11, 12 First, in TIR-FCCS the molecules are only excited near the glass-sample interface, which simplifies the discrimination between freely diffusing and immobilized particles. Second, the most important prerequisite for efficient single-molecule detection is a high fluorescence count rate per molecule (CPM). Using TIR excitation and exploiting the anisotropic emission of the fluorescence generated at the glass-sample interface, much higher can be extracted from the investigated molecules.13

In this work, we present for the first time a dual-color excitation scheme for TIR single-molecule detection, which enables us to perform FCCS at glass-sample interfaces with high sensitivity. The improved performance of TIR-FCCS was verified on a synthetic binding assay, including dual-labeled double-stranded DNA molecules in aqueous solution, demonstrating its potential for biological and cellular applications.

Figure 1 shows our dual-color TIR-FCCS setup. Two linear polarized laser beams were expanded to an e2 diameter of 2mm and collinearly aligned to the microscope objective (BE & BS; beam expansion 2.5× ). The laser powers were controlled by neutral density filters. An achromatic lens focused the beams into the back-focal plane (BFP) of the high numerical aperture (NA) oil immersion objective ( α -Plan-Fluar 100×1.45 with Immersoil™ 518F, Carl Zeiss Jena, Jena, Germany), which resulted in circular spots with e2 diameters at the cover slide-sample interface of 16μm (blue) and 20μm (red), respectively. In the BFP, a beam foci offset of 2.3mm from the optical axis resulted in a super-critical angle illumination, i.e., a z -confinement below 100nm resulting from the evanescent field excitation.

Fig. 1

Dual-color TIR-FCCS setup. HeNe 633: 18-mW HeNe gas laser (LHRP-1701, Laser 2000, Weßling, Germany). Pr 488: 22-mW solid-state laser (Protera™ 488-15, Novalux, Sunnyvale, CA). BE & BS: 2.5×beam expanders and beam steerers. CF: laser-line cleanup filters [Chroma Z488/10x (blue); Chroma Z633/10x (red); Chroma Technology Corp., Brattleboro, VT]. BC: dichroic beam combiner (Chroma Z488bcm). FL: focusing lens (achromat f=130mm ). L4: λ4 plate oriented for maximum fluorescence (OWIS, Staufen, Germany). DM: dual-band dichroic mirror (Omega DM488/633, Omega Optical Inc., Brattleboro, VT). Sample: droplet on a 150-μm -thick glass cover slide mounted on a xyz -translation stage (ULTRAlign 561D with μDrive Controller ESA-C, Newport Corp., Darmstadt, Germany). BS: dichroic beam splitter (Omega DML625). BF: emission band-pass filters [Chroma HQ540/80m and Omega 520DF40 (green); Chroma HQ690/80m and Omega 685DF70 (red)]. APD: detectors (Color online).


Due to the epi-illumination, the fluorescent light was collected with the same high NA objective toward the pinholes and the single-photon detectors. The pinholes were realized by two multimode fibers with a core diameter of 50μm . Dichroic mirrors and bandpass filters blocked the back-reflected laser light by more than OD10 and provided an excellent filtering of the green and red fluorescence. The fluorescence photons were detected by fiber-coupled single photon counting modules (SPCM-AQR-14-FC, PerkinElmer Optoelectronics, Wiesbaden, Germany; ASY50/105 silica fibers, Thorlabs Inc., Grünberg, Germany), whose signals (number of photons over time) were recorded and correlated with a USB hardware correlator (Flex02-08D,, Bridgewater, New Jersey). A focusing lens, a λ4 plate, and a dichroic mirror were moved in one block by a linear translator for adjusting the position of the beam foci in the BFP of the objective. In this way, the excitation angle could be adjusted without altering the optical path length between the focusing lens and the objective. The configuration can easily be changed to a conventional confocal epi-illumination by removing the focusing lens and by centering the collimated beams in the BFP.

As a synthetic binding assay, we used free rhodamine green (RhG) and Cy5 fluophores, and a 40 base pair double-stranded DNA labeled with Alexa488 and Cy5 (Zeiss cross-correlation standard). The laser powers were optimized with the dsDNA sample and the background was measured with a NaCl/EDTA/TRIS pH 8.0 buffer. The cross talk between the detection channels was calibrated with free 10-nM RhG and 50-nM Cy5, respectively. For cross-correlation measurements, different mixtures of the double-labeled dsDNA sample and free 9-nM RhG/ 50-nM Cy5 solutions were used and investigated with identical laser powers during a time interval of 20s . Plasma cleaning of the cover slides improved the results due to a strong suppression of unspecific binding at the glass surface.

For free diffusion the following model equation was used:13

Here, indices m and n represent the green and red detection channels; hence Ggg and Grr are the autocorrelations of the signals in the green and red detection channels, respectively, and Ggr is the cross-correlation of both signals. The index i represents the diffusing species: g for RhG, r for Cy5, and c for the dsDNA. Also, B is the measured background count rate, I is the total count rate, Qmi is the CPM in channel m of species i , Ni are the number of molecules in the effective sampling volume, Gmnt and τmnt are the triplet amplitudes and the triplet correlation times, respectively, Gmn1 are the offsets at infinite lag time τ , and Dmni describes the diffusion and is given by13
where τiz and τixy are the axial and lateral diffusion times of species i , respectively, and the scaled complementary error function is erfcx(x)=exp(x2)×erfc(x) . A multidimensional least-squares Gaussian-Newtonion algorithm was used to fit the experimental data to these model equations.

For these first experiments, only relative concentrations of the species were of interest. Therefore, a unique diffusion time was used, neglecting channel differences due to an imperfect overlap of the excitation and detection volumes. The triplet term accounts for the triplet contribution in the initial correlation amplitude G(0) .

The measured background signals were Bg=7.54kHz and Br=19.6kHz , respectively. Typically, autofluorescence from the immersion oil as well as from the glass slide were the main contributions to Bg and Br , respectively. The crosstalk CPM of RhG in the red channel was found to be Qrg=0.032×Qgg and assumed constant during the following evaluations. Cy5 did not show any crosstalk in the green channel, hence Qgr=0 . Therefore, the CPM were replaced by the crosstalk factor QrgQgg=0.032 for simplification.

Figure 2 shows the correlation results for a mixture of the free fluorophores (negative control). The count rates of this control sample were Ig=40.2kHz and Ir=66.0kHz . The CPMs were Qgg=(IgBg)Ng=13.1kHz and Qrr=[IrBr0.032×(IgBg)]Nr=18.9kHz , respectively. Figure 3 shows the results for the Cy5 and Alexa488 labeled dsDNA correlation (positive control). We measured Ig=39.3kHz and Ir=44.2kHz , from which Qgc=13.3kHz and Qrc=27.0kHz were extracted. The increased count rate for bound Cy5 is attributed to a reduction of conformational changes leading to higher fluorescence emission.14, 15 For τ> 10μs , the fit residuals were lower than 102 . For smaller lag times, shot noise and afterpulsing reduced the signal-to-noise ratio.

Fig. 2

Negative control ( 9-nM RhG/ 50-nM Cy5). Circles: green autocorrelation. Bold points: red autocorrelation. Dots: green-red cross-correlation. Inset: fit residuals GfitG(τ)1 (Color online).


Fig. 3

Positive control (dsDNA). Circles: green autocorrelation. Bold points: red autocorrelation. Dots: green-red cross-correlation. Inset: fit residuals (Color online).


Figure 4 summarizes the TIR-FCCS measurements on a titration series of double-labeled dsDNA in a mixture of free 9-nM RhG/ 50-nM Cy5, such that the total fluorophore concentration was approximately constant. The measured fraction of dsDNA scaled linearly with the mixed fraction from 1% (negative control) to 28% (positive control). The measurements showed a relative scatter of ±15% . The measured fraction was at best one-third of the mixed fraction. This can be explained by an excess of molecules with a single green label, possibly further enhanced by photobleaching of the red label during the two-color excitation.15 The overlap of the sampling volumes was estimated to 60% , which is close to the theoretical maximum. To reduce the influence of photobleaching, we used excitation intensities of 10μWμm2 . The measured CPM were about two times higher than with a confocal epi-illumination and a 1.20 NA water immersion objective at identical excitation intensities.13, 16

Fig. 4

Measured fraction of dsDNA versus mixed fraction


The diffusion times of the dsDNA were τz=51μs axially and τxy=2.3ms laterally, respectively. Given a penetration depth of 80nm and a waist radius of 370nm , the diffusion constant was calculated to D1.5×107cm2s , which is about 22% of the estimation DDNA6.8×107cm2s for a rodlike molecule with a diameter of 24Å and a length of 140Å . We attribute this to an increased hydrodynamic drag near the interface and weak unspecific binding, respectively.

This study demonstrates for the first time single-molecule FCCS measurements based on an epi-illumination TIR concept. This TIR-FCCS concept offers distinct advantages to confocal FCCS for coincidence assays at solid/liquid surfaces, in particular by virtue of the much higher fluorescence collection efficiencies and the confinement of the excitation field to the surface of interest.


We thank Aram Mooradian of Novalux for the Protera laser and his continous interest in our work, Klaus Weisshart of Carl Zeiss for contributing the cross-correlation sample, and Kai Hassler and Rudolf Rigler for many expert discussions. This work was supported by the Swiss National Science Foundation (SNSF) Grant No. 200021-103333. Jerker Widengren acknowledges support from the Swedish Strategic Research Foundation (SSF, BioX).



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© (2006) Society of Photo-Optical Instrumentation Engineers (SPIE)
Marcel Leutenegger, Michael Gösch, Rainer Andreas Leitgeb, Theo Lasser, Hans Blom, Jerker Widengren, and Christian Eggeling "Dual-color total internal reflection fluorescence cross-correlation spectroscopy," Journal of Biomedical Optics 11(4), 040502 (1 July 2006).
Published: 1 July 2006

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