Spatial fluorescence cross-correlation spectroscopy

Jaffiol, Rodolphe; Blancquaert, Yoann; Delon, Antoine; Derouard, Jacques

doi:10.1364/ao.45.001225

Cited by 31 publications

(23 citation statements)

References 25 publications

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“…Among these attempts where FCS in front of dielectric mirrors, [22] standing wave FCS, [23] total internal reflectance fluorescence (TIRF)-FCS, [24] or spatial correlation FCS between two detection volumes generated by detecting fluorescence through two laterally shifted pinholes. [25] The external ruler was provided either by the known modulation length of a standing light wave, or the estimated distance between the detection volumes. However, all the proposed methods suffer from the problem that for a precise quantification of the diffusion coefficient one still needs precise knowledge of the overall shape of the MDF.…”

Section: Introductionmentioning

confidence: 99%

Two‐Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements

et al. 2007

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We present a new method to measure absolute diffusion coefficients at nanomolar concentrations with high precision. Based on a modified fluorescence correlation spectroscopy (FCS)-setup, this method is improved by introducing an external ruler for measuring the diffusion time by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by a new two-parameter model to describe the molecule detection function (MDF). We present a recorded MDF and show the excellent agreement with the fitting model. We measure the diffusion coefficient of the red fluorescent dye Atto655 under various conditions and compare these values with a value achieved by gradient pulsed field NMR (GPF NMR). From these measurements we conclude, that the new measurement scheme is robust against optical and photophysical artefacts which are inherent to standard FCS. With two-focus-FCS, the diffusion coefficient of 4.26 x 10(-6) cm2s(-1) for Atto655 in water at 25 degrees C compares well with the GPF NMR value of 4.28 x 10(-6) cm2s(-1).

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Section: Introductionmentioning

confidence: 99%

Two‐Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements

et al. 2007

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“…A simplified scheme was to use an extended excitation volume which contains two separate detection volumes defined by two different pinholes. 32 However, this scheme sacrifices the spatial resolution of FCS and thus limits its applications. Here we present single pinhole spatial FCCS performed on the FCM for microfluidic flow as described in the optical setup.…”

Section: Single Pinhole Spatial Fccs For Flowmentioning

confidence: 99%

Multifunctional fluorescence correlation microscope for intracellular and microfluidic measurements

Pan

Foo

Lim

et al. 2007

Review of Scientific Instruments

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A modified fluorescence correlation microscope (FCM) was built on a commercial confocal laser scanning microscope (CLSM) by adding two sensitive detectors to perform fluorescence correlation spectroscopy (FCS). A single pinhole for both imaging and spectroscopy and a simple slider switch between the two modes thus facilitate the accurate positioning of the FCS observation volume after the confocal image acquisition. Due to the use of a single pinhole for CLSM and FCS the identity of imaged and spectroscopically observed positions is guaranteed. The presented FCM system has the capability to position the FCS observation volume at any point within the inner 30% of the field of view without loss in performance and in the inner 60% of the field of view with changes of FCS parameters of less than 10%. A single pinhole scheme for spatial fluorescence cross correlation spectroscopy performed on the FCM system is proposed to determine microfluidic flow angles. To show the applicability and versatility of the system, we measured the translational diffusion coefficients on the upper and lower membranes of Chinese hamster ovary cells. Two-photon excitation FCS was also realized by coupling a pulsed Ti: sapphire laser into the microscope and used for flow direction characterization in microchannels.

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“…To determine the theoretical expression of the fluorescence signal recorded with a confocal microscope, we closely followed the 2D scalar approach previously published [17]. The fluorescence signal recorded just after the pinhole, noted F(t), can be written as a fraction of the total signal in the pinhole plane, denoted fi(ri,zi,t) (we use the subscript i for the image space):…”

Section: Fluorescence Saturation In Confocal Microscopymentioning

confidence: 99%

Single molecule fluorescence saturation spectroscopy

Jaffiol¹,

Winckler²

2014

SPIE Proceedings

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Saturation spectroscopy is a powerful method to investigate photophysical parameters of single fluorescent molecules. Nevertheless, the impact of a gradual increase, over a broad range, of the laser excitation on the intramolecular dynamics is not completely understood, particularly concerning their fluorescence emission (the so-called brightness). As we have presented in a previous paper [1], we interpret the evolution of the brightness with the laser power by cascade absorption of two and three photons within a five-level molecular system. This multi-photon consecutive absorption leads us to reconsider the common expression of the saturation curve of fluorescent molecule. Furthermore, this multi-photon absorption process also affects the observation volume of microscope. So, in this paper we propose to interpret the size increase of the confocal observation volume according to simulations based upon two often used expressions of the Point Spread Functions (PSF) in fluorescence microscopy.

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Spatial fluorescence cross-correlation spectroscopy

Cited by 31 publications

References 25 publications

Two‐Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements

Two‐Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements

Multifunctional fluorescence correlation microscope for intracellular and microfluidic measurements

Single molecule fluorescence saturation spectroscopy

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