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Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule

Kai Hassler, Marcel Leutenegger, Per Rigler, Ramachandra Rao, Rudolf Rigler, Michael Gösch, and Theo Lasser

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Abstract

We designed a fluorescence correlation spectroscopy (FCS) system for measurements on surfaces. The system consists of an objective-type total internal reflection fluorescence (TIRF) microscopy setup, adapted to measure FCS. Here, the fluorescence exciting evanescent wave is generated by epi-illumination through the periphery of a high NA oil-immersion objective. The main advantages with respect to conventional FCS systems are an improvement in terms of counts per molecule (cpm) and a high signal to background ratio. This is demonstrated by investigating diffusion as well as binding and release of single molecules on a glass surface. Furthermore, the size and shape of the molecule detection efficiency (MDE) function was calculated, using a wave-vectorial approach and taking into account the influence of the dielectric interface on the emission properties of fluorophores.

©2005 Optical Society of America

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Figures (4)
Fig. 1.
Fig. 1. Schematic representation of the ‘objective-type TIR-FCS’ setup (left). L1 - L4: lenses; F1, F2: fluorescence filters; GP: glass plate; D: dichroic mirror; Obj: microscope objective; bfp: back focal plane of the objective; MS: motorized scanning stage. An enlargement of the ray-path inside the microscope objective (right). d: evanescent wave depth; Θ c : critical angle.

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Fig. 2.
Fig. 2. The normalized MDEs for confocal FCS (left) and TIR-FCS (right). The confocal FCS case was calculated for a 1.15 NA, 40 × water-immersion objective. For the TIR-FCS case a 1.45 NA, 100 × oil-immersion objective was considered. A diameter of the pinhole (core of the fiber) of 50μm was assumed in both cases. The excitation wavelength was 488 nm and the fluorescence emission wavelength was 542 nm.

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Fig. 3.
Fig. 3. Typical time trace for single rhodamine 6G molecules binding to a microscope slide (left). The right picture shows an enlargement of the two highest bursts. The binning time is 100μs.

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Fig. 4.
Fig. 4. Autocorrelation for diffusing rhodamine 6G molecules (upper left) and time trace (right). The overall measurement time was 30 s. Fitting the data with the model represented by equation 6 yields the following parameters: N = 1.2, τ z = 21.1μs, ω =0.38, p=15.4%, τt =1.6μs and cpm = 1.77 MHz. The red curve represents the fit to the autocorrelation data.

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Tables (1)

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Table 1. Parameter estimates for measurements with different pinhole diameters. pd: diameter of the pinhole. D: diffusion coefficient. ωt : theoretically obtained structure parameter. For other symbols refer to the discussion of equation 6. The parameter τ t was fixed to 1μs.

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Equations (7)

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MDE ( r ) = c CEF ( r ) I ( r ) .
d = λ 4 π ( n 2 2 sin 2 ( θ ) − n 1 2 ) − 1 2 .
CEF ( r ) = P ( z ) ∫ S circ ( q ′ a ) PSF ( q − q ′ , z ) d q ′ .
W n ≡ ∫ V MDE n ( r ) d r ,
MDE a ( x , y , z ) = exp ( − 2 ( x 2 + y 2 ) ω x y 2 ) exp ( − z h ) .
G ( τ ) = 1 + γ N [ 1 + p 1 − p exp ( − τ τ t ) ] ( 1 + τ ω 2 τ z ) − 1
× [ ( 1 − τ 2 τ z ) w ( i τ 4 τ z ) + τ π τ z ] .
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