Abstract
We present a new technique, polarization-modulation dual-focus fluorescence correlation spectroscopy (pmFCS), based on the recently intro-duced dual-focus fluorescence correlation spectroscopy (2fFCS) to measure the absolute value of diffusion coefficients of fluorescent molecules at pico- to nanomolar concentrations. Analogous to 2fFCS, the new technique is robust against optical saturation in yielding correct values of the diffusion coefficient. This is in stark contrast to conventional FCS where optical saturation leads to an apparent decrease in the determined diffusion coefficient with increasing excitation power. However, compared to 2fFCS, the new technique is simpler to implement into a conventional confocal microscope setup and is compatible with cw-excitation, only needing as add-ons an electro-optical modulator and a differential interference contrast prism. With pmFCS, the measured diffusion coefficient (D) for Atto655 maleimide in water at 25?C is determined to be equal to (4.09 +/- 0.07) x 10(-6)cm(2)/s, in good agreement with the value of 4.04 x 10-6cm2/s as measured by 2fFCS.
Highlights
Precise diffusion measurement of biomolecules at pico- to nanomolar concentrations is a difficult but important task with many potential applications
The original 2fFCS set-up used two pulsed (50 ps pulse width, 40 MHz overall repetition rate), spectrally identical lasers for generating the two foci, and employed pulsed interleaved excitation (PIE) [11] and time-correlated single-photon counting (TCSPC) [12] to temporally distinguish the fluorescence signals originating from each focus
We demonstrate the technique by measuring the diffusion coefficient of a reference dye with known diffusion coefficient, and compare the result with results of 2fFCS using PIE
Summary
Precise diffusion measurement of biomolecules at pico- to nanomolar concentrations is a difficult but important task with many potential applications. The original 2fFCS set-up used two pulsed (50 ps pulse width, 40 MHz overall repetition rate), spectrally identical lasers for generating the two foci, and employed pulsed interleaved excitation (PIE) [11] and time-correlated single-photon counting (TCSPC) [12] to temporally distinguish the fluorescence signals originating from each focus. This technique works exceptionally well, one needs an expensive pulse-interleaved laser excitation system and TCSPC detection electronics and signal processing, which restricts the wider dissemination of the 2fFCS idea to existing FCS set-ups employing single laser cw-excitation without TCSCP capabilities. We demonstrate the technique by measuring the diffusion coefficient of a reference dye with known diffusion coefficient, and compare the result with results of 2fFCS using PIE
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